AJP: Renal Physiology recent issues

Cell biology and physiology of the uroepithelium

Khandelwal, P., Abraham, S. N., Apodaca, G. — Tue, 24 Nov 2009 14:09:56 PST

The uroepithelium sits at the interface between the urinary space and underlying tissues, where it forms a high-resistance barrier to ion, solute, and water flux, as well as pathogens. However, the uroepithelium is not simply a passive barrier; it can modulate the composition of the urine, and it functions as an integral part of a sensory web in which it receives, amplifies, and transmits information about its external milieu to the underlying nervous and muscular systems. This review examines our understanding of uroepithelial regeneration and how specializations of the outermost umbrella cell layer, including tight junctions, surface uroplakins, and dynamic apical membrane exocytosis/endocytosis, contribute to barrier function and how they are co-opted by uropathogenic bacteria to infect the uroepithelium. Furthermore, we discuss the presence and possible functions of aquaporins, urea transporters, and multiple ion channels in the uroepithelium. Finally, we describe potential mechanisms by which the uroepithelium can transmit information about the urinary space to the other tissues in the bladder proper.

A new role for vitamin D receptor activation in chronic kidney disease

Valdivielso, J. M., Cannata-Andia, J., Coll, B., Fernandez, E. — Tue, 24 Nov 2009 14:09:56 PST

Vitamin D has proven to be much more than a simple "calcium hormone." The fact that the vitamin D receptor has been found in cells not related to mineral metabolism supports that statement. The interest of nephrologists in vitamin D and its effects beyond mineral metabolism has increased over the last few years, evidencing the importance of this so-called "sunshine hormone." In the present review, we highlight the most recent developments in the traditional use of vitamin D in chronic kidney disease (CKD) patients, namely, the control of secondary hyperparathyroidism (sHPT). Furthermore, we also explore the data available regarding the new possible therapeutic uses of vitamin D for the treatment of other complications present in CKD patients, such as vascular calcification, left ventricular hypertrophy, or proteinuria. Finally, some still scarce but very promising data regarding a possible role of vitamin D in kidney transplant patients also are reviewed. The available data point to a potential beneficial effect of vitamin D in CKD patients beyond the control of mineral metabolism.

Tissue-type plasminogen activator deficiency attenuates peritoneal fibrosis in mice

Tue, 24 Nov 2009 14:09:56 PST

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis therapy. The present study was performed to examine the mechanisms of PF in view of the plasminogen activator (PA)/plasmin/matrix metalloproteinase (MMP) cascade. PF was induced in tissue-type PA (tPA) deficient mice and wild-type mice by intraperitoneal injection of chlorhexidine gluconate. Mice were killed on day 21, and tissue samples were taken. Histopathological studies were performed. Plasmin activity, gelatinases activity, and the levels of tPA, transforming growth factor-β1 (TGF-β1), and MMP-2 mRNA were determined. Protein levels of MMP-3, tissue inhibitor of metalloproteinases (TIMP)-1, -2, and -3, phospho-Smad3, membrane-type 1 (MT1)-MMP, and MT3-MMP were also studied. On day 21, tPA +/+ mice showed severe PF, whereas tPA –/– mice showed milder change. Submesothelial basement membranes were dissolved in tPA +/+ mice while they were relatively preserved in tPA –/– mice. The levels of macrophage infiltration, staining for -smooth muscle actin (-SMA) and collagen type III, and vascular density were all significantly lower in tPA –/– mice than in tPA +/+ mice. The levels of plasmin activity, pro- and active MMP-2, mRNA expression of tPA and TGF-β1, and phospho-Smad3 protein were also lower in tPA –/– mice. No difference was observed between the two groups concerning the protein levels of MMP-3, TIMP-1, TIMP-2, TIMP-3, MT1-MMP, or MT3-MMP. These results indicate that the presence of tPA enhances inflammation, angiogenesis, and fibrogenesis in the peritoneum of the PF model mice. Activation of the PA/plasmin/MMP cascade may play a pivotal role in the pathogenesis of PF.

Diverse vasopressin V2 receptor functionality underlying partial congenital nephrogenic diabetes insipidus

Tue, 24 Nov 2009 14:09:56 PST

X-linked congenital nephrogenic diabetes insipidus (CNDI) is characterized by a defective renal response to the antidiuretic hormone (AVP) due to variations in the arginine vasopressin receptor 2 (AVPR2) gene. In a unique group of patients, the renal insensitivity to the effects of AVP is incomplete resulting in a partial phenotype. To investigate the molecular defects, two previously published variations in the AVPR2 gene, known to cause a partial CNDI phenotype, were expressed in transiently transfected human embryonic kidney cells. One variation (p.Arg104Cys) is located in the first extracellular loop and the other variation (p.Ser329Arg) is located in the intracellular COOH terminal of the receptor protein. Western blotting showed almost equal amounts of WT-V2R and Arg104Cys-V2R protein at steady state, whereas the level of Ser329Arg-V2R protein was lower. Confocal microscopy established that WT-V2R and Arg104Cys-V2R are localized on the cellular surface while the Ser329Arg-V2R primarily accumulates within the endoplasmic reticulum resulting in reduced surface expression. Ligand binding analysis demonstrated that the B_max for cells expressing Arg104Cys-V2R and Ser329Arg-V2R were 14.8- and 2.5-fold lower than B_max for WT-V2R, respectively. AVP affinity (1/K_d) for WT-V2R and the Ser329Arg-V2R was similar while 1/K_d for Arg104Cys-V2R was increased. cAMP assay revealed that cells expressing p.Arg104Cys-V2R or p.Ser329Arg-V2R produced 1.7- and 6.8-fold lower amounts of cAMP compared with WT-V2R, respectively. In conclusion, ligand binding and signal transduction capability are dependent on localization of the amino acid variation. Striking divergences at the level of receptor functionality may thus underlie similar clinical phenotypes in CNDI.

Aberrant planar cell polarity induced by urinary tract obstruction

Tue, 24 Nov 2009 14:09:56 PST

Flow sensing by primary cilia of the epithelial cells is involved in cystogenesis in polycystic kidney disease. We investigate whether a similar mechanism applies to the pathogenesis of cyst-like tubular dilatation induced by ureteral obstruction in mice. Robust proliferation occurs in the obstructed tubules when urine flow is interrupted as well as in the repairing tubules when urine flow is reestablished after relief of the obstruction, suggesting a urine flow-independent mechanism of proliferation. In the urothelium, proliferation is only detected above the obstruction, although urine flow ceased both above and below the obstruction. Our results support mechanical strain- rather than flow-mediated proliferation in obstructive uropathy. To understand the mechanism of cell proliferation leading to increased tubular diameter in cyst-like tubular dilatation, we examine planar cell polarity (PCP), which is necessary for oriented cell division and maintenance of tubular diameter. In dilated tubules, the orientation of cell division is randomized, atypical PKC (aPKC) is mislocalized, and the pattern of the expression of a core PCP protein, Frizzled3 (Fz3), is altered. In addition, the level of Fz3 expression is increased. These results indicate that aberrant PCP may contribute to cyst-like tubular dilatation in obstructive uropathy. Interestingly, the orientation of cell division, localization of aPKC, and Fz3 expression return to normal when obstruction is relieved, which suggest a role of normal PCP signaling in tubular repair.

Carbachol-induced rabbit bladder smooth muscle contraction: roles of protein kinase C and Rho kinase

Wang, T., Kendig, D. M., Smolock, E. M., Moreland, R. S. — Tue, 24 Nov 2009 14:09:56 PST

Smooth muscle contraction is regulated by phosphorylation of the myosin light chain (MLC) catalyzed by MLC kinase and dephosphorylation catalyzed by MLC phosphatase. Agonist stimulation of smooth muscle results in the inhibition of MLC phosphatase activity and a net increase in MLC phosphorylation and therefore force. The two pathways believed to be primarily important for inhibition of MLC phosphatase activity are protein kinase C (PKC)-catalyzed CPI-17 phosphorylation and Rho kinase (ROCK)-catalyzed myosin phosphatase-targeting subunit (MYPT1) phosphorylation. The goal of this study was to determine the roles of PKC and ROCK and their downstream effectors in regulating MLC phosphorylation levels and force during the phasic and sustained phases of carbachol-stimulated contraction in intact bladder smooth muscle. These studies were performed in the presence and absence of the PKC inhibitor bisindolylmaleimide-1 (Bis) or the ROCK inhibitor H-1152. Phosphorylation levels of Thr³⁸-CPI-17 and Thr⁶⁹⁶/Thr⁸⁵⁰-MYPT1 were measured at different times during carbachol stimulation using site-specific antibodies. Thr³⁸-CPI-17 phosphorylation increased concurrently with carbachol-stimulated force generation. This increase was reduced by inhibition of PKC during the entire contraction but was only reduced by ROCK inhibition during the sustained phase of contraction. MYPT1 showed high basal phosphorylation levels at both sites; however, only Thr⁸⁵⁰ phosphorylation increased with carbachol stimulation; the increase was abolished by the inhibition of either ROCK or PKC. Our results suggest that during agonist stimulation, PKC regulates MLC phosphatase activity through phosphorylation of CPI-17. In contrast, ROCK phosphorylates both Thr⁸⁵⁰-MYPT1 and CPI-17, possibly through cross talk with a PKC pathway, but is only significant during the sustained phase of contraction. Last, our results demonstrate that there is a constitutively activate pool of ROCK that phosphorylates MYPT1 in the basal state, which may account for the high resting levels of MLC phosphorylation measured in rabbit bladder smooth muscle.

Inflammation compromises renal dopamine D1 receptor function in rats

Asghar, M., Chugh, G., Lokhandwala, M. F. — Tue, 24 Nov 2009 14:09:56 PST

We tested the effects of inflammation on renal dopamine D1 receptor signaling cascade, a key pathway that maintains sodium homeostasis and blood pressure during increased salt intake. Inflammation was produced by administering lipopolysaccharide (LPS; 4 mg/kg ip) to rats provided without (normal salt) and with 1% NaCl in drinking water for 2 wk (high salt). Control rats had saline injection and received tap water. We found that LPS increased the levels of inflammatory cytokines, interleukin-6, and tumor necrosis factor- in the rats given either normal- or high-salt intake. Also, these rats had higher levels of oxidative stress markers, malondialdehyde and nitrotyrosine, and lower levels of antioxidant enzyme superoxide dismutase in the renal proximal tubules (RPTs). The nuclear levels of transcription factors NF-B increased and Nrf2 decreased in the RPTs in response to LPS in rats given normal and high salt. Furthermore, D1 receptor numbers, D1 receptor proteins, and D1 receptor agonist (SKF38393)-mediated ³⁵S-GTPS binding decreased in the RPTs in these rats. The basal activities of Na-K-ATPase in the RPTs were similar in control and LPS-treated rats given normal and high salt. SKF38393 caused inhibition of Na-K-ATPase activity in the primary cultures of RPTs treated with vehicle but not in the cultures treated with LPS. Furthermore, LPS caused an increase in blood pressure in the rats given high salt but not in the rats given normal salt. These results suggest that LPS differentially regulates NF-B and Nrf2, produces inflammation, decreases antioxidant enzyme, increases oxidative stress, and causes D1 receptor dysfunction in the RPTs. The LPS-induced dysfunction of renal D1 receptors alters salt handling and causes hypertension in rats during salt overload.

