bluuebunny/crossref_metadata_2025 huggingface dataset:
Contributions of angiotensin II and tumor necrosis factor-α to the development of renal fibrosis
Authors: Guo Guangjie , Morrissey Jeremiah , McCracken Ruth , Tolley Timothy , Liapis Helen , Klahr Saulo
Published: December 2017

Angiotensin II upregulates tumor necrosis factor-α (TNF-α) in the rat kidney with unilateral ureteral obstruction (UUO). In a mouse model of UUO, we found that tubulointerstitial fibrosis is blunted when the TNF-α receptor, TNFR1, is functionally knocked out. In this study, we used mutant mice with UUO in which the angiotensin II receptor AT1aor the TNF-α receptors TNFR1 and TNFR2 were knocked out to elucidate interactions between the two systems. The contribution of both systems to renal fibrosis was assessed by treating TNFR1/TNFR2-double knockout (KO) mice with an angiotensin-converting enzyme inhibitor, enalapril. The increased interstitial volume (Vvint) in the C57BI/6 wild-type mouse was decreased in the AT1aKO from 32.8 ± 4.0 to 21.0 ± 3.7% ( P < 0.005) or in the TNFR1/TNFR2 KO to 22.3 ± 2.1% ( P < 0.005). The Vvintof the TNFR1/TNFR2 KO was further decreased to 15.2 ± 3.7% ( P < 0.01) by enalapril compared with no treatment. The induction of TNF-α mRNA and transforming growth factor-β1 (TGF-β1) mRNA in the kidney with UUO was significantly blunted in the AT1aor TNFR1/TNFR2 KO mice compared with the wild-type mice. Treatment of the TNFR1/TNFR2 KO mouse with enalapril reduced both TNF-α and TGF-β1 mRNA and their proteins to near normal levels. Also, α-smooth muscle actin expression and myofibroblast proliferation were significantly inhibited in the AT1aor TNFR1/TNFR2 KO mice, and they were further inhibited in enalapril-treated TNFR1/TNFR2 KO mice. Incapacitating the angiotensin II or the TNF-α systems individually leads to partial blunting of fibrosis. Incapacitating both systems, by using a combination of genetic and pharmacological means, further inhibited interstitial fibrosis and tubule atrophy in obstructive nephropathy.
Effects of P-glycoprotein on cell volume regulation in mouse proximal tubule
Authors: Miyata Yukio , Asano Yasushi , Muto Shigeaki
Published: December 2017

The role of P-glycoprotein (P-gp) in cell volume regulation was examined in isolated nonperfused proximal tubule S2 segments from wild-type (WT) mice and those in which both mdr1a and mdr1b genes were knocked out (KO). When the osmolality of the bathing solution was rapidly decreased from 300 to 180 mosmol/kgH2O, the tubules from both the WT and KO mice exhibited regulatory volume decrease (RVD) by a similar magnitude after the initial cell swelling. The peritubular addition of two P-pg inhibitors (verapamil and cyclosporin A) to either group of the tubules had no effect on RVD. When the tubules from the WT mice were rapidly exposed to a hyperosmotic solution (500 mosmol/kgH2O) including 200 mM mannitol, they abruptly shrank to 82.1% of their control volume but remained in a shrunken state during the experimental period, indicating a lack of regulatory volume increase (RVI). The addition of the two P-gp inhibitors, but not the inhibitor of the renal organic cation transport system (tetraethylammonium), to the tubules from the WT mice resulted in RVI. Surprisingly, when the tubules from the KO mice were exposed to the hyperosmotic solution, they abruptly shrank to 79.9% of their control volume, and then gradually swelled to 87.7% of their control volume, showing RVI. However, exposure of the tubules from the KO mice to the hyperosmotic solution in the presence of the two P-gp inhibitors had no effect on RVI. When the tubules of the WT mice were exposed to the hyperosmotic solution including either of the two P-gp inhibitors, in the absence of peritubular Na+or in the presence of peritubular ethylisopropylamiloride (EIPA; the specific inhibitor of Na+/H+exchange), they did not exhibit RVI. In the tubules of the KO mice, both removing peritubular Na+and adding peritubular EIPA inhibited RVI induced by the hyperosmotic solution. We conclude that 1) in mouse proximal tubule, P-gp modulates RVI during hyperosmotic stress but not RVD during hyposmotic stress and 2) basolateral membrane Na+/H+exchange partly contributes to the P-gp-induced modulation of RVI under hyperosmotic stress.
1,25(OH)2D3stimulates Mg2+uptake into MDCT cells: modulation by extracellular Ca2+and Mg2+
Authors: Ritchie Gordon , Kerstan Dirk , Dai Long-Jun , Kang Hyung Sub , Canaff Lucie , Hendy Geoffrey N. , Quamme Gary A.
Published: December 2017

