bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of NHE3, NKCC2, and NCC abundance in kidney during aldosterone escape phenomenon: role of NO
Authors: Turban Sharon , Wang Xiao-Yan , Knepper Mark A.
Published: March 2015

Escape from aldosterone-induced renal NaCl retention is an important homeostatic mechanism in pathophysiological states in which plasma aldosterone levels are inappropriately elevated, e.g., in primary aldosteronism. Our previous studies demonstrated that the escape process occurs largely as a result of a marked suppression of the abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule but have also demonstrated a paradoxical increase in the protein abundance of the apical Na/H exchanger of the proximal tubule (NHE3). In the present study, we confirmed the increase in NHE3 and also showed that a similar increase in NHE3 protein abundance occurs in escape from ANG II-mediated NaCl retention. To investigate the potential role of nitric oxide (NO) in the observed upregulation of NHE3, we repeated the aldosterone escape experiment with a superimposed infusion of a NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME). l-NAME infusion abolished the increase in NHE3 protein abundance. Furthermore, in a different experiment, NO synthase inhibition uncovered an associated decrease in the abundance of the Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb, not seen with simple aldosterone escape. However, NO synthase inhibition did not block the decrease in NCC abundance normally seen with aldosterone escape. Furthermore, l-NAME infusion in aldosterone-treated rats markedly decreased both NHE3 and NKCC2 protein abundance, without changes in the corresponding mRNA levels. We conclude that NHE3 and NKCC2 protein abundances in kidney are positively regulated by NO and that the increase in NHE3 abundance seen in the aldosterone escape phenomenon is NO dependent.
PTH and DA regulate Na-K ATPase through divergent pathways
Authors: Khundmiri Syed Jalal , Lederer Eleanor
Published: March 2015

Parathyroid hormone (PTH) and dopamine (DA) inhibit Na-K ATPase activity and sodium-phosphate cotransport in proximal tubular cells. We previously showed that PTH and DA inhibit phosphate transport in opossum kidney (OK) cells through different signaling pathways. Therefore, we hypothesized that PTH and DA also inhibit Na-K ATPase through divergent pathways. We measured PTH and DA inhibition of Na-K ATPase activity in the presence of inhibitors of signaling pathways. PTH and DA inhibited Na-K ATPase in a biphasic manner, the early inhibition through protein kinase C (PKC)- and phospholipase A2(PLA2)-dependent pathways and the late inhibition through protein kinase A- and PLA2-dependent pathways. Inhibition of extracellular signal-regulated kinase (ERK) activation blocked early and late inhibition of Na-K ATPase by PTH but not by DA. Pertussis toxin blocked early and late inhibition by DA but not by PTH. Treatment with DA, but not PTH, resulted in an early downregulation of basolateral membrane expression of the α-subunit, whereas total cellular expression remained constant for both agonists. We conclude that PTH and DA regulate Na-K ATPase by different mechanisms through activation of divergent pathways.
Bradykinin reduces growth factor-induced glomerular ERK1/2 phosphorylation
Authors: Cellier Eric , Mage Marilyne , Duchêne Johan , Pécher Christiane , Couture Réjean , Bascands Jean-Loup , Girolami Jean-Pierre
Published: March 2015

Several experimental data report both mitogenic and antimitogenic effects of bradykinin (BK). To conciliate these apparent opposite effects, we hypothesized that, depending on cell context activation, BK could reduce the mitogenic effect of growth factors. Therefore, in the present study we assessed the existence of possible negative cross talk between BK and potential pathogenic growth factors in freshly isolated rat glomeruli (IG). Next, we determined whether this cross talk could be pharmacologically recruited during angiotensin-converting enzyme (ACE) inhibition in the diabetic rat. In IG from normal rats, BK, via activation of the B2kinin receptor (B2R), causes a transient stimulation of ERK1/2 phosphorylation, whereas it inhibits ERK1/2 phosphorylation induced by IGF-1, PDGF-BB, VEGF, or basic FGF. The reduction of growth factor-induced ERK1/2 phosphorylation is abolished by an inhibitor of tyrosine phosphatase. In glomeruli from diabetic rats, hyperglycemia increased the phosphorylation level of ERK-1/2 as well as oxidative stress. The reversal of these events by ACE inhibition is mediated via B2R activation. These observations are consistent with a potential therapeutic role of BK and B2R during glomerulosclerosis.
Role of internalization in AT1Areceptor function in proximal tubule epithelium
Authors: Thekkumkara Thomas , Linas Stuart L.
Published: March 2015