Aggravated renal inflammatory responses in TRPV1 gene knockout mice subjected to DOCA-salt hypertension

Wang, Y., Wang, D. H. — Tue, 24 Nov 2009 14:09:56 PST

To test the hypothesis that deletion of the transient receptor potential vanilloid type 1 (TRPV1) channel exaggerates hypertension-induced renal inflammatory response, wild-type (WT) or TRPV1-null mutant (TRPV1^–/–) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for 4 wk. Mean arterial pressure (MAP) determined by radiotelemetry increased in DOCA-salt-treated WT or TRPV1^–/– mice, whereas there was no difference in MAP between two strains at the baseline or after DOCA-salt treatment. DOCA-salt treatment increased urinary excretion of albumin and 8-isoprostane in both WT and TRPV1^–/– mice, and the increases were greater in magnitude in the latter strain. Periodic acid-Schiff and Mason's trichrome staining showed that kidneys of DOCA-salt-treated TRPV1^–/– mice exhibited more severe glomerulosclerosis and tubulointerstitial injury compared with DOCA-salt-treated WT mice. NF-B assay showed that DOCA-salt treatment increased renal activated NF-B concentrations in TRPV1^–/– mice compared with WT mice. Immunostaining and ELISA assay revealed that DOCA-salt-treated TRPV1^–/– mice had enhanced renal infiltration of monocyte/macrophage and lymphocyte, as well as increased renal levels of proinflammatory cytokine (TNF-, IL-6) and chemokine (MCP-1) compared with DOCA-salt-treated WT mice. Renal ICAM-1 but not VCAM-1 expression was also greater in DOCA-salt-treated TRPV1^–/– than WT mice. Dexamethasone (DEXA), an immunosuppressive drug, conveyed a renoprotective effect that was greater in DOCA-salt-treated TRPV1^–/– compared with WT mice. These data show that renal inflammation is exacerbated in DOCA-salt hypertension when TRPV1 gene is deleted and that the deterioration is ameliorated by DEXA treatment, indicating that TRPV1 may act as a potential regulator of the inflammatory process to lessen renal injury in DOCA-salt hypertension.

PTH transiently increases the percent mobile fraction of Npt2a in OK cells as determined by FRAP

Tue, 24 Nov 2009 14:09:56 PST

Renal sodium-dependent phosphate transporter 2a (Npt2a) binds to a number of PDZ adaptor proteins including sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), which regulates its retention in the apical membrane of renal proximal tubule cells and the response to parathyroid hormone (PTH). The present experiments were designed to study the lateral mobility of enhanced green fluorescent protein (EGFP)-Npt2a in proximal tubule-like opossum kidney (OK) cells using fluorescence recovery after photobleaching (FRAP) and to determine the role of PDZ binding proteins in mediating the effects of PTH. The mobile fraction of wild-type Npt2a (EGFP-Npt2a-TRL) under basal conditions was ~17%. Treatment of the cells with Bis(sulfosuccinimidyl) suberate, a water-soluble cross-linker, abolished recovery nearly completely, indicating that recovery represented lateral diffusion in the plasma membrane and not the exocytosis or synthesis of unbleached transporter. Substitution of the C-terminal amino acid PDZ binding sequence TRL with AAA (EGFP-Npt2a-AAA) resulted in a nearly twofold increase in percent mobile fraction of Npt2a. Treatment of cells with PTH resulted in a rapid increase in the percent mobile fraction to >30% followed by a time-dependent decrease to baseline or below. PTH had no effect on the mobility of EGFP-Npt2a-AAA expressed in native OK cells or on wild-type EGFP-Npt2a-TRL expressed in OK-H cells deficient in NHERF-1. These findings indicate that the association of Npt2a with PDZ binding proteins limits the lateral mobility of the transporter in the apical membrane of renal proximal tubule cells. Treatment with PTH, presumably by dissociating NHERF-1/Npt2a complexes, transiently increases the mobility of Npt2a, suggesting that freeing of Npt2a from the cytoskeleton precedes PTH-mediated endocytosis.

A new function for parietal epithelial cells: a second glomerular barrier

Tue, 24 Nov 2009 14:09:56 PST

The functional role of glomerular parietal epithelial cells (PECs) remains poorly understood. To test the hypothesis that PECs form an impermeable barrier to filtered protein through the formation of tight junctions (TJ), studies were performed in normal animals and in the anti-glomerular basement membrane (GBM) model of crescentic nephritis. Electron microscopy showed well-defined TJ between PECs in normal mice, which no longer could be identified when these cells became extensively damaged or detached from their underlying Bowman's basement membrane. The TJ proteins claudin-1, zonula occludens-1, and occludin stained positive in PECs; however, staining decreased in anti-GBM disease. To show that these events were associated with increased permeability across the PEC-Bowman's basement membrane barrier, control and diseased animals were injected intravenously with either Texas red-conjugated dextran (3 kDa) or ovalbumin (45 kDa) tracers. As expected, both tracers were readily filtered across the glomerular filtration barrier and taken up by proximal tubular cells. However, when the glomerular filtration barrier was injured in anti-GBM disease, tracers were taken up by podocytes and PECs. Moreover, tracers were also detected between PECs and the underlying Bowman's basement membrane, and in many instances were detected in the extraglomerular space. We propose that together with its underlying Bowman's basement membrane, the TJ of PECs serve as a second barrier to protein. When disturbed following PEC injury, the increase in permeability of this layer to filtered protein is a mechanism underlying periglomerular inflammation characteristic of anti-GBM disease.

Angiotensin II and hypertonicity modulate proximal tubular aquaporin 1 expression

Tue, 24 Nov 2009 14:09:56 PST

Aquaporin 1 (AQP1) is the major water channel in the renal proximal tubule (PT) and thin descending limb of Henle, but its regulation remains elusive. Here, we investigated the effect of ANG II, a key mediator of body water homeostasis, on AQP1 expression in immortalized rat proximal tubule cells (IRPTC) and rat kidney. Real-time PCR on IRPTC exposed to ANG II for 12 h revealed a biphasic effect AQP1 mRNA increased dose dependently in response to 10^–12 to 10^–8 M ANG II but decreased by 50% with 10^–7 M ANG II. The twofold increase of AQP1 mRNA in the presence of 10^–8 M ANG II was abolished by the AT₁ receptor blocker losartan. Hypertonicity due to either NaCl or mannitol also upregulated AQP1 mRNA by three- and twofold, respectively. Immunocytochemistry and Western blotting revealed a two- to threefold increase in AQP1 protein expression in IRPTC exposed concomitantly to ANG II (10^–8M) and hypertonic medium (either NaCl or mannitol), indicating that these stimuli were not additive. Three-dimensional reconstruction of confocal images suggested that AQP1 expression was increased by ANG II in both the apical and basolateral poles of IRPTC. In vivo studies showed that short-term ANG II infusion had a diuretic effect, while this effect was attenuated after several days of ANG II infusion. After 10 days, we observed a twofold increase in AQP1 expression in the PT and thin descending limb of Henle of ANG II-infused rats that was abolished when rats were treated with the selective AT₁-receptor antagonist olmesartan. Thus ANG II increases AQP1 expression in vitro and in vivo via direct interaction with the AT₁ receptor, providing an important regulatory mechanism to link PT water reabsorption to body fluid homeostasis via the renin-angiotensin system.

The farnesoid X receptor modulates renal lipid metabolism and diet-induced renal inflammation, fibrosis, and proteinuria

Tue, 24 Nov 2009 14:09:56 PST

Diet-induced obesity is associated with proteinuria and glomerular disease in humans and rodents. We have shown that in mice fed a high-fat diet, increased renal expression of the transcriptional factor sterol-regulatory element binding protein-1 (SREBP-1) plays a critical role in renal lipid accumulation and increases the activity of proinflammatory cytokines and profibrotic growth factors. In the current study, we have determined a key role of the farnesoid X receptor (FXR) in modulating renal SREBP-1 activity, glomerular lesions, and proteinuria. We found that feeding a Western-style diet to DBA/2J mice results in proteinuria, podocyte loss, mesangial expansion, renal lipid accumulation, and increased expression of proinflammatory factors, oxidative stress, and profibrotic growth factors. Treatment of these mice with the highly selective and potent FXR-activating ligand 6--ethyl-chenodeoxycholic acid (INT-747) ameliorates triglyceride accumulation by modulating fatty acid synthesis and oxidation, improves proteinuria, prevents podocyte loss, mesangial expansion, accumulation of extracellular matrix proteins, and increased expression of profibrotic growth factors and fibrosis markers, and modulates inflammation and oxidative stress. Our results therefore indicate that FXR activation could represent an effective therapy for treatment of abnormal renal lipid metabolism with associated inflammation, oxidative stress, and kidney pathology in patients affected by obesity.

Pulse mTOR inhibitor treatment effectively controls cyst growth but leads to severe parenchymal and glomerular hypertrophy in rat polycystic kidney disease

Tue, 24 Nov 2009 14:09:56 PST

The efficacy of mammalian target of rapamycin (mTOR) inhibitors is currently tested in patients affected by autosomal dominant polycystic kidney disease. Treatment with mTOR inhibitors has been associated with numerous side effects. However, the renal-specific effect of mTOR inhibitor treatment cessation in polycystic kidney disease is currently unknown. Therefore, we compared pulse and continuous everolimus treatment in Han:SPRD rats. Four-week-old male heterozygous polycystic and wild-type rats were administered everolimus or vehicle by gavage feeding for 5 wk, followed by 7 wk without treatment, or continuously for 12 wk. Cessation of everolimus did not result in the appearance of renal cysts up to 7 wk postwithdrawal despite the reemergence of S6 kinase activity coupled with an overall increase in cell proliferation. Pulse everolimus treatment resulted in striking noncystic renal parenchymal enlargement and glomerular hypertrophy that was not associated with compromised kidney function. Both treatment regimens ameliorated kidney function, preserved the glomerular-tubular connection, and reduced proteinuria. Pulse treatment at an early age delays cyst development but leads to striking glomerular and parenchymal hypertrophy. Our data might have an impact when long-term treatment using mTOR inhibitors in patients with autosomal dominant polycystic kidney disease is being considered.

Cholesterol induces renal vasoconstriction and anti-natriuresis by inhibiting nitric oxide production in anesthetized rats

Kopkan, L., Khan, M. A. H., Lis, A., Awayda, M. S., Majid, D. S. A. — Tue, 24 Nov 2009 14:09:56 PST

Although hypercholesterolemia is implicated in the pathophysiology of many renal disorders as well as hypertension, its direct actions in the kidney are not yet clearly understood. In the present study, we evaluated renal responses to administration of cholesterol (8 µg·min^–1·100 g body wt^–1; bound by polyethylene glycol) into the renal artery of anesthetized male Sprague-Dawley rats. Total renal blood flow (RBF) was measured by a Transonic flow probe, and glomerular filtration rate (GFR) was determined by Inulin clearance. In control rats (n = 8), cholesterol induced reductions of 10 ± 2% in RBF [baseline (b) 7.6 ± 0.3 µg·min^–1·100 g^–1], 17 ± 3% in urine flow (b, 10.6 ± 0.9 µg·min^–1·100 g^–1), 29 ± 3% in sodium excretion (b, 0.96 ± 0.05 µmol·min^–1·100 g^–1) and 24 ± 2% in nitrite/nitrate excretion (b, 0.22 ± 0.01 nmol·min^–1·100 g^–1) without an appreciable change in GFR (b, 0.87 ± 0.03 ml·min^–1·100 g^–1). These renal vasoconstrictor and anti-natriuretic responses to cholesterol were absent in rats pretreated with nitric oxide (NO) synthase inhibitor, nitro-l-arginine methylester (0.5 µg·min^–1·100 g^–1; n = 6). In rats pretreated with superoxide (O₂^–) scavenger tempol (50 µg·min^–1·100 g^–1; n = 6), the cholesterol-induced renal responses remained mostly unchanged, although there was a slight attenuation in anti-natriuretic response. This anti-natriuretic response to cholesterol was abolished in furosemide-pretreated rats (0.3 µg·min^–1·100 g^–1; n = 6) but remained unchanged in amiloride-pretreated rats (0.2 µg·min^–1·100 g^–1; n = 5), indicating that Na⁺/K⁺/2Cl^– cotransport is the dominant mediator of this effect. These data demonstrate that cholesterol-induced acute renal vasoconstrictor and antinatriuretic responses are mediated by a decrease in NO production. These data also indicate that tubular effect of cholesterol on sodium reabsorption is mediated by the furosemide sensitive Na⁺/K⁺/2Cl^– cotransporter.