The distal convoluted tubule plays a significant role in renal magnesium conservation. Although the cells of the distal convoluted tubule possess the vitamin D receptor, little is known about the effects of 1α,25-dihydroxyvitamin D [1,25(OH)2D3] on magnesium transport. In this study, we examined the effect of 1,25(OH)2D3on distal cellular magnesium uptake and the modulation of this response by extracellular Ca2+and Mg2+in an immortalized mouse distal convoluted tubule (MDCT) cell line. MDCT cells possess the divalent cation-sensing receptor (CaSR) that responds to elevation of extracellular Ca2+and Mg2+concentrations to diminish peptide hormone-stimulated Mg2+uptake. Mg2+uptake rates were determined by microfluorescence in Mg2+-depleted MDCT cells. Treatment of MDCT cells with 1,25(OH)2D3for 16–24 h stimulated basal Mg2+uptake in a concentration-dependent manner from basal levels of 164 ± 5 to 210 ± 11 nM/s, representing a 28 ± 3% change. Pretreatment with actinomycin D or cycloheximide abolished 1,25(OH)2D3-stimulated.Mg2+uptake (154 ± 18 nM/s), suggesting that 1,25(OH)2D3stimulates Mg2+uptake through gene activation and protein synthesis. Elevation of extracellular Ca2+inhibited 1,25(OH)2D3-stimulated Mg2+uptake (143 ± 5 nM/s). Preincubation of the cells with an antibody to the CaSR prevented the inhibition by elevated extracellular Ca2+of 1,25(OH)2D3-stimulated Mg2+uptake (202 ± 8 nM/s). Treatment with an antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of extracellular Ca2+on 1,25(OH)2D3-responsive Mg2+entry. This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary, 1,25(OH)2D3stimulated Mg2+uptake in MDCT cells, and this is dependent on de novo protein synthesis. Elevation of extracellular Ca2+, acting via the CaSR, inhibited 1,25(OH)2D3-stimulated Mg2+entry. These data indicate that 1,25(OH)2D3has important effects on the control of magnesium entry in MDCT cells and these responses can be modulated by extracellular divalent cations.
Urea inhibits hypertonicity-inducible TonEBP expression and action
Authors: Tian Wei , Cohen David M.
Published: December 2017

Tonicity-responsive genes are regulated by the TonE enhancer element and the tonicity-responsive enhancer binding protein (TonEBP) transcription factor with which it interacts. Urea, a permeant solute coexistent with hypertonic NaCl in the mammalian renal medulla, activates a characteristic set of signaling events that may serve to counteract the effects of NaCl in some contexts. Urea inhibited the ability of hypertonic stressors to increase expression of TonEBP mRNA and also inhibited tonicity-inducible TonE-dependent reporter gene activity. The permeant solute glycerol failed to reproduce these effects, as did cell activators including peptide mitogens and phorbol ester. The inhibitory effect of urea was evident as late as 2 h after the application of hypertonicity. Pharmacological inhibitors of known urea-inducible signaling pathways failed to abolish the inhibitory effect of urea. TonEBP action is incompletely understood, but evidence supports a role for proteasome function and p38 action in regulation; urea failed to inhibit proteasome function or p38 signaling in response to hypertonicity. Consistent with its effect on TonEBP expression and action, urea pretreatment inhibited the effect of hypertonicity on expression of the physiological effector gene, aldose reductase. Taken together, these data 1) define a molecular mechanism of urea-mediated inhibition of tonicity-dependent signaling, and 2) underscore a role for TonEBP abundance in regulating TonE-mediated gene transcription.
Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC
Authors: White Steven M. , Imig John D. , Kim Thu-Thuy , Hauschild Benjamin C. , Inscho Edward W.
Published: December 2017

This study tested the hypothesis that P2X receptor activation increases intracellular Ca2+concentration ([Ca2+]i) in preglomerular microvascular smooth muscle cells (MVSMC) by evoking voltage-dependent calcium influx. MVSMC were obtained and loaded with the calcium-sensitive dye fura 2 and studied by using single-cell fluorescence microscopy. The effect of P2X receptor activation on [Ca2+]iwas assessed by using the P2X receptor-selective agonist α,β-methylene-ATP and was compared with responses elicited by the endogenous P2 receptor agonist ATP. α,β-Methylene-ATP increased [Ca2+]idose dependently. Peak increases in [Ca2+]iaveraged 37 ± 11, 73 ± 15, and 103 ± 21 nM at agonist concentrations of 0.1, 1, and 10 μM, respectively. The average peak response elicited by 10 μM α,β-methylene-ATP was ∼34% of the response obtained with 10 μM ATP. α,β-Methylene-ATP induced a transient increase in [Ca2+]ibefore [Ca2+]ireturned to baseline, whereas ATP induced a biphasic response including a peak response followed by a sustained plateau. In Ca2+-free medium, ATP induced a sharp transient increase in [Ca2+]i, whereas the response to α,β-methylene-ATP was abolished. Ca2+channel blockade with 10 μM diltiazem or nifedipine attenuated the response to α,β-methylene-ATP, whereas nonspecific blockade of Ca2+influx pathways with 5 mM Ni2+abolished the response. Blockade of P2X receptors with the novel P2X receptor antagonist NF-279 completely but reversibly abolished the response to α,β-methylene-ATP. These results indicate that P2X receptor activation by α,β-methylene-ATP increases [Ca2+]iin preglomerular MVSMC, in part, by stimulating voltage-dependent Ca2+influx through L-type Ca2+channels.
P2 receptors in regulation of renal microvascular function
Authors: Inscho Edward W.
Published: December 2017