Angiotensin II (ANG II), acting through angiotensin type I (AT1) receptors on apical and basolateral surfaces of proximal tubule epithelial cells, increases sodium reabsorption in proximal tubules. Apical and basolateral receptors internalize after exposure to ANG II, but the role of internalization in receptor signaling and transport is not well defined. To determine the role of receptor internalization in ANG II-mediated receptor signaling and sodium transport, we stably expressed full-length and truncated AT1Areceptors in opossum kidney cells. After stimulation with ANG II, wild-type receptors on apical and basolateral surfaces rapidly internalized, inhibited adenylate cyclase, and increased transcellular sodium transport. Truncation of the cytoplasmic tail of the AT1Areceptor (TL314) resulted in receptors that were expressed on apical and basolateral surfaces but did not internalize, inhibit adenylate cyclase, or increase sodium transport. Because the cytoplasmic tail contains putative G protein coupling sites, mutant receptors that leave G protein interaction sites intact were designed. Cells expressing the truncation (TK333) or deletion (Del 315–329) also failed to internalize. When ANG II was added to basolateral surfaces of TK333 or Del 315–329, adenylate cyclase activity was inhibited and sodium transport was increased. In contrast, apical addition of ANG II was not associated with decreases in adenylate cyclase or increases in sodium transport. In conclusion, internalization pathways are important for AT1Areceptor function in polarized proximal tubule epithelial cells. Apical AT1Areceptors internalize before they interact with G proteins and signal cAMP. In contrast, basolateral AT1Areceptors interact with G proteins and signal cAMP without internalizing.
Renal microvascular actions of angiotensin II fragments
Authors: van Rodijnen William F. , van Lambalgen Ton A. , van Wijhe Michiel H. , Tangelder Geert-Jan , ter Wee Piet M.
Published: March 2015

In the present study, we investigated renal microvascular responses to ANG-(1–7) and ANG IV. Diameter changes of small interlobular arteries, afferent arterioles, and efferent arterioles were assessed by using isolated perfused hydronephrotic rat kidneys. ANG-(1–7) and ANG IV concentration dependently decreased the diameters of all investigated renal microvessel, however, with a much lower potency than ANG II. The ANG II type 1 receptor blocker irbesartan completely reversed the responses to ANG-(1–7) and ANG IV, whereas the ANG II type 2 receptor blocker PD-123319 had no effect. Both ANG-(1–7) and ANG IV failed to alter renal microvascular constriction induced by ANG II. In addition, subnanomolar concentrations of ANG-(1–7) had no effect on the myogenic-induced tone of interlobular arteries and afferent arterioles. Thus our data indicate that at high concentrations, ANG-(1–7) and ANG IV are able to activate the ANG II type 1 receptor, thereby inducing renal microvascular constriction. The failure of ANG-(1–7) and ANG IV to reduce ANG II- and pressure-induced constrictions suggests that these fragments do not exert a vasodilator and/or ANG II antagonistic action in the kidney.
Endothelin-1 activates mesangial cell ERK1/2 via EGF-receptor transactivation and caveolin-1 interaction
Authors: Hua Hong , Munk Snezana , Whiteside Catharine I.
Published: March 2015

Endothelin-1 (ET-1) stimulates glomerular mesangial cell proliferation and extracellular matrix protein transcription through an ERK1/2-dependent pathway. In this study, we determined whether ET-1 activation of glomerular mesangial cell ERK1/2 is mediated through EGF receptor (EGF-R) transactivation and whether intact caveolae are required. We showed that ET-1 stimulated tyrosine phosphorylation of the EGF-R in primary cultured, growth-arrested rat mesangial cells. In response to ET-1, ERK1/2 phosphorylation was increased by 27 ± 1-fold and attenuated by AG-1478, a specific EGF-R inhibitor, to 9 ± 1-fold. Moreover, filipin III and β-cyclodextrin, two cholesterol-depleting drugs known to disrupt caveolae, significantly reduced ET-1-induced phosphorylation of ERK1/2. In addition, preincubation of mesangial cells with a myristoylated peptide that binds to the caveolin-1 scaffolding domain diminished ET-1 activation of ERK1/2. ET-1 caused interaction of caveolin-1 with phosphorylated ERK1/2 identified by coimmunoprecipitation. Activation of ERK1/2 and its interaction with caveolin-1 were reduced by AG-1478, β-cyclodextrin, or inhibition of PKC. Phosphorylated ERK1/2 localized at focal adhesion complexes along with phospho-caveolin-1, suggesting specific sites of compartmentalization of these signaling molecules. Hence, ET-1 activates mesangial cell ERK1/2 predominantly through a pathway involving EGF-R transactivation, leading to a mechanism involving attachment to caveolin-1, presumably in caveolae.
Role of macula densa cyclooxygenase-2 in renovascular hypertension
Authors: Hartner Andrea , Cordasic Nada , Goppelt-Struebe Margarete , Veelken Roland , Hilgers Karl F.
Published: March 2015