Low-dose indomethacin after ischemic acute kidney injury prevents downregulation of Oat1/3 and improves renal outcome

Tue, 24 Nov 2009 14:09:56 PST

We have previously shown that expression of renal organic anion transporters Oat1 and Oat3 is diminished by prostaglandin E₂ (PGE₂) and that both transporters are downregulated after renal ischemia. Because PGE₂ is increased after renal ischemia and is generated by cyclooxygenases (COX), we investigated the effect of the COX inhibitor indomethacin on expression of Oat1/3 after ischemic acute kidney injury (iAKI). iAKI was induced in rats by bilateral clamping of renal arteries for 45 min. Indomethacin (1 mg/kg) was given intraperitoneally as soon as reperfusion started. Sham-treated animals served as controls. Oat1/3 were determined by qPCR and Western blot. PGE₂ in blood and urine was measured by enzyme-linked immunosorbent assay. Invasion of monocytes/macrophages was determined. Glomerular filtration rate and renal plasma flow were determined. All parameters were detected 24 h after ischemia. PAH net secretion, as well as clearance and secretion of PGE₂ were calculated. In clamped animals, indomethacin restored expression of Oat1/3, as well as PAH net secretion, PGE₂ clearance, or PGE₂ secretion. Additionally, indomethacin substantially improved kidney function as measured by glomerular filtration and PAH clearance. Indomethacin did not affect ischemia-induced invasion of monocytes/macrophages. In conclusion, our study indicates that low-dose indomethacin applied after ischemia prevents ischemia-induced downregulation of Oat1/3 during reperfusion and has a substantial protective effect on kidney function after iAKI. The beneficial effect of low-dose indomethacin on renal outcome is likely due to an effect different from inhibition of inflammation. In accordance to the decreased PAH net secretion, renal excretion of an endogenous organic anion (PGE₂) is also impaired after ischemia and reperfusion.

Oxidative stress-induced JNK activation contributes to proinflammatory phenotype of aging diabetic mesangial cells

Wu, J., Mei, C., Vlassara, H., Striker, G. E., Zheng, F. — Tue, 24 Nov 2009 14:09:56 PST

Chronic inflammation and increased oxidative stress (OS) play an important role in diabetic nephropathy progression. Herein, we show that mesangial cells from streptozotocin-induced aging diabetic mice, a model of progressive diabetic nephropathy, exhibited increased OS and a proinflammatory phenotype characterized by elevated chemokines and ICAM-1 expression. This phenotypic change was consistent with the extensive inflammatory lesions present in aging diabetic kidneys and was not found in mesangial cells from old and young controls or young diabetic mice. Activation of the c-Jun NH₂-terminal kinase (JNK) pathway was a likely contributor to the proinflammatory phenotype of aging diabetic mesangial cells since 1) phosphorylated JNK levels and JNK kinase activity were increased in these cells, 2) suppression of JNK significantly decreased monocyte chemoattractant protein-1 (MCP-1) production in these cells, and 3) activation of JNK in normal mesangial cells induced inflammation. Elevated OS in aging diabetic mesangial cells may be a cause of JNK activation and inflammation, because antioxidant treatment decreased JNK phosphorylation and MCP-1 production. Additionally, decreased expression of mitogen-activated protein kinase phosphatase 5 (MKP5) may also contribute to increased JNK and inflammation in aging diabetic mesangial cells since overexpression of MKP5 in these cells normalized phosphorylated JNK levels and reversed the proinflammatory phenotype. Moreover, knocking down of MKP5 expression in old control mesangial cells resulted in JNK activation and MCP-1 production, a phenotype seen in aging diabetic mesangial cells. Interestingly, MKP5 phosphatase activity was diminished by free radicals in vitro. Thus, OS may induce inflammation in mesangial cells by activating JNK through either a direct activation of JNK or indirectly by suppression of MKP5 activity. Proinflammatory phenotype of mesangial cells may contribute to chronic inflammatory lesions and disease progression of aging diabetic mice.

Regulation of the mitochondrial permeability transition in kidney proximal tubules and its alteration during hypoxia-reoxygenation

Tue, 24 Nov 2009 14:09:56 PST

Development of the mitochondrial permeability transition (MPT) can importantly contribute to lethal cell injury from both necrosis and apoptosis, but its role varies considerably with both the type of cell and type of injury, and it can be strongly opposed by the normally abundant endogenous metabolites ADP and Mg²⁺. To better characterize the MPT in kidney proximal tubule cells and assess its contribution to injury to them, we have refined and validated approaches to follow the process in whole kidney proximal tubules and studied its regulation in normoxic tubules and after hypoxia-reoxygenation (H/R). Physiological levels of ADP and Mg²⁺ greatly decreased sensitivity to the MPT. Inhibition of cyclophilin D by cyclosporine A (CsA) effectively opposed the MPT only in the presence of ADP and/or Mg²⁺. Nonesterified fatty acids (NEFA) had a large role in the decreased resistance to the MPT seen after H/R irrespective of the available substrate or the presence of ADP, Mg²⁺, or CsA, but removal of NEFA was less effective at restoring normal resistance to the MPT in the presence of electron transport complex I-dependent substrates than with succinate. The data indicate that the NEFA accumulation that occurs during both hypoxia in vitro and ischemic acute kidney injury in vivo is a critical sensitizing factor for the MPT that overcomes the antagonistic effect of endogenous metabolites and cyclophilin D inhibition, particularly in the presence of complex I-dependent substrates, which predominate in vivo.

Regulation of Na+/H+ exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix, L. R., Malnic, G., Girardi, A. C. C. — Tue, 24 Nov 2009 14:09:56 PST

The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na⁺/H⁺ exchanger NHE3 in LLC-PK₁ cells. We found that NHE3-mediated Na⁺-dependent intracellular pH (pH_i) recovery decreased ~50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pH_i recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

The balance of autocrine VEGF-A and VEGF-C determines podocyte survival

Tue, 24 Nov 2009 14:09:56 PST

Podocytes are an important component of the glomerular filtration barrier and are the major source of vascular endothelial growth factor (VEGF) in the glomerulus. The role of VEGF for the phenotype of the glomerular endothelium has been intensely studied; however, the direct effects of autocrine VEGF on the podocyte are largely unknown. In this study we characterized the expression of VEGF isoforms and VEGF receptors in cultured human podocytes and examined direct effects on cell signaling and apoptosis after stimulation with exogenous VEGF or ablation of autocrine VEGF. We identified VEGF-A and VEGF-C as the dominant isoforms in human podocytes and showed that autocrine levels of both are important for the intracellular activation of antiapoptotic phosphoinositol 3-kinase/AKT and suppression of the proapoptotic p38MAPK via VEGFR-2. We demonstrated that ablation of VEGF-A or VEGF-C as well as treatment with bevacizumab or a VEGFR-2/-3 tyrosine kinase inhibitor led to reduced podocyte survival. In contrast, ablation of VEGF-B had no effect on podocyte survival. Treatment with exogenous VEGF-C reversed the effect of VEGF-A neutralization, and exogenous VEGF-A abrogated the effect of VEGF-C ablation in human podocytes. Our results underline the importance of autocrine VEGF for podocyte survival and indicate the delicate balance of VEGF-A and VEGF-C to influence progression of glomerular diseases.

Is nephrogenesis affected by preterm birth? Studies in a non-human primate model

Tue, 24 Nov 2009 14:09:56 PST

Nephrogenesis occurs predominantly in late gestation at a time when preterm infants are already delivered. The aims of this study were to assess the effect of preterm birth and the effect of antenatal glucocorticoid treatment on nephrogenesis. Preterm baboons, which were delivered at 125 days gestation and ventilated for up to 21 days postnatally, were compared with gestational controls. A cohort of preterm baboons that had been exposed to antenatal glucocorticoids were compared with unexposed preterm baboons. The number of glomerular generations was estimated using a medullary ray glomerular-counting method, and glomerular number was estimated using unbiased stereology. CD31 and WT-1 localization was examined using immunohistochemistry and VEGF was localized using in situ hybridization. The number of glomerular generations was not affected by preterm birth, and total glomerular numbers were within the normal range. Kidneys were significantly enlarged in preterm baboons with a significant decrease in glomerular density (number of glomeruli per gram of kidney) in the preterm kidney compared with gestational controls. Neonates exposed to antenatal steroids had an increased kidney-to-body weight ratio and also more developed glomeruli compared with unexposed controls. Abnormal glomeruli, with a cystic Bowman's space and shrunken glomerular tuft, were often present in the superficial renal cortex of both the steroid-exposed and unexposed preterm kidneys; steroid exposure had no significant effect on the proportion of abnormal glomeruli. The proportion of abnormal glomeruli in the preterm kidneys ranged from 0.2 to 18%. In conclusion, although nephrogenesis is ongoing in the extrauterine environment, our findings demonstrate that preterm birth, independent of steroid exposure, is associated with a high proportion of abnormal glomeruli in some, but not all neonatal kidneys. Whether final nephron endowment is affected in those kidneys exhibiting a high proportion of abnormal glomeruli is yet to be confirmed.

Changes of renal AQP2, ENaC, and NHE3 in experimentally induced heart failure: response to angiotensin II AT1 receptor blockade

Tue, 24 Nov 2009 14:09:56 PST

Heart failure (HF) was induced by ligation of the left anterior descending artery (LAD). Left ventricular end-diastolic pressure (LVEDP) >25 mmHg (at day 23 after LAD ligation) was the inclusion criterion. The rats were divided into three groups: sham-operated (Sham, n = 23, LVEDP: 5.6 ± 0.6 mmHg), HF (n = 14, LVEDP: 29.4 ± 1.4 mmHg), and candesartan (1 mg·kg^–1·day^–1 sc)-treated HF (HF + Can, n = 9, LVEDP: 29.2 ± 1.2 mmHg). After 7 days (i.e., 29 days after LAD ligation) semiquantitative immunoblotting revealed increased abundance of inner medulla aquaporin-2 (AQP2) and AQP2 phosphorylated at Ser²⁵⁶ (p-AQP2) in HF. There was also markedly enhanced apical targeting of AQP2 and p-AQP2 in inner medullary collecting duct (IMCD) in HF compared with Sham rats, shown by immunocytochemistry. Candesartan treatment significantly reversed the increases in both AQP2 and p-AQP2 expression and targeting. In contrast, there were only modest changes in other collecting duct segments. Semiquantitative immunoblots revealed increased expression of type 3 Na⁺/H⁺ exchanger (NHE3) and Na⁺-K⁺-2Cl^– cotransporter (NKCC2) in kidneys from HF compared with Sham rats: both effects were reversed or prevented by candesartan treatment. The protein abundance of -epithelial sodium channel (-ENaC) was increased while β-ENaC and -ENaC expression was decreased in the cortex and outer stripe of the outer medulla in HF compared with Sham rats, which was partially reversed by candesartan treatment. These findings strongly support an important role of angiotensin II in the pathophysiology of renal water and sodium retention associated with HF.