In the last 10–15 years, interest in the physiological role of P2 receptors has grown rapidly. Cellular, tissue, and organ responses to P2 receptor activation have been described in numerous in vivo and in vitro models. The purpose of this review is to provide an update of the recent advances made in determining the involvement of P2 receptors in the control of renal hemodynamics and the renal microcirculation. Special attention will be paid to work published in the last 5–6 years directed at understanding the role of P2 receptors in the physiological control of renal microvascular function. Several investigators have begun to evaluate the effects of P2 receptor activation on renal microvascular function across several species. In vivo and in vitro evidence consistently supports the hypothesis that P2 receptor activation by locally released extracellular nucleotides influences microvascular function. Extracellular nucleotides selectively influence preglomerular resistance without having an effect on postglomerular tone. P2 receptor inactivation blocks autoregulatory behavior whereas responsiveness to other vasoconstrictor agonists is retained. P2 receptor stimulation activates multiple intracellular signal transduction pathways in preglomerular smooth muscle cells and mesangial cells. Renal microvascular cells and mesangial cells express multiple subtypes of P2 receptors; however, the specific role each plays in regulating vascular and mesangial cell function remains unclear. Accordingly, the results of studies performed to date provide strong support for the hypothesis that P2 receptors are important contributors to the physiological regulation of renal microvascular and/or glomerular function.
Effect of COX-2 inhibitor NS-398 on expression of PGE2receptor subtypes in M-1 mouse CCD cells
Authors: Nasrallah Rania , Laneuville Odette , Ferguson Shawn , Hébert Richard L.
Published: December 2017

Our present study has investigated the effect of cyclooxygenase-2 (COX-2) inhibition on prostaglandin E2(PGE2) receptor expression in M-1 cortical collecting duct cells and measured their response to PGE2. Using a semiquantitative titration analysis method, we show that following the addition of the COX-2-specific inhibitor NS-398, E-prostanoid receptor subtype (EP3and EP4) mRNA expression was found to increase threefold each vs. the vehicle-treated control. We also observed that EP1but not EP2is expressed in M-1 cells and EP2levels are not induced by NS-398. To determine the status of the PGE2response on exposure to NS-398, we measured cAMP levels in cells after stimulation with varying concentrations of PGE2, then pretreated the cells with 10 μM NS-398 before PGE2exposure and found a significant rise in the stimulatory effect of PGE2on cAMP production. Finally, Western blot analysis of the levels of the EP4receptor protein in control vs. NS-398-treated cells revealed an induction in protein levels in these cells, correlating with the induction in EP4mRNA. We conclude that NS-398 upregulates the expression of EP3and EP4mRNA in M-1 cells. Also, EP4protein levels are increased, resulting in an increased stimulation of cAMP production by PGE2.
Apical membrane of native OMCDi cells has nonselective cation channels
Authors: Xia Shen-Ling , Noh Seung-Hyun , Verlander Jill W. , Gelband Craig H. , Wingo Charles S.
Published: December 2017

The purpose of this study was to examine cation channel activity in the apical membrane of the outer medullary collecting duct of the inner stripe (OMCDi) using the patch-clamp technique. In freshly isolated and lumen-opened rabbit OMCDi, we have observed a single channel conductance of 23.3 ± 0.6 pS ( n = 17) in cell-attached (c/a) patches with high KCl in the bath and in the pipette at room temperature. Channel open probability varied among patches from 0.06 ± 0.01 at −60 mV ( n = 5) to 0.31 ± 0.04 at 60 mV ( n = 6) and consistently increased upon membrane depolarization. In inside-out (i/o) patches with symmetrical KCl solutions, the channel conductance (22.8 ± 0.8 pS; n = 10) was similar as in the c/a configuration. Substitution of the majority of Cl− with gluconate from KCl solution in the pipette and bath did not significantly alter reversal potential ( E rev) or the channel conductance (19.7 ± 1.1 pS in asymmetrical potassium gluconate, n = 4; 21.4 ± 0.5 pS in symmetrical potassium gluconate, n = 3). Experiments with 10-fold lower KCl concentration in bath solution in i/o patches shifted E rev to near the E rev of K+. The estimated permeability of K+ vs. Cl− was over 10, and the conductance was 13.4 ± 0.1 pS ( n = 3). The channel did not discriminate between K+ and Na+, as evidenced by a lack of a shift in the E rev with different K+ and Na+ concentration solutions in i/o patches ( n = 3). The current studies demonstrate the presence of cation channels in the apical membrane of native OMCDicells that could participate in K+ secretion or Na+ absorption.
Effect of dietary K intake on apical small-conductance K channel in CCD: role of protein tyrosine kinase
Authors: Wei Yuan , Bloom Peter , Lin Daohong , Gu Ruimin , Wang Wen Hui
Published: December 2017