Upregulation of the inducible cyclooxygenase (COX-2) in the macula densa accompanies the activation of the juxtaglomerular apparatus in many high-renin conditions. The functional role of COX-2 in these disease states is poorly understood. We tested whether COX-2 is required to increase renin in renovascular hypertension. Rats with established two-kidney, one-clip (2K1C) hypertension were treated for 2 wk with two different inhibitors of COX-2, NS-398 and rofecoxib, respectively. Hypertension in 2K1C rats was not affected or slightly enhanced by COX-2 inhibition, as measured intra-arterially in conscious animals. The increase in plasma renin activity was also unchanged by both rofecoxib and NS-398. The number of glomeruli with a renin-positive juxtaglomerular apparatus was elevated in clipped kidneys and decreased in contralateral kidneys of 2K1C rats. This pattern was unaltered by COX-2 inhibition. To test the effects of COX-2 blockade on a primarily macula densa-mediated stimulus, we studied salt depletion for comparison. A low-salt diet induced a significant increase in plasma renin activity, which was partially inhibited by treatment with NS-398. We conclude that inhibition of COX-2 in established renovascular hypertension does not affect renin synthesis or release. Thus either COX-2 is not necessary for the macula densa mechanism or the macula densa is not important for maintaining high renin in renovascular hypertension.
Distribution and oligomeric association of splice forms of Na+-K+-ATPase regulatory γ-subunit in rat kidney
Authors: Arystarkhova Elena , Wetzel Randall K. , Sweadner Kathleen J.
Published: March 2015

Renal Na+-K+-ATPase is associated with the γ-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, γaand γb. Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the α-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na+-K+-ATPase conformation. In the cortex, the proportion of the γb-subunit was markedly lower, and the γa-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the γb-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na+-K+-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the γb-subunit. Because different mechanisms maintain and regulate Na+homeostasis in different nephron segments, the splice forms of the γ-subunit may have evolved to control the renal Na+pump through pump properties, gene expression, or both.
Dietary potassium restriction stimulates endocytosis of ROMK channel in rat cortical collecting duct
Authors: Chu Po-Yin , Quigley Raymond , Babich Victor , Huang Chou-Long
Published: March 2015

ROMK potassium channels are present in the cortical collecting ducts (CCDs) of the kidney and serve as the exit pathways for K+secretion in this nephron segment. Dietary K+restriction reduces the abundance of ROMK in the kidney. We have previously shown that ROMK undergoes endocytosis via clathrin-coated vesicles in Xenopus laevis oocytes and in cultured cells. Here, we examined the effect of dietary K+restriction on endocytosis of ROMK in CCDs using double-labeling immunofluorescent staining and confocal microscopic imaging in whole kidney sections as well as in individually isolated tubules. We found that ROMK abundance in kidney cortex and CCDs was reduced in rats fed a K+-restricted diet compared with rats fed the control K+diet. In the control animals, ROMK staining was preferentially localized to the apical membrane of CCDs. Compared with control tubules, ROMK staining in CCDs was markedly shifted toward intracellular locations in animals fed a K+-deficient diet for 48 h. Some of the intracellular distribution of ROMK colocalized with an early endosomal marker, early endosomal antigen-1 or with a late endosomal/lysosomal marker, lysosomal membrane glycoprotein-120. These results suggest that K+restriction reduces the abundance of ROMK in CCDs by increasing endocytosis and degradation of the channel protein. This decrease in the abundance of ROMK is likely important for maintaining K+homeostasis during K+deficiency.
Structure/function analysis of Na+-K+-ATPase central isoform-specific region: involvement in PKC regulation
Authors: Pierre Sandrine V. , Duran Marie-Josée , Carr Deborah L. , Pressley Thomas A.
Published: March 2015

Specific functions served by the various Na+-K+-ATPase α-isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH2terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH2terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat α1- and α2-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K+deocclusion pattern and their response to PKC activation. Comparisons with rat full-length α1- and α2-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K+deocclusion.
Delayed administration of hepatocyte growth factor reduces renal fibrosis in obstructive nephropathy
Authors: Yang Junwei , Liu Youhua
Published: March 2015