Mice lacking mPGES-1 are resistant to lithium-induced polyuria

Jia, Z., Wang, H., Yang, T. — Tue, 24 Nov 2009 14:09:56 PST

Cyclooxygenase-2 activity is required for the development of lithium-induced polyuria. However, the involvement of a specific, terminal prostaglandin (PG) isomerase has not been evaluated. The present study was undertaken to assess lithium-induced polyuria in mice deficient in microsomal prostaglandin E synthase-1 (mPGES-1). A 2-wk administration of LiCl (4 mmol·kg^–1·day^–1 ip) in mPGES-1 +/+ mice led to a marked polyuria with hyposmotic urine. This was associated with elevated renal mPGES-1 protein expression and increased urine PGE₂ excretion. In contrast, mPGES-1 –/– mice were largely resistant to lithium-induced polyuria and a urine concentrating defect, accompanied by nearly complete blockade of high urine PGE₂ and cAMP output. Immunoblotting, immunohistochemistry, and quantitative (q) RT-PCR consistently detected a significant decrease in aquaporin-2 (AQP2) protein expression in both the renal cortex and medulla of lithium-treated +/+ mice. This decrease was significantly attenuated in the –/– mice. qRT-PCR detected similar patterns of changes in AQP2 mRNA in the medulla but not in the cortex. Similarly, the total protein abundance of the Na-K-2Cl cotransporter (NKCC2) in the medulla but not in the cortex of the +/+ mice was significantly reduced by lithium treatment. In contrast, the dowregulation of renal medullary NKCC2 expression was significantly attenuated in the –/– mice. We conclude that mPGES-1-derived PGE₂ mediates lithium-induced polyuria likely via inhibition of AQP2 and NKCC2 expression.

Accelerated senescence in kidneys of low-birth-weight rats after catch-up growth

Luyckx, V. A., Compston, C. A., Simmen, T., Mueller, T. F. — Tue, 24 Nov 2009 14:09:56 PST

Epidemiological studies show a strong association between low birth weight and hypertension, renal, and cardiovascular disease, especially after catch-up growth. Senescence is an important contributor to the progression of chronic disease. Developmentally programmed premature senescence may be a link among low birth weight, catch-up growth, and adult disease. Low birth weight was induced by feeding pregnant rats a low-protein diet from day 12 of gestation to 10 days postdelivery. Low- and normal-birth-weight male offspring were weaned onto regular or high-calorie diets to enhance catch-up growth. Kidneys and hearts of offspring were analyzed for RNA and protein markers of stress-induced senescence (p16, p21, p53, Rb). Markers of mitochondrial stress (p66Shc) and activation of endoplasmic reticulum protein secretion (Ero1) were analyzed as regulators of reactive oxygen species generation. Reactive oxygen species are known to be associated with premature aging. Senescence markers were not different in low- or normal-birth-weight kidneys at birth. During rapid catch-up growth, p16 and p21 increased significantly in low-birth-weight kidneys and hearts (P < 0.01). Renal p16 levels increased progressively and were significantly higher in low-birth-weight kidneys at 3 and 6 mo (P ≤ 0.02). Renal p66Shc and Ero1 were significantly higher in low- compared with normal- birth-weight kidneys at 6 mo, suggesting reactive oxygen species generation (P ≤ 0.03). Low-birth-weight rats exhibit accelerated senescence in kidneys and hearts after rapid catch-up growth, a likely important link between early growth and subsequent hypertension, renal, and cardiovascular disease.

Corrigendum

Tue, 24 Nov 2009 14:09:57 PST

Heme oxygenase: the key to renal function regulation

Abraham, N. G., Cao, J., Sacerdoti, D., Li, X., Drummond, G. — Fri, 30 Oct 2009 10:16:29 PDT

Heme oxygenase (HO) plays a critical role in attenuating the production of reactive oxygen species through its ability to degrade heme in an enzymatic process that leads to the production of equimolar amounts of carbon monoxide and biliverdin/bilirubin and the release of free iron. The present review examines the beneficial role of HO-1 (inducible form of HO) that is achieved by increased expression of this enzyme in renal tissue. The influence of the HO system on renal physiology, obesity, vascular dysfunction, and blood pressure regulation is reviewed, and the clinical potential of increased levels of HO-1 protein, HO activity, and HO-derived end products of heme degradation is discussed relative to renal disease. The use of pharmacological and genetic approaches to investigate the role of the HO system in the kidney is key to the development of therapeutic approaches to prevent the adverse effects that accrue due to an impairment in renal function.

Regulation of mRNA translation in renal physiology and disease

Fri, 30 Oct 2009 10:16:29 PDT

Translation, a process of generating a peptide from the codons present in messenger RNA, can be a site of independent regulation of protein synthesis; it has not been well studied in the kidney. Translation occurs in three stages (initiation, elongation, and termination), each with its own set of regulatory factors. Mechanisms controlling translation include small inhibitory RNAs such as microRNAs, binding proteins, and signaling reactions. Role of translation in renal injury in diabetes, endoplasmic reticulum stress, acute kidney injury, and, in physiological adaptation to loss of nephrons is reviewed here. Contribution of mRNA translation to physiology and disease is not well understood. Because it is involved in such diverse areas as development and cancer, it should prove a fertile field for investigation in renal science.

Purinoceptor regulation of renal tubular transport is coming of age

Inscho, E. W. — Fri, 30 Oct 2009 10:16:29 PDT

Extracellular ATP inhibits transport in medullary thick ascending limbs: role of P2X receptors

Silva, G. B., Garvin, J. L. — Fri, 30 Oct 2009 10:16:29 PDT

Absorption of NaCl by the thick ascending limb (TAL) involves active transport and therefore depends on oxidative phosphorylation. Extracellular ATP has pleiotropic effects, including both stimulation and inhibition of transport and inhibition of oxidative phosphorylation. However, it is unclear whether ATP alters TAL transport and how this occurs. We hypothesized that ATP inhibits TAL Na absorption by reducing Na entry. We measured oxygen consumption in TAL suspensions. ATP reduced oxygen consumption in a concentration-dependent manner. The purinergic (P2) receptor antagonist suramin (300 µM) blocked the effect of ATP on TAL oxygen consumption (147 ± 15 vs. 146 ± 16 nmol O₂·min^–1·mg protein^–1). In contrast, the adenosine receptor antagonist theophylline did not block the effect of ATP on oxygen consumption. When Na-K-2Cl cotransport and Na/H exchange were blocked with furosemide (100 µM) plus dimethyl amiloride (100 µM), ATP did not inhibit TAL oxygen consumption (from 78 ± 13 to 98 ± 5 nmol O₂·min^–1·mg protein^–1). The Na ionophore nystatin (200 U/ml) increased TAL oxygen consumption to a similar extent in both ATP- and vehicle-treated samples (368 ± 41 vs. 397 ± 47 nmol O₂·min^–1·mg protein^–1). The nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester (3 mM) blocked the ATP effects on TAL oxygen consumption (157 ± 10 vs. 165 ± 15 nmol O₂·min^–1·mg protein^–1). The P2X-selective receptor antagonist NF023 blocked the effect of ATP on oxygen consumption, whereas the P2X-selective agonist β--Me-ATP reduced oxygen consumption in a concentration-dependent manner. We conclude that ATP inhibits Na transport-related oxygen consumption in TALs by reducing Na entry and P2X receptors and nitric oxide mediate this effect.

Exercise activates redox-sensitive transcription factors and restores renal D1 receptor function in old rats

George, L., Lokhandwala, M. F., Asghar, M. — Fri, 30 Oct 2009 10:16:29 PDT

We have previously reported that age-associated oxidative stress via protein kinase C (PKC) increases D₁ receptor (D1R) phosphorylation and causes D1R-G protein uncoupling in renal proximal tubules (RPTs) of old Fischer 344 rats. This results in reduced ability of D1R agonist SKF-38393 to inhibit Na⁺-K⁺-ATPase in RPTs of old rats. Here, we studied the effect of treadmill exercise on markers of oxidative stress, PKC, D1R phosphorylation, D1R-G protein coupling, and Na⁺-K⁺-ATPase activity in RPTs of adult and old rats. We found increased levels of malondialdehyde, a marker of oxidative stress, in RPTs of old rats, which decreased during exercise. Nuclear levels of nuclear erythroid-related factor (Nrf)-2 and nuclear factor (NF)-B in RPTs, transcription factors involved in antioxidant enzyme gene transcription, increased in exercised old rats. This was accompanied by an increase in the activity and expression of antioxidant enzymes, superoxide dismutase and heme oxygenase-1. Age-related decrease in the levels of D1R mRNAs and proteins was attenuated during exercise. Furthermore, exercise in old rats decreased PKC activity and D1R phosphorylation and increased SKF-38393-mediated [³⁵S]guanosine 5'-O-(3-thiotriphosphate) binding (an index of D1R-G protein coupling). SKF-38393 also caused inhibition of Na⁺-K⁺-ATPase in these animals. Also, exercise caused a decrease in proteinuria and increase in phosphaturia in old rats. These results suggest beneficial effects of exercise in terms of increasing antioxidant defenses, decreasing oxidative stress, and improving kidney function in general and D1R function in particular in aging. Both Nrf-2 and NF-B seem to play key role in this phenomenon.

Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2 in renal papillary collecting duct cells

Fri, 30 Oct 2009 10:16:29 PDT

Focal adhesions (FAs) are structures of cell attachment to the extracellular matrix. We previously demonstrated that the intrarenal hormone bradykinin (BK) induces the restructuring of FAs in papillary collecting duct cells by dissipation of vinculin, but not talin, from FAs through a mechanism that involves PLCβ activation, and that it also induces actin cytoskeleton reorganization. In the present study we investigated the mechanism by which BK induces the dissipation of vinculin-stained FAs in collecting duct cells. We found that BK induces the internalization of vinculin by a noncaveolar and independent pinocytic pathway and that at least a fraction of this protein is delivered to the recycling endosomal compartment, where it colocalizes with the transferrin receptor. Regarding the reassembly of vinculin-stained FAs, we found that BK induces the formation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂]-enriched vinculin-containing vesicles, which, by following a polarized exocytic route, transport vinculin to the site of FA assembly, an action that depends on actin filaments. The present study, which was carried out with cells that were not genetically manipulated, shows for the first time that BK induces the formation of vesicle-like structures containing vinculin and PtdIns(4,5)P₂, which transport vinculin to the site of FA assembly. Therefore, the modulation of the formation of these vesicle-like structures could be a physiological mechanism through which the cell can reuse the BK-induced internalized vinculin to be delivered for newly forming FAs in renal papillary collecting duct cells.

Deletion of the vitamin D receptor specifically in the parathyroid demonstrates a limited role for the receptor in parathyroid physiology

Fri, 30 Oct 2009 10:16:29 PDT

1,25(OH)₂D₃ decreases parathyroid hormone (PTH) gene transcription through the vitamin D receptor (VDR). Total body VDR^–/– mice have high PTH levels, hypocalcemia, hypophosphatemia, and bone malformations. To investigate PTH regulation by the VDR specifically in the parathyroid, we generated parathyroid-specific VDR knockout mice (PT-VDR^–/–). In both strains, there was a decrease in parathyroid calcium receptor (CaR) levels. The number of proliferating parathyroid cells was increased in the VDR^–/– mice but not in the PT-VDR^–/– mice. Serum PTH levels were moderately but significantly increased in the PT-VDR^–/– mice with normal serum calcium levels. The sensitivity of the parathyroid glands of the PT-VDR^–/– mice to calcium was intact as measured by serum PTH levels after changes in serum calcium. This indicates that the reduced CaR in the PT-VDR^–/– mice enables a physiologic response to serum calcium. Serum C-terminal collagen crosslinks, a marker of bone resorption, were increased in the PT-VDR^–/– mice with no change in the bone formation marker, serum osteocalcin, consistent with a resorptive effect due to the increased serum PTH levels in the PT-VDR^–/– mice. Therefore, deletion of the VDR specifically in the parathyroid decreases parathyroid CaR expression and only moderately increases basal PTH levels, suggesting that the VDR has a limited role in parathyroid physiology.