We have used Western blot to examine the expression of cSrc protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP)-1D in the renal cortex, and the patch-clamp technique to determine the role of PTK in mediating the effect of dietary K intake on the small-conductance K (SK) channel in the cortical collecting duct (CCD). When rats were on a K-deficient (KD) diet for 1, 3, 5, and 7 days, the expression of cSrc increased by 40, 90, 140, and 135%, respectively. In contrast, the expression of cSrc in the renal cortex from rats on a high-K (HK) diet for 1, 2, and 3 days decreased by 40, 60, and 75%, respectively. However, the protein level of PTP-1D was not significantly changed by dietary K intake. The addition of 1 μM herbimycin A increased NP o, a product of channel number ( N) and open probability ( P o) in the CCD from rats on a normal diet or on a KD diet. The increase in NP o was 0.30 (normal), 0.45 (1-day KD), 0.65 (3-day KD), 1.55 (5-day KD), and 1.85 (7-day KD), respectively. Treatment of the CCD with herbimycin A from rats on a KD diet increased NP o per patch from the control value (0.7) to 1.4 (1-day KD), 1.6 (3-day KD), 2.6 (5-day KD), and 3.5 (7-day KD), respectively. In contrast, HK intake for as short as 1 day abolished the effect of herbimycin A. Furthermore, the expression of ROMK channels in the renal cortex was the same between rats on a KD diet or on a HK diet. Moreover, treatment with herbimycin A did not further increase NP o in the CCDs from rats on a HK diet. We conclude that dietary K intake plays a key role in regulating the activity of the SK channels and that PTK is involved in mediating the effect of the K intake on channel activity in the CCD.
Transport of NH3/NH 4 + in oocytes expressing aquaporin-1
Authors: Nakhoul Nazih L. , Hering-Smith Kathleen S. , Abdulnour-Nakhoul Solange M. , Hamm L. Lee
Published: December 2017

The aim of this study was to determine whether expressing aquaporin (AQP)-1 could affect transport of NH3. Using ion-selective microelectrodes, the experiments were conducted on frog oocytes (cells characterized by low NH3permeability) expressing AQP1. In H2O-injected oocytes, exposure to NH3/NH[Formula: see text] (20 mM, pH 7.5) caused a sustained cell acidification and no initial increase in pHi(as expected from NH3influx), and the cell depolarized to near 0 mV. The absence of Na+, the presence of Ba2+, or raising bath pH (pHB) did not inhibit the magnitude of the pHidecrease or result in an initial increase in pHiwhen NH3/NH[Formula: see text] was added. However, after the cell was acidified (because of NH3/NH[Formula: see text]), raising pHBto 8.0 caused a slow increase in pHibut had no effect on membrane potential. The changes in pHiwith raising pHBdid not occur in the absence of NH3/NH[Formula: see text]. In AQP1 oocytes, exposure to NH3/NH[Formula: see text]usually resulted in little or no change in pHi, and in the absence of Na+there was a small increase in pHi(the cell still depolarized to near 0 mV). However, after exposure to NH3/NH[Formula: see text], raising pHBto 8.0 caused pHito increase more than two times faster than in control oocytes. This increase in pHiis likely the result of increased NH3entry and not the result of NH[Formula: see text] transport. These results indicate that 1) the oocyte membrane, although highly permeable to NH[Formula: see text], has a significant NH3permeability and 2) NH3permeability is enhanced by AQP1.
Essential role of NHE3 in facilitating formate-dependent NaCl absorption in the proximal tubule
Authors: Wang Tong , Yang Chao-Ling , Abbiati Thecla , Shull Gary E. , Giebisch Gerhard , Aronson Peter S.
Published: December 2017

The absorption of NaCl in the proximal tubule is markedly stimulated by formate. This stimulation of NaCl transport is consistent with a cell model involving Cl−-formate exchange in parallel with pH-coupled formate recycling due to nonionic diffusion of formic acid or H+-formate cotransport. The formate recycling process requires H+ secretion. Although Na+-H+ exchanger isoform NHE3 accounts for the largest component of H+ secretion in the proximal tubule, 40–50% of the rates of HCO[Formula: see text] absorption or cellular H+ extrusion persist in NHE3 null mice. The purpose of the present investigation is to use NHE3 null mice to directly test the role of apical membrane NHE3 in mediating NaCl absorption stimulated by formate. We demonstrate that formate stimulates NaCl absorption in the mouse proximal tubule microperfused in vivo, but the component of NaCl absorption stimulated by formate is absent in NHE3 null mice. In contrast, stimulation of NaCl absorption by oxalate is preserved in NHE3 null mice, indicating that oxalate-stimulated NaCl absorption is independent of Na+-H+ exchange. The virtually complete dependence of formate-induced NaCl absorption on NHE3 activity raises the possibility that NHE3 and the formate transporters are functionally coupled in the brush border membrane.
Inducible nitric oxide synthase and glomerular hemodynamics in rats with liver cirrhosis
Authors: Porst Markus , Hartner Andrea , Krause Holger , Hilgers Karl F. , Veelken Roland
Published: December 2017

This study was designed to test the hypothesis that glomerular de novo expression of inducible nitric oxide synthase (iNOS) contributes to renal hemodynamic abnormalities in liver cirrhosis developed 3 wk after common bile duct ligature (CBDL). De novo expression of iNOS mRNA was detected by RT-PCR in RNA extracts from isolated CBDL rat glomeruli whereas no iNOS mRNA was found in control rat glomerular RNA. Immunohistochemical staining for iNOS was negative in control animals whereas, in CBDL rats, positive iNOS staining was detected in an apparently mesangial pattern in all glomeruli. Western blots of protein extracts from isolated glomeruli of CBDL rats, but not control animals, showed a prominent iNOS band of 130 kDa. Mean arterial pressure (MAP), renal plasma flow (RPF; p-aminohippurate clearance), and glomerular filtration rate (GFR; inulin clearance) were unaltered in CBDL rats, but the application of 4 mg/kgl- N(6)-(1-iminoethyl)lysine, a specific inhibitor of iNOS, reduced GFR and RPF significantly in CBDL rats, whereas control animals were not affected. Similar results were obtained with lipopolysaccharide (LPS)-pretreated animals, which were studied as a positive control for iNOS expression and as a model for recent iNOS induction. We conclude that de novo expression of iNOS occurs in glomeruli of rats with liver cirrhosis and that nitric oxide, generated by iNOS, contributes to the maintenance of glomerular filtration in the early state of this disease.
Immunohistochemical localization of H-K-ATPase α2c-subunit in rabbit kidney
Authors: Verlander Jill W. , Moudy Robin M. , Campbell W. Grady , Cain Brian D. , Wingo Charles S.
Published: December 2017