Hepatocyte growth factor (HGF) is a renotropic protein that elicits antifibrogenic activity by preventing the activation of matrix-producing myofibroblast cells in animal models of chronic renal diseases. However, whether a delayed administration of HGF can still attenuate renal fibrosis remains uncertain. In this study, we examined the therapeutic potential of exogenous HGF on an established renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Three days after UUO, the obstructed kidneys displayed interstitial fibrotic lesions with characteristic features of an established renal fibrosis, as manifested by myofibroblast activation, fibronectin overexpression, interstitial matrix deposition, and transforming growth factor-β1 upregulation. Beginning at this time point, administration of recombinant HGF into mice by intravenous injections for 11 days markedly suppressed the progression of renal interstitial fibrosis. HGF significantly suppressed renal α-smooth muscle actin expression, total kidney collagen contents, interstitial matrix components, such as fibronectin, and renal expression of transforming growth factor-β1 and its type I receptor. Compared with the starting point (3 days after UUO), HGF treatment largely blunted the progression of myofibroblast accumulation and collagen deposition but did not reverse it. Delayed administration of HGF also suppressed the myofibroblastic transdifferentiation from tubular epithelial cells in vitro, as demonstrated by a decline in α-smooth muscle actin and fibronectin expression. These results suggest that exogenous HGF exhibits potent therapeutic effects on retarding the progression of an established renal fibrosis.
A mathematical model of rat collecting duct I. Flow effects on transport and urinary acidification
Authors: Weinstein Alan M.
Published: March 2015

A mathematical model of the rat collecting duct (CD) has been developed by concatenating previously published models of cortical (Weinstein AM. Am J Physiol Renal Physiol 280: F1072–F1092, 2001); outer medullary (Weinstein AM. Am J Physiol Renal Physiol 279: F24–F45, 2000); and inner medullary segments (Weinstein AM. Am J Physiol Renal Physiol 274: F841–F855, 1998). Starting with end-distal tubular flow rate and composition, plus interstitial solute profiles, the model predicts urinary solute flows, including the buffer concentrations required to assess net acid excretion. In the model CD, the interstitial corticomedullary osmotic gradient provides the basis for the flow effect on the transport of several solutes. For substances that have an interstitial accumulation and that can have diffusive secretion (e.g., urea and NH[Formula: see text]), enhanced luminal flow increases excretion by decreasing luminal accumulation. For substances that are reabsorbed (e.g., K+ and HCO[Formula: see text]), and for which luminal accumulation can enhance reabsorption, increasing luminal flow again increases excretion by decreasing luminal solute concentration. In model calculations, flow-dependent increases in HCO[Formula: see text] and NH[Formula: see text] approximately balance, so net acid excretion is little changed by flow, albeit at a higher urinary pH. The model identifies delivery flow rate to the CD as a potent determinant of urinary pH, with high flows blunting maximal acidification. At even modestly high flows (9 nl · min−1 · tubule−1, with 6% of filtered Na+ entering the CD), the model cannot achieve a urinary pH <5.5 unless the delivered HCO[Formula: see text]concentration is extremely low (<2 mM). Nevertheless, simulation of Na2SO4 diuresis does yield both an increase in net acid excretion and a decrease in urinary HCO[Formula: see text](i.e., a decrease in pH) despite the increase in urinary flow. This model should provide a tool for examining hypotheses regarding transport defects underlying distal renal tubular acidosis.
A mathematical model of rat collecting duct II. Effect of buffer delivery on urinary acidification
Authors: Weinstein Alan M.
Published: March 2015

A mathematical model of the rat collecting duct (CD) is used to examine the effect of delivered load of bicarbonate and nonbicarbonate buffer on urinary acidification. Increasing the delivered load of HCO[Formula: see text] produces bicarbonaturia, and, with luminal carbonic anhydrase absent, induces a disequilibrium luminal pH and a postequilibration increase in urinary Pco 2. At baseline flows, this disequilibrium disappears when luminal carbonic anhydrase rate coefficients reach 1% of full catalysis. The magnitude of the equilibration Pco 2 depends on the product of urinary acid phosphate concentration and the disequilibrium pH. Thus, although increasing phosphate delivery to the CD decreases the disequilibrium pH, the increase in urinary phosphate concentration yields an overall increase in postequilibration Pco 2. In simulations of experimental HCO[Formula: see text] loading in the rat, model predictions of urinary Pco 2 exceed the measured Pco 2 of bladder urine. In part, the higher model predictions for urinary Pco 2 may reflect higher urinary flow rates and lower urinary phosphate concentrations in the experimental preparations. However, when simulation of CD function during HCO[Formula: see text] loading acknowledges the high ambient renal medullary Pco 2 (5), the predicted urinary Pco 2 of the model CD is yet that much greater. This discrepancy cannot be resolved within the model but requires additional experimental data, namely, concomitant determination of urinary buffer concentrations within the tubule fluid sampled for Pco 2 and pH. This model should provide a means for simulating formal testing of urinary acidification and thus for examining hypotheses regarding transport defects underlying distal renal tubular acidosis.
Kidney injury molecule-1 expression in murine polycystic kidney disease
Authors: Kuehn E. Wolfgang , Park Kwon Moo , Somlo Stefan , Bonventre Joseph V.
Published: March 2015