Maternal nutrient restriction during early fetal kidney development attenuates the renal innate inflammatory response in obese young adult offspring

Sharkey, D., Gardner, D. S., Symonds, M. E., Budge, H. — Fri, 30 Oct 2009 10:16:29 PDT

Obesity is an independent risk factor for developing chronic kidney disease. Toll-like receptor 4 (TLR4), interleukin (IL)-18, and uncoupling protein 2 (UCP2) are important components of the innate immune system mediating inflammatory renal damage. Early to midgestation maternal nutrient restriction appears to protect the kidney from the deleterious effects of early onset obesity, although the mechanisms remain unclear. We examined the combined effects of gestational maternal nutrient restriction during early fetal kidney development and early onset obesity on the renal innate immune response in offspring. Pregnant sheep were randomly assigned to a normal (control, 100%) or nutrient-restricted (NR, 50%) diet from days 30 to 80 gestation and 100% thereafter. Offspring were killed humanely at 7 days or, following rearing in an obesogenic environment, at 1 yr of age, and renal tissues were collected. IL-18 and TLR4 expression were strongly correlated irrespective of intervention. Seven-day NR offspring had significantly lower relative renal mass and IL-18 mRNA expression. At 1 yr of age, obesity resulted in increased mRNA abundance of TLR4, IL-18, and UCP2, coupled with tubular atrophy and greater immunohistological staining of glomerular IL-6 and medullary tumor necrosis factor (TNF)-. NR obese offspring had a marked reduction of TLR4 abundance and renal IL-6 staining. In conclusion, maternal nutrient restriction during early fetal kidney development attenuates the effects of early onset obesity-related nephropathy, in part, through the downregulation of the innate inflammatory response. A better understanding of maternal nutrition and the in utero nutritional environment may offer therapeutic strategies aimed at reducing the burden of later kidney disease.

Deletion of Frs2{alpha} from the ureteric epithelium causes renal hypoplasia

Fri, 30 Oct 2009 10:16:29 PDT

Fibroblast growth factor receptor 2 (Fgfr2) signaling is critical in maintaining ureteric branching architecture and mesenchymal stromal morphogenesis in the kidney. Fibroblast growth factor receptor substrate 2 (Frs2) is a major docking protein for Fgfr2 with downstream targets including Ets variant (Etv) 4 and Etv5 in other systems. Furthermore, global deletion of Frs2 causes early embryonic lethality. The purpose of the study was to determine the role of Frs2 in mediating Fgfr2 signaling in the ureteric epithelium. To that end, we generated mice with conditional deletion of Frs2 in the ureteric epithelium (Frs2^UB–/–) and mice with point mutations in the Frs2 binding site of Fgfr2 (Fgfr2^LR/LR). Frs2^UB–/– mice developed mild renal hypoplasia characterized by decreased ureteric branching morphogenesis but maintained normal overall branching architecture and had normal mesenchymal stromal development. Reduced nephron endowment in postnatal mutant mice was observed, corresponding with the reduction in branching morphogenesis. Furthermore, there were no apparent renal abnormalities in Fgfr2^LR/LR mice. Interestingly, Etv4 and Etv5 expression was unaltered in Frs2^UB–/– mice, as was Sprouty1, an antagonist of Frs2 signaling. However, Ret and Wnt11 (molecules critical for ureteric branching morphogenesis) mRNA levels were lower in mutants vs. controls. Taken together, these findings suggest that Fgfr2 signals through adapter molecules other than Frs2 in the ureteric epithelium. Furthermore, Frs2 may transmit signals through other receptor kinases present in ureteric epithelium. Finally, the renal hypoplasia observed in Frs2^UB–/– mice is likely secondary to decreased Ret and Wnt11 expression.

PKC-dependent superoxide production by the renal medullary thick ascending limb from diabetic rats

Yang, J., Lane, P. H., Pollock, J. S., Carmines, P. K — Fri, 30 Oct 2009 10:16:29 PDT

Type 1 diabetes (T1D) is a state of oxidative stress accompanied by PKC activation in many tissues. The primary site of O₂^•– production by the normal rat kidney is the medullary thick ascending limb (mTAL). We hypothesized that T1D increases O₂^•– production by the mTAL through a PKC-dependent mechanism involving increased expression and translocation of one or more PKC isoforms. mTAL suspensions were prepared from rats with streptozotocin-induced T1D (STZ mTALs) and from normal or sham rats (normal/sham mTALs). O₂^•– production by STZ mTALs was fivefold higher than normal/sham mTALs (P < 0.05). PMA (30 min) mimicked the effect of T1D on O₂^•– production. Exposure to calphostin C or chelerythrine (PKC inhibitors), Gö6976 (PKC/β inhibitor), or rottlerin (PKC inhibitor) decreased O₂^•– production to <20% of untreated baseline in both normal/sham and STZ mTALs. PKCβ inhibitors had no effect. PKC activity was increased in STZ mTALs (P < 0.05 vs. normal/sham mTALs) and was unaltered by antioxidant exposure (tempol). PKC protein levels were increased by 70% in STZ mTALs, with a ~30% increase in the fraction associated with the membrane (both P < 0.05 vs. sham). PKCβ protein levels were elevated by 29% in STZ mTALs (P < 0.05 vs. sham) with no change in the membrane-bound fraction. Neither PKC protein levels nor its membrane-bound fraction differed between groups. Thus STZ mTALs display PKC activation, upregulation of PKC and PKCβ protein levels, increased PKC translocation to the membrane, and accelerated O₂^•– production that is eradicated by inhibition of PKC or PKC (but not PKCβ). We conclude that increased PKC expression and activity are primarily responsible for PKC-dependent O₂^•– production by the mTAL during T1D.

Mesangial cell-derived factors alter monocyte activation and function through inflammatory pathways: possible pathogenic role in diabetic nephropathy

Min, D., Lyons, J. G., Bonner, J., Twigg, S. M., Yue, D. K., McLennan, S. V. — Fri, 30 Oct 2009 10:16:29 PDT

Infiltration of macrophages to the kidney is a feature of early diabetic nephropathy. For this to happen monocytes must become activated, migrate from the circulation, and infiltrate the mesangium. This process involves degradation of extracellular matrix, a process mediated by matrix metalloproteinases (MMPs). In the present study we investigate the expression of proinflammatory cytokines TNF-, IL-6, and MMP-9 in glomeruli of control and diabetic rodents and use an in vitro coculture system to examine whether factors secreted by mesangial cells in response to a diabetic milieu can induce monocyte MMP-9 expression and infiltration. After 8 wk of diabetes, the glomerular level of TNF-, IL-6, and macrophage number and colocalization of MMP-9 with macrophage were increased (P < 0.01). Coculture of THP1 monocytes and glomerular mesangial cells in 5 or 25 mM glucose increased MMP-9 (5 mM: 65% and 25 mM: 112%; P < 0.05) and conditioned media degradative activity (5 mM: 30.0% and 25 mM: 33.5%: P < 0.05). These effects were reproduced by addition of mesangial cell conditioned medium to THP1 cells. High glucose (25 mM) increased TNF-, IL-6, and monocyte chemoattractant protein-1 in mesangial cell conditioned medium. These cytokines all increased adhesion and differentiation of THP1 cells (P < 0.05), but only TNF- and IL-6 increased MMP-9 expression (50- and 60-fold, respectively; P < 0.05). Our results show that mesangial cell-secreted factors increase monocyte adhesion, differentiation, MMP expression, and degradative capacity. High glucose could augment these effects by increasing mesangial cell proinflammatory cytokine secretion. This mesangial cell-monocyte interaction may be important in activating monocytes to migrate from the circulation to the kidney in the early stages of diabetic nephropathy.

Differential regulation of E-cadherin and {alpha}-smooth muscle actin by BMP 7 in human renal proximal tubule epithelial cells and its implication in renal fibrosis

Fri, 30 Oct 2009 10:16:30 PDT

Chronic kidney diseases are characterized by progressive tubulointerstitial fibrosis, and TGFβ1 plays a crucial role in its development. Bone morphogenic protein 7 (BMP 7), another member of the TGF superfamily, antagonized the profibrotic effects of TGFβ1, including epithelial mesenchymal transition and E-cadherin loss, in the previous studies from animal models. We investigated the effect of BMP 7 on TGFβ1-mediated E-cadherin loss in two different transformed human adult proximal tubule epithelia. We found that BMP 7 not only failed to prevent TGFβ1-mediated E-cadherin loss but itself downregulated E-cadherin levels and that it had an additive effect with TGFβ1 in inducing E-cadherin loss. The downregulation of E-cadherin by BMP 7 was mediated through the Smad1/5 pathway. BMP 7-mediated E-cadherin loss was not followed by de novo -smooth muscle actin (-SMA) expression (a marker of myofibroblastic phenotype), which was due to the concurrent induction of Inhibitor of DNA binding 1 (Id1, a basic helix loop helix class transcriptional regulator) through a non-Smad pathway. Concurrent treatment of BMP 7 and TGFβ1 prevented TGFβ1-mediated -SMA induction. In summary, our results suggest that E-cadherin loss, the key feature of epithelial mesenchymal transition, will not necessarily be followed by total phenotype change; rather, cells may undergo some loss of phenotypic marker in a ligand-dependent manner and participate in reparative processes. The inhibition of de novo expression of -SMA could explain the antifibrotic effect of BMP 7. Id1 might play a crucial role in maintaining proximal tubule epithelial cell phenotype and its signaling regulation could be a potential therapeutic target.

Surface expression of sodium channels and transporters in rat kidney: effects of dietary sodium

Frindt, G., Palmer, L. G. — Fri, 30 Oct 2009 10:16:30 PDT

The abundance of Na transport proteins in the luminal membrane of the rat kidney was assessed using in situ biotinylation and immunoblotting. When animals were fed an Na-deficient diet for 1 wk, the amounts of epithelial Na channel (ENaC) β-subunit (β-ENaC) and -subunit (-ENaC) and Na-Cl cotransporter (NCC) protein in the surface fraction increased relative to controls by 1.9-, 3.5-, and 1.5-fold, respectively. The amounts of the luminal Na/H exchanger (NHE3) and the luminal Na-K-2Cl cotransporter (NKCC2) did not change significantly. The increases in ENaC subunits were mimicked by administration of aldosterone for 1 wk, but the increase in NCC was not. When the animals were fed a high-Na (5% NaCl) diet for 1 wk, the surface expression of β-ENaC increased by 50%, whereas that of the other membrane proteins did not change, relative to controls. The biochemical parameter most strongly affected by dietary Na was the abundance of the 65-kDa cleaved form of -ENaC at the surface. This increased by 8.5-fold with Na depletion and decreased by 40% with Na loading. The overall 14-fold change reflected regulation of the total abundance of the subunit as well as the fraction of the subunit protein in the cleaved form. We conclude that cleavage of -ENaC and its expression at the apical surface play a major role in the regulation of renal Na reabsorption.