The rabbit kidney possesses mRNA for the H-K-ATPase α1-subunit (HKα1) and two splice variants of the H-K-ATPase α2-subunit (HKα2). The purpose of this study was to determine the specific distribution of one of these, the H-K-ATPase α2c-subunit isoform (HKα2c), in rabbit kidney by immunohistochemistry. Chicken polyclonal antibodies against a peptide based on the NH2terminus of HKα2cwere used to detect HKα2cimmunoreactivity in tissue sections. Immunohistochemical localization of HKα2crevealed intense apical immunoreactivity in a subpopulation of cells in the connecting segment, cortical collecting duct, and outer medullary collecting duct in both the outer and inner stripe. An additional population of cells exhibited a thin apical band of immunolabel. Immunohistochemical colocalization of HKα2cwith carbonic anhydrase II, the Cl−/HCO[Formula: see text] exchanger AE1, and HKα1indicated that both type A and type B intercalated cells possessed intense apical HKα2cimmunoreactivity, whereas principal cells and connecting segment cells had only a thin apical band of HKα2c. Labeled cells were evident through the middle third of the inner medullary collecting duct in the majority of animals. Immunolabel was also present in papillary surface epithelial cells, cells in the cortical thick ascending limb of Henle's loop (cTAL), and the macula densa. Thus in the rabbit kidney, apical HKα2cis present and may contribute to acid secretion or potassium uptake throughout the connecting segment and collecting duct in both type A and type B intercalated cells, principal cells, and connecting segment cells, as well as in cells in papillary surface epithelium, cTAL, and macula densa.
Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na+/H+exchange in OK cells
Authors: Weinman Edward J. , Steplock Deborah , Wade James B. , Shenolikar Shirish
Published: December 2017

Na+/H+exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na+/H+exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1–355), or a truncated mNHERF(1–325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) at 10−4M inhibited Na+/H+exchange activity in control and OK cells expressing wild-type mNHERF(1–355) by >60% but by <10% in cells expressing mNHERF(1–325). NHE3 coimmunoprecipitated with mNHERF(1–325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1–325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3- O-methyl-d-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.
Trafficking and cell surface stability of ENaC
Authors: Rotin Daniela , Kanelis Voula , Schild Laurent
Published: December 2017

The epithelial Na+channel (ENaC) plays a key role in the regulation of Na+and water absorption in several epithelia, including those of the distal nephron, distal colon, and lung. Accordingly, mutations in ENaC leading to reduced or increased channel activity cause human diseases such as pseudohypoaldosteronism type I or Liddle's syndrome, respectively. The gain of ENaC function in Liddle's syndrome is associated with increased activity and stability of the channel at the plasma membrane. Thus understanding the regulation of channel processing and trafficking to and stability at the cell surface is of fundamental importance. This review describes some of the recent advances in our understanding of ENaC trafficking, including the role of glycosylation, ENaC solubility in nonionic detergent, targeting signal(s) and hormones. It also describes the regulation of ENaC stability at the cell surface and the roles of the ubiquitin ligase Nedd4 (and ubiquitination) and clathrin-mediated endocytosis in that regulation.
Role of guanylyl cyclase and cytochrome P-450 on renal response to nitric oxide
Authors: López Bernardo , Moreno Carol , Salom Miguel García , Roman Richard J. , Fenoy Francisco J.
Published: December 2017

The present study evaluated whether inhibition of guanylyl cyclase (GC) with 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) and methylene blue (MB) or inhibition of the renal metabolism of arachidonic acid by cytochrome P-450 (CYP450) enzymes with 1-aminobenzotriazole (ABT) and N-hydroxy- N′-(4 butyl-2-methyl phenyl)formamidine (HET0016) alters the renal tubular and vascular effects of a nitric oxide (NO) donor in vivo. Intrarenal infusion of ODQ or MB at a dose of 170 nmol · kg−1 · min−1 lowered renal blood flow (RBF) by 30 and 15%, respectively; glomerular filtration rate (GFR) by 26 and 18%, respectively; and sodium and water excretion by ∼35%. In rats pretreated with nitro-l-arginine methyl ester (37 nmol · kg−1 · min−1) to block the endogenous production of NO, intrarenal infusion of the NO donor S-nitroso- N-acetylcysteine (S-NO-NAC; 50 nmol · kg−1 · min−1) increased RBF (18%), sodium (73%), and water excretion (61%). ODQ or MB administration blocked the effect of S-NO-NAC on RBF but not the diuretic and natriuretic response. Pretreatment of rats with ABT or HET0016 also abolished the renal vasodilatory response to the NO donor and reduced its diuretic and natriuretic effect. These results indicate that both activation of GC and inhibition of CYP450 enzymes contribute to the renal vascular actions of NO, whereas the natriuretic and diuretic actions of NO appear to be largely CYP450 dependent.
Vitamin D reduces renal NaPi-2 in PTH-infused rats: complexity of vitamin D action on renal Pihandling
Authors: Friedlaender M. M. , Wald H. , Dranitzki-Elhalel M. , Zajicek H. K. , Levi M. , Popovtzer M. M.
Published: December 2017