Kidney injury molecule-1 (Kim-1) is a type 1 membrane protein maximally upregulated in proliferating and dedifferentiated tubular cells after renal ischemia. Because epithelial dedifferentiation, proliferation, and local ischemia may play a role in the pathophysiology of autosomal dominant polycystic kidney disease, we investigated Kim-1 expression in a mouse model of this disease. In the Pkd2WS25/−mouse model for autosomal dominant polycystic kidney disease, cystic kidneys show markedly upregulated Kim-1 levels compared with noncystic control kidneys. Kim-1 is present in a subset of cysts of different sizes and segmental origins and in clusters of proximal tubules near cysts. Kim-1-expressing tubular cells show decreased complexity and quantity of basolateral staining for Na-K-ATPase. Other changes in polarity characteristic of ischemic injury are not present in Kim-1-expressing pericystic tubules. Polycystin-2 expression is preserved in Kim-1-expressing tubules. The interstitium surrounding Kim-1-expressing tubules shows high proliferative activity and staining for smooth muscle α-actin, characteristic of myofibroblasts. Although the functional role of the protein in cysts remains unknown, Kim-1 expression in tubules is strongly associated with partial dedifferentiation of epithelial cells and may play a role in the development of interstitial fibrosis.
Mechanical stretch regulates cell survival in human bladder smooth muscle cells in vitro
Authors: Galvin David J. , Watson R. William G. , Gillespie James I. , Brady Hugh , Fitzpatrick John M.
Published: March 2015

Our understanding of the pathophysiology of the overactive bladder is poor. It has been proposed that localized contractions result in the abnormal stretching of bladder smooth muscle. We hypothesize that stretch regulates the cellular processes that determine tissue size. The purpose of this study was to investigate the effect of stretch on apoptosis, proliferation, cell hypertrophy, and growth factor production in human bladder smooth muscle cells in vitro. Normal human detrusor muscle was obtained from patients undergoing radical cystectomy for invasive bladder cancer, and primary cultures were established. Cells were mechanically stretched on flexible plates at a range of pressures and times. Apoptosis was assessed by propidium iodide incorporation and flow cytometry. Radiolabeled thymidine and amino acid incorporation were used to assess proliferation and cell hypertrophy. ELISA and RT-PCR were used to assess growth factor production. Mechanical stretch inhibits apoptosis in a time- and dose-dependent manner and was associated with increases in the antiapoptotic proteins heat shock protein-70 and cIAP-1. Stretch also increases smooth muscle cell proliferation and hypertrophy, but hypertrophy is the more dominant response. These changes were associated with increases in IGF-1 and basic FGF and a decrease in transforming growth factor-β1. Mechanical stretch regulates apoptosis, proliferation, and cell hypertrophy in human bladder smooth muscle cells.
Molecular and functional characterization of a urea transporter from the kidney of the Atlantic stingray
Authors: Janech Michael G. , Fitzgibbon Wayne R. , Chen Ruihua , Nowak Mark W. , Miller Donald H. , Paul Richard V. , Ploth David W.
Published: March 2015

In general, marine elasmobranch fishes (sharks, skates, and rays) maintain body fluid osmolality above seawater, principally by retaining large amounts of urea. Maintenance of the high urea concentration is due in large part to efficient renal urea reabsorption. Regulation of renal urea reabsorption also appears to play a role in maintenance of fluid homeostasis of elasmobranchs that move between habitats of different salinities. We identified and cloned a novel 2.7-kb cDNA from the kidney of the euryhaline Atlantic stingray Dasyatis sabina (GenBank accession no. AF443781 ). This cDNA putatively encoded a 431-amino acid protein (strUT-1) that had a high degree of sequence identity (71%) to the shark kidney facilitated urea transporter (UT). However, the predicted COOH-terminal region of strUT-1 appears to contain an additional sequence that is unique among cloned renal UTs. Injection of strUT-1 cRNA into Xenopusoocytes induced a 33-fold increase in [14C]urea uptake that was inhibited by phloretin. Four mRNA bands were detected in kidney by Northern blot: a transcript at 2.8 kb corresponding to the expected size of strUT-1 mRNA and bands at 3.8, 4.5, and 5.5 kb. Identification of a facilitated UT in the kidney of the Atlantic stingray provides further support for the proposal that passive mechanisms contribute to urea reabsorption by elasmobranch kidney.
Hypotonic induction of SGK1 and Na+transport in A6 cells
Authors: Rozansky David J. , Wang Jian , Doan Ninh , Purdy Timothy , Faulk Tonya , Bhargava Aditi , Dawson Kevin , Pearce David
Published: March 2015