Enhanced tubuloglomerular feedback in mice with vascular overexpression of A1 adenosine receptors

Fri, 30 Oct 2009 10:16:30 PDT

Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.38-kb fragment of the rat smooth muscle -actin promoter and first intron (12). Two founder lines with highest expression in the kidney [353 ± 42 and 575 ± 43% compared with the wild type (WT)] were used in the experiments. Enhanced expression of A1AR at the expected site in these lines was confirmed by augmented constrictor responses of isolated afferent arterioles to administration of the A1AR agonist N⁶-cyclohexyladenosine. Maximum TGF responses (0–30 nl/min flow step) were increased from 8.4 ± 0.9 mmHg in WT (n = 21) to 14.2 ± 0.7 mmHg in A1AR-transgene (tg) 4 (n = 22; P < 0.0001), and to 12.6 ± 1.2 mmHg in A1AR-tg7 (n = 12; P < 0.02). Stepwise changes in perfusion flow caused greater numerical TGF responses in A1AR-tg than WT in all flow ranges with differences reaching levels of significance in the intermediate flow ranges of 7.5–10 and 10–15 nl/min. Proximal-distal single-nephron glomerular filtration rate (SNGFR) differences (free-flow micropuncture) were also increased in A1AR-tg, averaging 6.25 ± 1.5 nl/min compared with 2.6 ± 0.51 nl/min in WT (P = 0.034). Basal plasma renin concentrations as well as the suppression of renin secretion after volume expansion were similar in A1AR-tg and WT mice, suggesting lack of transgene expression in juxtaglomerular cells. These data indicate that A1AR expression in vascular smooth muscle cells is a critical component for TGF signaling and that changes in renal vascular A1AR expression may determine the magnitude of TGF responses.

Proinsulin C-peptide reduces diabetes-induced glomerular hyperfiltration via efferent arteriole dilation and inhibition of tubular sodium reabsorption

Nordquist, L., Brown, R., Fasching, A., Persson, P., Palm, F. — Fri, 30 Oct 2009 10:16:30 PDT

C-peptide reduces diabetes-induced glomerular hyperfiltration in diabetic patients and experimental animal models. However, the mechanisms mediating the beneficial effect of C-peptide remain unclear. We investigated whether altered renal afferent-efferent arteriole tonus or alterations in tubular Na⁺ transport (T_Na) in response to C-peptide administration mediate the reduction of diabetes-induced glomerular hyperfiltration. Glomerular filtration rate, filtration fraction, total and cortical renal blood flow, total kidney O₂ consumption (Qo₂), T_Na, fractional Na⁺ and Li⁺ excretions, and tubular free-flow and stop-flow pressures were measured in anesthetized adult male normoglycemic and streptozotocin-diabetic Sprague-Dawley rats. The specific effect of C-peptide on transport-dependent Qo₂ was investigated in vitro in freshly isolated proximal tubular cells. C-peptide reduced glomerular filtration rate (–24%), stop-flow pressure (–8%), and filtration fraction (–17%) exclusively in diabetic rats without altering renal blood flow. Diabetic rats had higher baseline T_Na (+40%), which was reduced by C-peptide. Similarly, C-peptide increased fractional Na⁺ (+80%) and Li⁺ (+47%) excretions only in the diabetic rats. None of these parameters was affected by vehicle treatments in either group. Baseline Qo₂ was 37% higher in proximal tubular cells from diabetic rats than controls and was normalized by C-peptide. C-peptide had no effect on ouabain-pretreated diabetic cells from diabetic rats. C-peptide reduced diabetes-induced hyperfiltration via a net dilation of the efferent arteriole and inhibition of tubular Na⁺ reabsorption, both potent regulators of the glomerular net filtration pressure. These findings provide new mechanistic insight into the beneficial effects of C-peptide on diabetic kidney function.

Morg1 heterozygous mice are protected from acute renal ischemia-reperfusion injury

Hammerschmidt, E., Loeffler, I., Wolf, G. — Fri, 30 Oct 2009 10:16:30 PDT

Renal ischemia and reperfusion injury leads to acute renal failure when proinflammatory and apoptotic processes in the kidney are activated. The increase in hypoxia-inducible transcription factor- (HIF-), an important transcription factor for several genes, can attenuate ischemic renal injury. We recently identified a novel WD-repeat protein designated Morg1 (MAPK organizer 1) that interacts with prolyl hydroxylase 3 (PHD3), an important enzyme involved in the regulation of HIF-1 and HIF-2 expression. While homozygous Morg1 –/– mice are embryonic lethal, heterozygous Morg1 +/– mice have a normal phenotype. We show here that Morg1 +/– were partially protected from renal ischemia-reperfusion injury compared with wild-type Morg1 +/+ animals. Morg1 +/– mice compared with wild-type animals revealed a stronger increase in HIF-1 and HIF-2 expression in the ischemic-reperfused kidney associated with enhanced serum erythropoietin levels. However, no significant expression of HIF-1 and HIF-2 was found in nonischemic kidneys without any difference between Morg1 +/– and Morg1 +/+ mice. Ischemic kidneys of Morg1 +/– mice expressed more erythropoietin mRNA than ischemic kidneys from wild-type animals. Renal ischemia in Morg1 +/– mice resulted in a decrease in renal inflammation and reduction of proinflammatory cytokines (MCP-1, IP-10, MIP-2) compared with wild-type mice. Furthermore, there was significantly less apoptosis and tubular damage in Morg1 +/– kidneys after ischemia-reperfusion, and this was also reflected in significantly improved renal function compared with wild-type. Thus Morg1 may be a novel therapeutic target to limit renal injury after ischemia-reperfusion.

Early monitoring of acute tubular necrosis in the rat kidney by 23Na-MRI

Fri, 30 Oct 2009 10:16:30 PDT

Reabsorption of water and other molecules is dependent on the corticomedullary sodium concentration gradient in the kidney. During the early course of acute tubular necrosis (ATN), this gradient is altered. Therefore, ²³Na magnetic resonance imaging (MRI) was used to study the alterations in renal sodium distribution in the rat kidney during ischemia and reperfusion (IR) injury, which induces ATN. In-magnet ischemia was induced for 0 (control), 10, 20, 30 or 50 min in Wistar rats. ²³Na images were collected every 10 min during baseline, ischemia, and 60-min reperfusion periods. T₁ and T₂ relaxation times were measured by both ²³Na-MRI and -MRS on a separate cohort of animals during ischemia and reperfusion for correction of relaxation-related tissue sodium concentration (TSC). A marked decrease was observed in the medulla and cortex ²³Na-MRI signal intensity (SI) during the early evolution of ATN caused by IR injury, with the sodium reabsorption function of the kidney being irreversibly damaged after 50 min of ischemia. Sodium relaxation time characteristics were similar in the medulla and cortex of normal kidney, but significantly decreased with IR. The changes in relaxation times in both compartments were identical; thus the medulla-to-cortex sodium SI ratio represents the TSC ratio of both compartments. The extent of IR damage observed with histological examination correlated with the ²³Na-MRI data. ²³Na-MRI has great potential for noninvasive, clinical diagnosis of evolving ATN in the setup of acute renal failure and in differentiating ATN from other causes of renal failure where tubular function is maintained.

Thrombospondin-2 therapy ameliorates experimental glomerulonephritis via inhibition of cell proliferation, inflammation, and TGF-{beta} activation

Daniel, C., Wagner, A., Hohenstein, B., Hugo, C. — Fri, 30 Oct 2009 10:16:30 PDT

We recently identified thrombospondin-2 (TSP-2) as an endogenous regulator of matrix remodelling and inflammation in experimental kidney disease by studying TSP-2-deficient mice. In this study, we asked whether systemic TSP-2 overexpression via thigh muscle transfection is able to ameliorate the time course of the anti-Thy1 glomerulonephritis model. After induction of anti-Thy1 nephritis, rats were transfected either with an overexpression plasmid for TSP-2 or lacZ as a control. Biopsies, urine, and blood samples were taken on days 1, 3, and 6 after disease induction. Muscular overexpression of TSP-2 reduced glomerular transforming growth factor (TGF)-β activation and glomerular extracellular matrix formation as determined by collagen IV and fibronectin. In addition, activation of mesangial cells to the myofibroblast-like phenotype was also significantly decreased in TSP-2-overexpressing animals. TSP-2 overexpression inhibited both glomerular endothelial and mesangial cell proliferation, resulting in a reduced glomerular cell number and glomerular tuft area. The inflammatory response, as monitored by T cells and antigen-presenting cells, was reduced significantly by TSP-2 overexpression, but influx of macrophages was unchanged. These data demonstrate TSP-2 as a potential therapeutic agent to inhibit the glomerular proliferative and inflammatory response as well as TGF-β activation and extracellular matrix accumulation in experimental mesangial proliferative glomerulonephritis.

Analysis of the cytoplasmic interaction between polycystin-1 and polycystin-2

Fri, 30 Oct 2009 10:16:30 PDT

Autosomal dominant polycystic kidney disease (ADPKD) arises following mutations of either Pkd1 or Pkd2. The proteins these genes encode, polycystin-1 (PC1) and polycystin-2 (PC2), form a signaling complex using direct intermolecular interactions. Two distinct domains in the C-terminal tail of PC2 have recently been identified, an EF-hand and a coiled-coil domain. Here, we show that the PC2 coiled-coil domain interacts with the C-terminal tail of PC1, but that the PC2 EF-hand domain does not. We measured the K_0.5 of the interaction between the C-terminal tails of PC1 and PC2 and showed that the direct interaction of these proteins is abrogated by a PC1 point mutation that was identified in ADPKD patients. Finally, we showed that overexpression of the PC1 C-terminal tail in MDCK cells alters the Ca²⁺ response, but that overexpression of the PC1 C-terminal tail containing the disease mutation does not. These results allow a more detailed understanding of the mechanism of pathogenic mutations in the cytoplasmic regions of PC1 and PC2.

Rac1 promotes TGF-{beta}-stimulated mesangial cell type I collagen expression through a PI3K/Akt-dependent mechanism

Hubchak, S. C., Sparks, E. E., Hayashida, T., Schnaper, H. W. — Fri, 30 Oct 2009 10:16:30 PDT

Transforming growth factor (TGF)-β is a central mediator in the progression of glomerulosclerosis, leading to accumulation of aberrant extracellular matrix proteins and inappropriate expression of smooth muscle -actin in the kidney. Previously, we reported that disrupting the cytoskeleton diminished TGF-β-stimulated type I collagen accumulation in human mesangial cells. As cytoskeletal signaling molecules, including the Rho-family GTPases, have been implicated in fibrogenesis, we sought to determine the respective roles of RhoA and Rac1 in HMC collagen I expression. TGF-β1 activated both RhoA and Rac1 within 5 min of treatment, and this activation was dependent on the kinase activity of the type I TGF-β receptor. TGF-β1-stimulated induction of type I collagen mRNA expression and promoter activity was diminished by inhibiting Rac1 activity and was increased by a constitutively active Rac1 mutant, whereas inhibiting RhoA activity had no such effect. Rac1 activation required phosphatidylinositol-3-kinase (PI3K) activity. Furthermore, the PI3K antagonist, LY294002, reduced TGF-β1-stimulated COL1A2 promoter activity and Rac1 activation. It also partially blocked active Rac1-stimulated collagen promoter activity, suggesting that PI3K activity contributes to both TGF-β activation of Rac1 and signal propagation downstream of Rac1. Thus, while both Rac1 and RhoA are rapidly activated in response to TGF-β1 in human mesangial cells, only Rac1 activation enhances events that contribute to mesangial cell collagen expression, through a positive feedback loop involving PI3K.