Acute administration of dihydroxycholecalciferol [1,25(OH)2D3] blunts phosphaturia and increases urinary cAMP excretion in parathyroid hormone (PTH)-infused parathyroidectomized (PTX) rats. Because chronic administration of 1,25(OH)2D3enhances the phosphaturic response to exogenous parathyroid hormone despite blunting of urinary cAMP excretion, we have examined the expression of the renal cortex type II Na-Picotransporter (NaPi-2) mRNA and protein in 1) chronic PTX Sabra rats, 2) PTX rats receiving a physiological dose of 1,25(OH)-2-D3, 3) PTX rats receiving 35 ng/h of PTH, and 4) rats receiving both PTH and 1,25(OH)2D3, for 7 days via osmotic minipumps. Our results confirm that there is increased phosphaturia in the PTH+1,25(OH)2D3-infused animals despite blunting of urinary cAMP excretion, a reduced filtered load of phosphate, and lack of a phosphaturic effect by 1,25(OH)2D3alone. Both PTH and 1,25(OH)2D3significantly reduced expression of renal cortex NaPi-2 mRNA and NaPi-2 protein, and the administration of PTH together with 1,25(OH)2D3had additive effects in further decreasing NaPi-2 mRNA and NaPi-2 protein levels. Expression of two other epithelial transporters, type 1 Na-sulfate and type 1 Na-glucose cotransporters, were not different between the groups, suggesting specificity of the effects of PTH and 1,25(OH)2D3on phosphate transport. The effect of chronic administration of 1,25(OH)2D3has not been noted previously, and the cellular mechanisms and signaling processes that mediate the decrease in NaPi-2 remain to be determined.
CFTR disruption impairs cAMP-dependent Cl−secretion in primary cultures of mouse cortical collecting ducts
Authors: Bens Marcelle , Van Huyen Jean-Paul Duong , Cluzeaud Françoise , Teulon Jacques , Vandewalle Alain
Published: December 2017

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-d-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(−/−)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+assessed by the short-circuit current ( Isc) method in both CFTR(+/+) and CFTR(−/−) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in Iscinduced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter Iscin BAm-treated CFTR(−/−) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of36Cl−in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal36Cl−flux in CFTR(−/−) CCDs but not the basal-to-apical36Cl−flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl−in mouse CCD.
Collagen IV promotes repair of renal cell physiological functions after toxicant injury
Authors: Nony Paul A. , Nowak Grazyna , Schnellmann Rick G.
Published: December 2017

Collagen IV is found in the renal proximal tubular cell (RPTC) basement membrane and is a mediator of renal development and function. Pharmacological concentrations ofl-ascorbic acid phosphate (AscP) promote the repair of physiological functions in RPTC sublethally injured by S-(1,2-dichlorovinyl)-l-cysteine (DCVC). We hypothesized that AscP promotes RPTC repair by stimulating collagen IV synthesis and/or deposition. RPTC exhibited increased synthesis but decreased deposition of collagen IV after DCVC exposure. In contrast, RPTC cultured in pharmacological concentrations of AscP maintained collagen IV deposition. The activity of prolyl hydroxylase was decreased in RPTC after DCVC injury, an effect that was partially attenuated in injured RPTC cultured in pharmacological concentrations of AscP. The addition of exogenous collagen IV to the culture media of DCVC-injured RPTC promoted the repair of mitochondrial function and Na+-K+-ATPase activity. However, neither collagen I, laminin, nor fibronectin promoted cell repair. These data demonstrate an association between AscP-stimulated deposition of collagen IV and exogenous collagen IV and repair of physiological functions, suggesting that collagen IV plays a specific role in RPTC repair after sublethal injury.
Ammonium interaction with the epithelial sodium channel
Authors: Nakhoul Nazih L. , Hering-Smith Kathleen S. , Abdulnour-Nakhoul Solange M. , Hamm L. Lee
Published: December 2017

The purpose of this study was to investigate the direct effect of NH3/NH[Formula: see text] on mouse epithelial Na+channels (mENaC) expressed in Xenopusoocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na+current, intracellular pH (pHi), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na+reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in the absence of 20 mM NH4Cl but only by 100 ± 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pHi(0.43 ± 0.07) similar to the NH4Cl-induced pHidecrease (0.47 ± 0.12). Removal of Na+in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pHi(in the absence of NH4Cl). Removal of external Na+resulted in an outward current of 3.7 ± 0.8 μA (at −60 mV). The magnitude of this change in current was only 2.7 ± 0.7 μA when Na+was removed in the presence of NH4Cl. In oocytes expressing mENaC, NH4Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na+were not different from zero. These results indicate that NH4Cl inhibits Na+transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH4Cl. Permeability and selectivity of ENaC to NH[Formula: see text] may play a role.
Immunocytochemical localization of Na-K-ATPase α- and γ-subunits in rat kidney
Authors: Wetzel Randall K. , Sweadner Kathleen J.
Published: December 2017