Serum and glucocorticoid-regulated kinase-1 (SGK1) is a serine-threonine kinase that is regulated at the transcriptional level by numerous regulatory inputs, including mineralocorticoids, glucocorticoids, follicle-stimulating hormone, and osmotic stress. In the distal nephron, SGK1 is induced by aldosterone and regulates epithelial Na+channel-mediated transepithelial Na+transport. In other tissues, including liver and shark rectal gland, SGK1 is regulated by hypertonic stress and is thought to modulate epithelial Na+channel- and Na+-K+-2Cl−cotransporter-mediated Na+transport. In this report, we examined the regulation of SGK1 mRNA and protein expression and Na+currents in response to osmotic stress in A6 cells, a cultured cell line derived from Xenopus laevis distal nephron. We found that in contrast to hepatocytes and rectal gland cells, hypotonic conditions stimulated SGK1 expression and Na+transport in A6 cells. Moreover, a correlation was found between SGK1 induction and the later phase of activation of Na+transport in response to hypotonic treatment. When A6 cells were pretreated with an inhibitor of phosphatidylinositol 3-kinase (PI3K), Na+transport was blunted and only inactive forms of SGK1 were expressed. Surprisingly, these results demonstrate that both hypertonic and hypotonic stimuli can induce SGK1 gene expression in a cell type-dependent fashion. Moreover, these data lend support to the view that SGK1 contributes to the defense of extracellular fluid volume and tonicity in amphibia by mediating a component of the hypotonic induction of distal nephron Na+transport.
Relevance of renal-specific oxidoreductase in tubulogenesis during mammalian nephron development
Authors: Kanwar Yashpal S. , Yang Qiwei , Tian Yufeng , Lin Sun , Wada Jun , Chugh Sumant , Srivastava Satish K.
Published: March 2015

Renal-specific oxidoreductase (RSOR), an enzyme relevant to diabetic nephropathy, is exclusively expressed in renal tubules. Studies were initiated to determine whether, like other tubule-specific proteins, it selectively modulates tubulogenesis. Northern blot analyses revealed a ∼1.5-kb transcript, and RSOR expression was detectable in mice embryonic kidneys at day 13, gradually increased by day 17, and extended into neo- and postnatal periods. RSOR mRNA and protein expression was confined to proximal tubules, commencing at gestational day 17 and increasing subsequently, but remained absent in glomeruli and medulla. Treatment with RSOR antisense oligodeoxynucleotide resulted in a dose-dependent dysmorphogenesis of metanephric explants harvested at gestational day 13. The explants were smaller and had expanded mesenchyme, and the population of tubules was markedly decreased. The glomeruli were unaffected, as assessed by mRNA expression of glomerular epithelial protein 1 and reactivity with wheat germ agglutinin. Antisense treatment led to a selective reduction of RSOR mRNA. Immunoprecipitation also indicated a selective translational blockade of RSOR. These findings suggest that RSOR is developmentally regulated, exhibits a distinct spatiotemporal distribution, and probably plays a role in tubulogenesis.
Angiotensin II stimulates cyclooxygenase-2 mRNA expression in renal tissue from rats with kidney failure
Authors: Hernández José , Astudillo Horacio , Escalante Bruno
Published: March 2015