Decreased renal perfusion rapidly increases plasma membrane Na-K-ATPase in rat cortex by an angiotensin II-dependent mechanism

Yingst, D. R., Araghi, A., Doci, T. M., Mattingly, R., Beierwaltes, W. H. — Fri, 30 Oct 2009 10:16:30 PDT

To understand how rapid changes in blood pressure can regulate Na-K-ATPase in the kidney cortex, we tested the hypothesis that a short-term (5 min) decrease in renal perfusion pressure will increase the amount of Na-K-ATPase in the plasma membranes by an angiotensin II-dependent mechanism. The abdominal aorta of anesthetized Sprague-Dawley rats was constricted with a ligature between the renal arteries, and pressure was monitored on either side during acute constriction. Left renal perfusion pressure was reduced to 70 ± 1 mmHg (n = 6), whereas right renal perfusion pressure was 112 ± 4 mmHg. In control (nonconstricted) rats (n = 5), pressure to both kidneys was similar at 119 ± 6 mmHg. After 5 min of reduced perfusion, femoral venous samples were taken for plasma renin activity (PRA) and the kidneys excised. The cortex was dissected, minced, sieved, and biotinylated. Lower perfusion left kidneys showed a 41% increase (P < 0.003) in the amount of Na-K-ATPase in the plasma membrane compared with right kidneys. In controls, there was no difference in cell surface Na-K-ATPase between left and right kidneys (P = 0.47). PRA was 57% higher in experimental animals compared with controls. To test the role of angiotensin II in mediating the increase in Na-K-ATPase, we repeated the experiments (n = 6) in rats treated with ramiprilat. When angiotensin-converting enzyme was inhibited, the cell surface Na-K-ATPase of the two kidneys was equal (P =0.46). These results confirm our hypothesis: rapid changes in blood pressure regulate trafficking of Na-K-ATPase in the kidney cortex.

The instructive role of metanephric mesenchyme in ureteric bud patterning, sculpting, and maturation and its potential ability to buffer ureteric bud branching defects

Fri, 30 Oct 2009 10:16:30 PDT

Kidney organogenesis depends on reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) to form the UB-derived collecting system and MM-derived nephron. With the advent of in vitro systems, it is clear that UB branching can occur independently of MM contact; however, little has been done to detail the role of MM cellular contact in this process. Here, a model system in which the cultured isolated UB is recombined with uninduced MM is used to isolate the effects of the MM progenitor tissue on the development and maturation of the collecting system. By morphometrics, we demonstrate that cellular contact with the MM is required for vectorial elongation of stalks and tapering of luminal caliber of UB-derived tubules. Expression analysis of developmentally significant genes indicates the cocultured tissue is most similar to an embryonic day 19 (E19) kidney. The likely major contributor to this is the functional maturation of the collecting duct and proximal nephron segments in the UB-induced MM, as measured by quantitative PCR, of the collecting duct-specific arginine vasopressin receptor and the nephron tubule segment-specific organic anion transporter OAT1, Na-P_i type 2 cotransporter, and Tamm-Horsfall protein gene expressions. However, expression of aquaporin-2 is upregulated similarly in isolated UB and cocultured tissue, suggesting that some aspects of functional maturation can occur independently of MM cellular contact. In addition to its sculpting effects, the MM normalized a "branchless" UB morphology induced by FGF7 or heregulin in isolated UB culture. The morphological changes induced by the MM were accompanied by a reassignment of GFR1 (a receptor for GDNF) to tips. Such "quality control" by the MM of UB morphology may provide resiliency to the branching program. This may help to explain a number of knockout phenotypes in which branching and/or cystic defects are less impressive than expected. A second hit in the MM may thus be necessary to make these defects fully apparent.

AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway

Li, X. C., Hopfer, U., Zhuo, J. L. — Fri, 30 Oct 2009 10:16:30 PDT

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT₁ (AT_1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT₁-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT₁ receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT₁-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT₁ siRNAs inhibited AT₁ expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by ~90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT₁-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in ~52% decreases in AT₁-mediated FITC-ANG II uptake and ~66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT₁ expression or clathrin-dependent transferrin uptake. Both losartan and AT_1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT₁ (AT_1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.

Presence of cyclic nucleotide-gated channels in the rat urethra and their involvement in nerve-mediated nitrergic relaxation

Triguero, D., Sancho, M., Garcia-Flores, M., Garcia-Pascual, A. — Fri, 30 Oct 2009 10:16:30 PDT

We have addressed the distribution of cGMP-gated channels (CNG) in the rat urethra for the first time, as well as their putative role in mediating of the relaxation elicited by electrical field stimulation of nitrergic nerves. Functional studies have shown that specifically blocking CNG with l-cis-diltiazem leads to the rapid inhibition of urethral relaxation induced either by nitric oxide (NO) released by the nerves or by soluble guanylate cyclase activated with YC-1. By contrast, nerve-mediated noradrenergic contractions were only slowly and mildly reduced by l-cis-diltiazem. This effect was mimicked by lower concentrations of the d-diltiazem isomer, probably due to the nonspecific inhibition of voltage-dependent calcium channels. However, d-diltiazem did not affect relaxation responses. The expression of heteromeric retinal-like CNGA1 channels was demonstrated by conventional PCR on mRNA from the rat urethra. These channels were located in a subpopulation of intramuscular interstitial cells of Cajal (ICC) as well as in smooth muscle cells, although they were less abundant in the latter. CNG channels could not be visualized in any nervous structure within the urethral wall, in agreement with the emerging view that a subset of ICC serves as a target for NO. These channels could provide a suitable ionic mechanism to associate the changes in cytosolic calcium with the activation of the nitric NO-cGMP pathway and relaxation although the precise mechanisms involved remain to be elucidated.

Transglutaminase-1 protects renal epithelial cells from hydrogen peroxide-induced apoptosis through activation of STAT3 and AKT signaling pathways

Fri, 30 Oct 2009 10:16:30 PDT

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. In this study, we investigated the role of TGase-1 in cell survival and the survival signaling pathways regulated by TGase-1 in RPTC following oxidant injury. Exposure of RPTC to hydrogen peroxide (H₂O₂) resulted in apoptosis and an increase in TGase activity. Inhibition of TGase activity with monodansylcadervine (MDC), a TGase inhibitor, or knockdown of TGase-1 with small interference (si)RNA enhanced apoptosis and decreased cell survival in H₂O₂-treated RPTC. Conversely, overexpression of TGase-1 rendered RPTC more resistant to H₂O₂ toxicity and MDC treatment blocked this response. Concurrent with RPTC apoptosis, phosphorylation of AKT, signal transducer and activator of transcription-3 (STAT3), and glucogen synthase kinase-3β (GSK-3β) were observed. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated H₂O₂-induced cell death and increased GSK-3β activity by dephosphorylation at serine 9. Furthermore, treatment with GSK-3β inhibitors reduced H₂O₂-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore, we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury.

Developmental renin expression in mice with a defective renin-angiotensin system

Fri, 30 Oct 2009 10:16:30 PDT

During nephrogenesis, renin expression shifts from the vessel walls of interlobular arteries to the terminal portions of afferent arterioles in a wavelike pattern. Since the mechanisms responsible for the developmental deactivation of renin expression are as yet unknown, we hypothesized that the developing renin-angiotensin system (RAS) may downregulate itself via negative feedback to prevent overactivity of renin. To test for a possible role of angiotensin II in the developmental deactivation of renin expression, we studied the development of intrarenal renin expression in mice lacking ANG II AT_1a, AT_1b, or AT₂ receptors and in animals with abolished circulating ANG II due to deletion of the gene for angiotensin I-converting enzyme (ACE). The development of intrarenal renin expression was normal in mice lacking ANG II AT_1b or AT₂ receptors. In animals lacking both ANG II AT_1a and AT_1b receptors, ACE, or ANG II AT_1a receptors, renin expression was normal early and renin disappeared from mature vessels until development of cortical interlobular and afferent arterioles began. The development of cortical vessels in these genotypes was accompanied by a markedly increased number of renin-expressing cells, many of which were ectopically located and attached in a grapelike fashion to the outer vessel perimeter. Although the number of renin-expressing cells declined during final maturation of the kidneys, the atypical distribution pattern of renin cells was maintained. These findings suggest that ANG II does not play a central role in the typical developmental shift in renin expression from the arcuate vessels to the afferent arterioles. During postnatal maturation of mouse kidneys, interruption of the RAS causes severe hyperplasia of renin cells via a mechanism that centrally involves AT_1a receptors. However, the distribution pattern of renin cells in adult kidneys with an interrupted RAS does not mimic any normal developmental stage since renin expression is frequently found in cells outside the arteriolar vessel walls in RAS mutants.

Activation of local aldosterone system within podocytes is involved in apoptosis under diabetic conditions

Fri, 30 Oct 2009 10:16:30 PDT

Previous studies have shown that mineralocorticoid receptor (MCR) blocker reduces proteinuria in diabetic nephropathy (DN), but the role of aldosterone in podocyte injury has never been explored in DN. This study was undertaken to elucidate whether a local aldosterone system existed in podocytes and to examine its role in podocyte apoptosis under diabetic conditions. In vitro, immortalized podocytes were exposed to 5.6 mM glucose (NG), NG + 24.4 mM mannitol, and 30 mM glucose (HG) with or without 10^–7 M spironolactone (SPR). In vivo, 32 Sprague-Dawley rats were injected with diluent (C, n = 16) or streptozotocin intraperitoneally [diabetes mellitus (DM), n = 16], and 8 rats from each group were treated with SPR for 3 mo. Aldosterone synthase (CYP11B2) and MCR mRNA and protein expression were determined by real-time PCR and Western blot, respectively, and aldosterone levels by radioimmunoassay. Western blot for apoptosis-related molecules, Hoechst 33342 staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine apoptosis. CYP11B2 and MCR expression were significantly higher in HG-stimulated podocytes and DM glomeruli compared with NG cells and C glomeruli, respectively, along with increased aldosterone levels. Western blot analysis revealed that cleaved caspase-3 and Bax expression was significantly increased, whereas Bcl-2 expression was significantly decreased in HG-stimulated podocytes and in DM glomeruli. Apoptosis determined by Hoechst 33342 staining and TUNEL assay were also significantly increased in podocytes under diabetic conditions. These changes in the expression of apoptosis-related proteins and the increase in apoptotic cells were inhibited by SPR treatment. These findings suggest that a local aldosterone system is activated and is involved in podocyte apoptosis under diabetic conditions.

Novel missense mutation of uromodulin in mice causes renal dysfunction with alterations in urea handling, energy, and bone metabolism

Fri, 30 Oct 2009 10:16:30 PDT

Uromodulin-associated kidney disease is a heritable renal disease in humans caused by mutations in the uromodulin (UMOD) gene. The pathogenesis of the disease is mostly unknown. In this study, we describe a novel chemically induced mutant mouse line termed Umod^A227T exhibiting impaired renal function. The A227T amino acid exchange may impair uromodulin trafficking, leading to dysfunction of thick ascending limb cells of Henle's loop of the kidney. As a consequence, homozygous mutant mice display azotemia, impaired urine concentration ability, reduced fractional excretion of uric acid, and a selective defect in concentrating urea. Osteopenia in mutant mice is presumably a result of chronic hypercalciuria. In addition, body composition, lipid, and energy metabolism are indirectly affected in heterozygous and homozygous mutant Umod^A227T mice, manifesting in reduced body weight, fat mass, and metabolic rate as well as reduced blood cholesterol, triglycerides, and nonesterified fatty acids. In conclusion, Umod^A227T might act as a gain-of-toxic-function mutation. Therefore, the Umod^A227T mouse line provides novel insights into consequences of disturbed uromodulin excretion regarding renal dysfunction as well as bone, energy, and lipid metabolism.