The γ-subunit of the Na-K-ATPase is a single-span membrane protein that alters the kinetic properties of the enzyme. It is expressed in the kidney, but our initial observations indicated that it is not present in all nephron segments (Arystarkhova E, Wetzel RK, Asinovski NK, and Sweadner KJ. J Biol Chem 274: 33183–33185, 1999). Here we used triple-label confocal immunofluorescence microscopy in rat kidney with antibodies to Na-K-ATPase α1- and γ-subunits and nephron segment-specific markers. Na-K-ATPase α1-subunit stain was low but unambiguous in proximal segments, moderate in macula densa, connecting tubules, and cortical collecting ducts, high in thick ascending limb and distal convoluted tubules, and nearly undetectable in glomeruli, descending and ascending thin limb, and medullary collecting ducts. The γ-subunit colocalized at staining levels similar to α1-subunit in basolateral membranes in all segments except cortical thick ascending limb and cortical collecting ducts, which had α1-subunit but no detectable γ-subunit stain. Selective γ-subunit expression may contribute to the variations in Na-K-ATPase properties in different renal segments.
SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells
Authors: Shukla Alok , Hager Henrik , Corydon Thomas Juhl , Bean Andrew J. , Dahl Ronald , Vajda Zsolt , Li Hui , Hoffmann Hans Jürgen , Nielsen Søren
Published: December 2017

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins ( N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK1cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.
Role of the extracellular cAMP-adenosine pathway in renal physiology
Authors: Jackson Edwin K. , Dubey Raghvendra K.
Published: December 2017

Adenosine exerts physiologically significant receptor-mediated effects on renal function. For example, adenosine participates in the regulation of preglomerular and postglomerular vascular resistances, glomerular filtration rate, renin release, epithelial transport, intrarenal inflammation, and growth of mesangial and vascular smooth muscle cells. It is important, therefore, to understand the mechanisms that generate extracellular adenosine within the kidney. In addition to three “classic” pathways of adenosine biosynthesis, contemporary studies are revealing a novel mechanism for renal adenosine production termed the “extracellular cAMP-adenosine pathway.” The extracellular cAMP-adenosine pathway is defined as the egress of cAMP from cells during activation of adenylyl cyclase, followed by the extracellular conversion of cAMP to adenosine by the serial actions of ecto-phosphodiesterase and ecto-5′-nucleotidase. This mechanism of extracellular adenosine production may provide hormonal control of adenosine levels in the cell-surface biophase in which adenosine receptors reside. Tight coupling of the site of adenosine production to the site of adenosine receptors would permit a low-capacity mechanism of adenosine biosynthesis to have a large impact on adenosine receptor activation. The purposes of this review are to summarize the physiological roles of adenosine in the kidney; to describe the classic pathways of renal adenosine biosynthesis; to review the evidence for the existence of the extracellular cAMP-adenosine pathway; and to describe possible physiological roles of the extracellular cAMP-adenosine pathway, with particular emphasis on the kidney.
Activation of PKC-βIin glomerular mesangial cells is associated with specific NF-κB subunit translocation
Authors: Kumar Anoop , Hawkins Karen S. , Hannan Meredith A. , Ganz Michael B.
Published: December 2017

Changes in expression and activity of protein kinase C (PKC) isoforms and early transcription factors may account for alterations in cell behavior seen in diabetes. We studied the expression of PKC-βIin rat glomerular mesangial cells (MCs) cultured in normal or high glucose and compared it with the temporal and spatial expression of dimeric transcription factor (NF-κB) p50 and p65. The results show that in unstimulated cells PKC-βIand NF-κB p50 are distributed in the cytosol and, on stimulation, their distribution is perinuclear and they are localized to the membrane. Serum-starved MCs cultured in high-glucose medium exhibit a predominantly cytosolic localization of PKC-βIand both p50 and p65 NF-κB. However, phorbol 12-myristate 13-acetate (PMA) stimulation of cells grown in the presence of high glucose resulted in membrane translocation of PKC-βIthat was associated with nuclear translocation of NF-κB p65, but not NF-κB p50. Moreover, the translocation to the nucleus for NF-κB p65 was significantly higher in MCs exposed to high glucose compared with those exposed to normal glucose. These observations indicate that the NF-κB p65, but not NF-κB p50, expression and translocation pattern mirrors that of PKC-βI, which may be one important pathway by which signaling is enhanced in the high-glucose state.
C/EBPβ contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK1-F+cells
Authors: Liu Xiangdong , Wall Quynh-Thu , Taylor Lynn , Curthoys Norman P.
Published: December 2017

Phospho enolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK−2300Luc reporter constructs in LLC-PK1-F+cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK1-F+cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPβ but not for C/EBPα or C/EBPδ. Treatment of cells with cAMP did not affect the expression of C/EBPβ, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK−2300Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPβ, but not of C/EBPα or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPβ functions as a dominant negative inhibitor. Thus the binding of C/EBPβ to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.
Genetic disruption of atrial natriuretic peptide receptor-A alters renin and angiotensin II levels
Authors: Shi Shang-Jin , Nguyen Huong T. , Sharma Guru Dutt , Navar L. Gabriel , Pandey Kailash N.
Published: December 2017