We have shown increased cyclooxygenase-2 (COX-2) expression in rats with kidney failure. Increased angiotensin II concentration, hypertension, and renal mass reduction have been described during development of kidney failure. Thus we explored each of these mechanisms, because any one of them could be responsible for COX-2 induction. Kidney failure increased systolic blood pressure from 104 ± 5 to 138 ± 2 mmHg, urinary PGE2from 74 ± 17 to 185 ± 25 ng/24 h, and COX-2 expression from 0.06 ± 0.04 to 0.17 ± 0.03 arbitraty units (AU). Treatment of the rats with ramipril or losartan prevented the increase in blood pressure, urinary PGE2, and COX-2 expression in the rats with kidney failure. Infusion of angiotensin II increased blood pressure from 101 ± 6 to 132 ± 6 mmHg, urinary PGE2excretion from 62 ± 15 to 155 ± 17 ng/24 h, and COX-2 expression from 0.23 ± 0.01 to 1.6 ± 0.3 AU. When the angiotensin II-infused rats were treated with nitrendipine, blood pressure decreased from 132 ± 6 to 115 ± 2 mmHg, and urinary PGE2excretion decreased from 152 ± 18 to 97 ± 12 ng/24 h, whereas COX-2 expression was 1.6 ± 0.7 and 1.7 ± 0.5 AU for rats with and without nitrendipine. Blood pressure of the rats with renal pole resection was similar to that in sham rats (97 ± 7 and 91 ± 4 mmHg, respectively), whereas COX-2 expression was increased in rats with renal pole resection, from 0.06 ± 0.04 to 0.12 ± 0.03 AU. We suggest that in kidney failure, the increase in angiotensin II concentration regulates COX-2 expression, thereby increasing prostaglandin synthesis, which contributes to the development of kidney failure.
Adenosine attenuates oxidant injury in human proximal tubular cells via A1and A2aadenosine receptors
Authors: Lee H. T. , Emala Charles W.
Published: March 2015

We have recently demonstrated protection against renal ischemic-reperfusion injury in vivo by A1- and A2a-adenosine receptor (AR) modulations. To further elucidate the signaling cascades of AR-induced cytoprotection against reperfusion/oxidant-mediated injury, immortalized human proximal tubule (HK-2) cells were treated with H2O2. H2O2caused dose- and time-dependent HK-2 cell death that was measured by lactate dehydrogenase release and trypan blue dye uptake. Adenosine protected against H2O2-induced HK-2 cell death by means of A1- and A2a-AR activation. A1-AR-mediated protection involves pertussis toxin-sensitive G proteins and protein kinase C, whereas A2a-AR-mediated protection involves protein kinase A activation by means of cAMP and activation of the cAMP response element binding protein. Moreover, protein kinase A activators (forskolin and Sp-isomer cAMP) also protected HK-2 cells against H2O2injury. De novo gene transcription and protein synthesis are required for both A1- and A2a-AR-mediated cytoprotection as actinomycin D and cycloheximide, respectively, blocked cytoprotection. Chronic treatments with a nonselective AR agonist abolished the protection by adenosine. Moreover, chronic treatments with a nonselective AR antagonist increased the endogenous tolerance of HK-2 cells against H2O2. We concluded that A1- and A2a-AR activation protects HK-2 cells against H2O2-induced injury by means of distinct signaling pathways that require new gene transcription and new protein synthesis.
Murine models of polycystic kidney disease: molecular and therapeutic insights
Authors: Guay-Woodford Lisa M.
Published: March 2015

Numerous murine (mouse and rat) models of polycystic kidney disease (PKD) have been described in which the mutant phenotype results from a spontaneous mutation or engineering via chemical mutagenesis, transgenic technologies, or gene-specific targeting in mouse orthologs of human PKD genes. These murine phenotypes closely resemble human PKD, with common abnormalities observed in tubular epithelia, the interstitial compartment, and the extracellular matrix of cystic kidneys. In both human and murine PKD, genetic background appears to modulate the renal cystic phenotype. In murine models, these putative modifying effects have been dissected into discrete factors called quantitative trait loci and genetically mapped. Several lines of experimental evidence support the hypothesis that PKD genes and their modifiers may define pathways involved in cystogenesis and PKD progression. Among the various pathway abnormalities described in murine PKD, recent provocative data indicate that structural and/or functional defects in the primary apical cilia of tubular epithelia may play a key role in PKD pathogenesis. This review describes the most widely studied murine models; highlights the data regarding specific gene defects and genetic modifiers; summarizes the data from these models that have advanced our understanding of PKD pathogenesis; and examines the effect of various therapeutic interventions in murine PKD.
Identification of developmentally regulated mesodermal-specific transcript in mouse embryonic metanephros
Authors: Kanwar Yashpal S. , Kumar Anil , Ota Kosuke , Lin Sun , Wada Jun , Chugh Sumant , Wallner Elisabeth I.
Published: March 2015