Oxalate-induced activation of PKC-{alpha} and -{delta} regulates NADPH oxidase-mediated oxidative injury in renal tubular epithelial cells

Thamilselvan, V., Menon, M., Thamilselvan, S. — Fri, 30 Oct 2009 10:16:30 PDT

Oxalate-induced oxidative stress contributes to cell injury and promotes renal deposition of calcium oxalate crystals. However, we do not know how oxalate stimulates reactive oxygen species (ROS) in renal tubular epithelial cells. We investigated the signaling mechanism of oxalate-induced ROS formation in these cells and found that oxalate significantly increased membrane-associated protein kinase C (PKC) activity while at the same time lowering cytosolic PKC activity. Oxalate markedly translocated PKC- and - from the cytosol to the cell membrane. Pretreatment of LLC-PK₁ cells with specific inhibitors of PKC- or - significantly blocked oxalate-induced generation of superoxide and hydrogen peroxide along with NADPH oxidase activity, LDH release, lipid hydroperoxide formation, and apoptosis. The PKC activator PMA mimicked oxalate's effect on oxidative stress in LLC-PK₁ cells as well as cytosol-to-membrane translocation of PKC- and -. Silencing of PKC- expression by PKC--specific small interfering RNA significantly attenuated oxalate-induced cell injury by decreasing hydrogen peroxide generation and LDH release. We believe this is the first demonstration that PKC-- and --dependent activation of NADPH oxidase is one of the mechanisms responsible for oxalate-induced oxidative injury in renal tubular epithelial cells. The study suggests that the therapeutic approach might be considered toward attenuating oxalate-induced PKC signaling-mediated oxidative injury in recurrent stone formers.

Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption

Bugaj, V., Pochynyuk, O., Stockand, J. D. — Fri, 30 Oct 2009 10:16:31 PDT

We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na⁺ reabsorption during water conservation vs. maintenance of Na⁺ balance. We find that vasopressin significantly increases ENaC activity within 2–3 min by increasing open probability (P_o). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18–24 h) and pretreatment of isolated CD with vasopressin (~30 min) resulted in a similar increase in P_o. In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P_o, increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na⁺ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na⁺ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.

Reduction of renal triglyceride accumulation: effects on proximal tubule Na+/H+ exchange and urinary acidification

Bobulescu, I. A., Dubree, M., Zhang, J., McLeroy, P., Moe, O. W. — Fri, 30 Oct 2009 10:16:31 PDT

One main pathophysiological mechanism underlying the increased risk for uric acid nephrolithiasis in humans with the metabolic syndrome is the excretion of unduly acidic urine, in part because of reduced excretion of the main urinary buffer, ammonium. The Zucker diabetic fatty (ZDF) rat, an established rodent model of the metabolic syndrome, has similar urinary abnormalities, attributed in part to lower expression and activity of the principal mediator of proximal tubule ammonium excretion, brush-border membrane Na⁺/H⁺ exchanger 3 (NHE3). These defects are associated with renal tubular steatosis in ZDF rats, but the causal relationship between renal steatosis and defective urinary acidification has not been investigated in vivo. We hypothesized that reduction of renal steatosis would commensurately normalize urinary acidification parameters. We treated ZDF rats with thiazolidinediones to reduce nonadipose tissue steatosis. Four weeks of treatment reduced renal triglyceride accumulation and restored urinary acidification parameters in ZDF rats to levels comparable to their lean littermates; urinary acidification was not affected by treatment in lean rats. To further document the direct effects of fat, we showed that functional abnormalities induced by fat loading in a cell culture model of proximal tubule steatosis and lipotoxicity can be reversed by fat removal but not by thiazolidinediones alone. Together, these findings support the causative role of renal steatosis in the pathogenesis of urinary acidification defects, demonstrate reversibility upon lipid removal, and highlight a potential therapeutic strategy for renal abnormalities in the metabolic syndrome.

Do distinct populations of dorsal root ganglion neurons account for the sensory peptidergic innervation of the kidney?

Fri, 30 Oct 2009 10:16:31 PDT

Peptidergic afferent renal nerves (PARN) have been linked to kidney damage in hypertension and nephritis. Neither the receptors nor the signals controlling local release of neurokinines [calcitonin gene-related peptide (CGRP) and substance P (SP)] and signal transmission to the brain are well-understood. We tested the hypothesis that PARN, compared with nonrenal afferents (Non-RN), are more sensitive to acidic stimulation via transient receptor potential vanilloid type 1 (TRPV1) channels and exhibit a distinctive firing pattern. PARN were distinguished from Non-RN by fluorescent labeling (DiI) and studied by in vitro patch-clamp techniques in dorsal root ganglion neurons (DRG; T11-L2). Acid-induced currents or firing due to current injection or acidic superfusion were studied in 252 neurons, harvested from 12 Sprague-Dawley rats. PARN showed higher acid-induced currents than Non-RN (transient: 15.9 ± 5.1 vs. 0.4 ± 0.2* pA/pF at pH 6; sustained: 20.0 ± 4.5 vs. 6.2 ± 1.2* pA/pF at pH 5; *P < 0.05). The TRPV1 antagonist capsazepine inhibited sustained, amiloride-transient currents. Forty-eight percent of PARN were classified as tonic neurons (TN = sustained firing during current injection), and 52% were phasic (PN = transient firing). Non-RN were rarely tonic (15%), but more frequently phasic (85%), than PARN (P < 0.001). TN were more frequently acid-sensitive than PN (50–70 vs. 2–20%, P < 0.01). Furthermore, renal PN were more frequently acid-sensitive than nonrenal PN (20 vs. 2%, P < 0.01). Confocal microscopy revealed innervation of renal vessels, tubules, and glomeruli by CGRP- and partly SP-positive fibers coexpressing TRPV1. Our data show that PARN are represented by a very distinct population of small-to-medium sized DRG neurons exhibiting more frequently tonic firing and TRPV1-mediated acid sensitivity. These very distinct DRG neurons might play a pivotal role in renal physiology and disease.

Developmental delays consistent with cochlear hypothyroidism contribute to failure to develop hearing in mice lacking Slc26a4/pendrin expression

Fri, 30 Oct 2009 10:16:31 PDT

Mutations of SLC26A4 cause an enlarged vestibular aqueduct, nonsyndromic deafness, and deafness as part of Pendred syndrome. SLC26A4 encodes pendrin, an anion exchanger located in the cochlea, thyroid, and kidney. The goal of the present study was to determine whether developmental delays, possibly mediated by systemic or local hypothyroidism, contribute to the failure to develop hearing in mice lacking Slc26a4 (Slc26a4^–/–). We evaluated thyroid function by voltage and pH measurements, by array-assisted gene expression analysis, and by determination of plasma thyroxine levels. Cochlear development was evaluated for signs of hypothyroidism by microscopy, in situ hybridization, and quantitative RT-PCR. No differences in plasma thyroxine levels were found in Slc26a4^–/– and sex-matched Slc26a4^+/– littermates between postnatal day 5 (P5) and P90. In adult Slc26a4^–/– mice, the transepithelial potential and the pH of thyroid follicles were reduced. No differences in the expression of genes that participate in thyroid hormone synthesis or ion transport were observed at P15, when plasma thyroxine levels peaked. Scala media of the cochlea was 10-fold enlarged, bulging into and thereby displacing fibrocytes, which express Dio2 to generate a cochlear thyroid hormone peak at P7. Cochlear development, including tunnel opening, arrival of efferent innervation at outer hair cells, endochondral and intramembraneous ossification, and developmental changes in the expression of Dio2, Dio3, and Tectb were delayed by 1–4 days. These data suggest that pendrin functions as a HCO₃^– transporter in the thyroid, that Slc26a4^–/– mice are systemically euthyroid, and that delays in cochlear development, possibly due to local hypothyroidism, lead to the failure to develop hearing.

Unlike each drug alone, lisinopril if combined with avosentan promotes regression of renal lesions in experimental diabetes

Fri, 30 Oct 2009 10:16:31 PDT

In the present study, we evaluated the effect of simultaneously blocking angiotensin II synthesis and endothelin (ET)-1 activity as a multimodal intervention to implement renoprotection in overt diabetic nephropathy. Mechanisms underlying combined therapy effectiveness were addressed by investigating podocyte structure and function and glomerular barrier size-selective properties. Uninephrectomized rats made diabetic by streptozotocin received orally placebo, lisinopril (12.5 mg/l), the ET_A receptor antagonist avosentan (30 mg/kg), or their combination from 4 (when animals had proteinuria) to 8 mo. Proteinuria, renal damage, podocyte number, nephrin expression, and glomerular size selectivity by graded-size Ficoll molecule fractional clearance were assessed. Combined therapy normalized proteinuria, provided complete protection from tubulointerstitial damage, and induced regression of glomerular lesions, while only a partial renoprotection was achieved by each drug alone. Lisinopril plus avosentan restored to normal values the number of podocytes. Single therapies only limited podocyte depletion. Defective nephrin expression of diabetes was prevented by each drug. Altered glomerular size selectivity to large macromolecules of diabetic rats was remarkably improved by lisinopril and the combined treatment. Avosentan ameliorated peritubular capillary architecture and reduced interstitial inflammation and fibrosis. The ACE inhibitor and ET_A receptor antagonist induced regression of glomerular lesions in overt diabetic nephropathy. Regression of renal disease was conceivably the result of the synergistic effect of the ACE inhibitor of preserving glomerular permselective properties and the ET_A antagonist in improving tubulointerstitial changes. These findings provide mechanistic insights to explain the antiproteinuric effect of this combined therapy in diabetes.

Early exposure to germs modifies kidney damage and inflammation after experimental ischemia-reperfusion injury

Jang, H. R., Gandolfo, M. T., Ko, G. J., Satpute, S., Racusen, L., Rabb, H. — Fri, 30 Oct 2009 10:16:31 PDT

Kidney ischemia-reperfusion injury (IRI) is, in part, mediated by immune and inflammatory factors. Since microbial stimuli are known to alter immune and inflammatory responses, we hypothesized that differences in perinatal microbial status would modify renal injury following IRI. We performed bilateral renal IRI on 6-wk-old germ-free and control mice and studied the effects on kidney lymphocyte trafficking, cytokines, function, and structure. Compared with control mice, normal kidneys of germ-free mice exhibited more NKT cells and lower IL-4 levels. Postischemia, more CD8 T cells trafficked into postischemic kidneys of germ-free mice compared with control mice. Renal structural injury and functional decline following IRI were more severe in germ-free mice compared with control mice. When germ-free mice were conventionalized with the addition of bacteria to their diet, the extent of renal injury after IRI became equivalent to age-matched control mice, with similar numbers and phenotypes of T cells and NKT cells, as well as cytokine expression in both normal kidneys and postischemic kidneys of conventionalized germ-free mice and age-matched control mice. Thus microbial stimuli influence the phenotype of renal lymphocytes and the expression of cytokines of normal kidneys and also modulate the outcome of IRI.

Regulation of rat intestinal Na-dependent phosphate transporters by dietary phosphate

Fri, 30 Oct 2009 10:16:31 PDT

Hyperphosphatemia associated with chronic kidney disease is one of the factors that can promote vascular calcification, and intestinal P_i absorption is one of the pharmacological targets that prevents it. The type II Na-P_i cotransporter NaPi-2b is the major transporter that mediates P_i reabsorption in the intestine. The potential role and regulation of other Na-P_i transporters remain unknown. We have identified expression of the type III Na-P_i cotransporter PiT-1 in the apical membrane of enterocytes. Na-P_i transport activity and NaPi-2b and PiT-1 proteins are mostly expressed in the duodenum and jejunum of rat small intestine; their expression is negligible in the ileum. In response to a chronic low-P_i diet, there is an adaptive response restricted to the jejunum, with increased brush border membrane (BBM) Na-P_i transport activity and NaPi-2b, but not PiT-1, protein and mRNA abundance. However, in rats acutely switched from a low- to a high-P_i diet, there is an increase in BBM Na-P_i transport activity in the duodenum that is associated with an increase in BBM NaPi-2b protein abundance. Acute adaptive upregulation is restricted to the duodenum and induces an increase in serum P_i that produces a transient postprandial hyperphosphatemia. Our study, therefore, indicates that Na-P_i transport activity and NaPi-2b protein expression are differentially regulated in the duodenum vs. the jejunum and that postprandial upregulation of NaPi-2b could be a potential target for treatment of hyperphosphatemia.

Corrigendum

Fri, 30 Oct 2009 10:16:31 PDT