We have studied cardiovascular and renal phenotypes in Npr1 (genetic determinant of natriuretic peptide receptor-A; NPRA) gene-disrupted mutant mouse model. The baseline systolic arterial pressure (SAP) in 0-copy mutant (−/−) mice (143 ± 2 mmHg) was significantly higher than in 2-copy wild-type (+/+) animals (104 ± 2 mmHg); however, the SAP in 1-copy heterozygotes (+/−) was at an intermediate value (120 ± 4 mmHg). To determine whether Npr1 gene function affects the renin-angiotensin-aldosterone system (RAAS), we measured the components of RAAS in plasma, kidney, and adrenal gland of 0-copy, 1-copy, and 2-copy male mice. Newborn (2 days after the birth) 0-copy pups showed 2.5-fold higher intrarenal renin contents compared with 2-copy wild-type counterparts (0-copy 72 ± 12 vs. 2-copy 30 ± 7 μg ANG I · mg protein−1· h−1, respectively). The intrarenal ANG II level in 0-copy pups was also higher than in 2-copy controls (0-copy 33 ± 5 vs. 2-copy 20 ± 2 pg/mg protein, respectively). However, both young (3 wk) and adult (16 wk) 0-copy mutant mice showed a dramatic 50–80% reduction in plasma renin concentrations (PRCs) and in expression of renal renin message compared with 2-copy control animals. In contrast, the adrenal renin content and mRNA expression levels were 1.5- to 2-fold higher in 0-copy adult mice than in 2-copy animals. The results suggest that inhibition of renal and systemic RAAS is a compensatory response that prevents greater increases in elevated arterial pressures in adult NPRA null mutant mice. However, the greater renin and ANG II levels seen in 0-copy newborn pups provide evidence that the direct effect of NPRA activation on renin is an inhibitory response.
Renal salt wasting in mice lacking NHE3 Na+/H+exchanger but not in mice lacking NHE2
Authors: Ledoussal Clara , Lorenz John N. , Nieman Michelle L. , Soleimani Manoocher , Schultheis Patrick J. , Shull Gary E.
Published: December 2017

To study the role of Na+/H+exchanger isoform 2 (NHE2) and isoform 3 (NHE3) in sodium-fluid volume homeostasis and renal Na+conservation, mice with Nhe2( Nhe2−/−) and/or Nhe3( Nhe3−/−) null mutations were fed a Na+-restricted diet, and urinary Na+excretion, blood pressure, systemic acid-base and electrolyte status, and renal function were analyzed. Na+-restricted Nhe2−/−mice, on either a wild-type or Nhe3 heterozygous mutant ( Nhe3+/−) background, did not exhibit excess urinary Na+excretion. After 15 days of Na+restriction, blood pressure, fractional excretion of Na+, and the glomerular filtration rate (GFR) of Nhe2−/−Nhe3+/−mice were similar to those of Nhe2+/+and Nhe3+/−mice, and no metabolic disturbances were observed. Nhe3−/−mice maintained on a Na+-restricted diet for 3 days exhibited hyperkalemia, urinary salt wasting, acidosis, sharply reduced blood pressure and GFR, and evidence of hypovolemic shock. These results negate the hypothesis that NHE2 plays an important renal function in sodium-fluid volume homeostasis; however, they demonstrate that NHE3 is critical for systemic electrolyte, acid-base, and fluid volume homeostasis during dietary Na+restriction and that its absence leads to renal salt wasting.
Detection and localization of H+-K+-ATPase isoforms in human kidney
Authors: Kraut Jeffrey A. , Helander Kerstin G. , Helander Herbert F. , Iroezi Ngozi D. , Marcus Elizabeth A. , Sachs George
Published: December 2017

An H+-K+-ATPase contributes to hydrogen secretion and potassium reabsorption by the rat and rabbit collecting ducts. Transport of these ions appears to be accomplished by one or both of two isoforms of the H+-K+-ATPase, HKα1and HKα2,because both isoforms are found in the collecting ducts and transport of hydrogen and potassium is attenuated by exposure to inhibitors of these transport proteins. To evaluate whether an H+-K+-ATPase is present in the human kidney, immunohistochemical studies were performed using normal human renal tissue probed with antibodies directed against epitopes of three of the known isoforms of the H+-K+-ATPase, HKα1, HKα2, and HKα4, and the V-type H+-ATPase. Cortical and medullary tissue probed with antibodies against HKα1showed cytoplasmic staining of intercalated cells that was less intense than that observed in the parietal cells of normal rat stomach stained with the same antibody. Also, weak immunoreactivity was detected in principal cells of the human collecting ducts. Cortical and medullary tissue probed with antibodies directed against HKα4revealed weak, diffuse staining of intercalated cells of the collecting ducts and occasional light staining of principal cells. Cortical and medullary tissue probed with antibodies directed against the H+-ATPase revealed staining of intercalated cells of the collecting ducts and some cells of the proximal convoluted tubules. By contrast, no discernible staining was noted with the use of the antibody against HKα2. These data indicate that HKα1and HKα4are present in the collecting ducts of the human kidney. In this location, these isoforms might contribute to hydrogen and potassium transport by the kidney.