Mesodermal-specific cDNA or transcript (MEST) was identified by suppression subtractive hybridization-PCR of cDNA isolated from embryonic day 13vs. newborn mice kidneys. At day 13 of mouse gestation, a high expression of MEST, with a single ∼2.7-kb transcript that was exclusively localized to the metanephric mesenchyme was observed. The MEST mRNA expression gradually decreased during the later stages and then abruptly decreased in the newborn kidneys and subsequent postnatal life, after which a very mild expression persisted in the glomerular mesangium. Regression in mRNA expression during embryonic renal development appears to be related to methylation of the MEST gene. Treatment of metanephroi, harvested at day 13 of gestation with MEST-specific antisense oligodeoxynucleotide resulted in a dose-dependent decrease in the size of the explants and the nephron population. This was associated with a selective decrease in MEST mRNA expression and accelerated apoptosis of the mesenchyme. These findings suggest that MEST, a gene with a putative mesenchymal cell-derived protein, conceivably plays a role in mammalian metanephric development.
Sp1 and Sp3 transcription factors synergistically regulate HGF receptor gene expression in kidney
Authors: Zhang Xianghong , Li Yingjian , Dai Chunsun , Yang Junwei , Mundel Peter , Liu Youhua
Published: March 2015

We investigated the expression pattern and underlying mechanism that controls hepatocyte growth factor (HGF) receptor (c-met) expression in normal kidney and a variety of kidney cells. Immunohistochemical staining showed widespread expression of c-met in mouse kidney, a pattern closely correlated with renal expression of Sp1 and Sp3 transcription factors. In vitro, all types of kidney cells tested expressed different levels of c-met, which was tightly proportional to the cellular abundances of Sp1 and Sp3. Both Sp1 and Sp3 bound to the multiple GC boxes in the promoter region of the c-met gene. Coimmunoprecipitation suggested a physical interaction between Sp1 and Sp3. Functionally, Sp1 markedly stimulated c-met promoter activity. Although Sp3 only weakly activated the c-met promoter, its combination with Sp1 synergistically stimulated c-met transcription. Conversely, deprivation of Sp proteins by transfection of decoy Sp1 oligonucleotide or blockade of Sp1 binding with mithramycin A inhibited c-met expression. The c-met receptor in all types of kidney cells was functional and induced protein kinase B/Akt phosphorylation in a distinctly dynamic pattern after HGF stimulation. These results indicate that members of the Sp family of transcription factors play an important role in regulating constitutive expression of the c-met gene in all types of renal cells. Our findings suggest that HGF may have a broader spectrum of target cells and possess wider implications in kidney structure and function than originally thought.
Extracellular cAMP inhibits proximal reabsorption: are plasma membrane cAMP receptors involved?
Authors: Bankir Lise , Ahloulay Mina , Devreotes Peter N. , Parent Carole A.
Published: March 2015

Glucagon binding to hepatocytes has been known for a long time to not only stimulate intracellular cAMP accumulation but also, intriguingly, induce a significant release of liver-borne cAMP in the blood. Recent experiments have shown that the well-documented but ill-understood natriuretic and phosphaturic actions of glucagon are actually mediated by this extracellular cAMP, which inhibits the reabsorption of sodium and phosphate in the renal proximal tubule. The existence of this “pancreato-hepatorenal cascade” indicates that proximal tubular reabsorption is permanently influenced by extracellular cAMP, the concentration of which is most probably largely dependent on the insulin-to-glucagon ratio. The possibility that renal cAMP receptors may be involved in this process is supported by the fact that cAMP has been shown to bind to brush-border membrane vesicles. In other cell types (i.e., adipocytes, erythrocytes, glial cells, cardiomyocytes), cAMP eggress and/or cAMP binding have also been shown to occur, suggesting additional paracrine effects of this nucleotide. Although not yet identified in mammals, cAMP receptors (cARs) are already well characterized in lower eukaryotes. The amoeba Dictyostelium discoideum expresses four different cARs during its development into a multicellular organism. cARs belong to the superfamily of seven transmembrane domain G protein-coupled receptors and exhibit a modest homology with the secretin receptor family (which includes PTH receptors). However, the existence of specific cAMP receptors in mammals remains to be demonstrated. Disturbances in the pancreato-hepatorenal cascade provide an adequate pathophysiological understanding of several unexplained observations, including the association of hyperinsulinemia and hypertension, the hepatorenal syndrome, and the hyperfiltration of diabetes mellitus. The observations reviewed in this paper show that cAMP should no longer be regarded only as an intracellular second messenger but also as a first messenger responsible for coordinated hepatorenal functions, and possibly for paracrine regulations in several other tissues.
Alternate promoters and variable splicing lead to hNedd4–2 isoforms with a C2 domain and varying number of WW domains
Authors: Itani Omar A. , Campbell Jason R. , Herrero Juan , Snyder Peter M. , Thomas Christie P.
Published: March 2015

Mutations that disrupt a PY motif in epithelial Na+channel (ENaC) subunits increase surface expression of Na+channels in the collecting duct, resulting in greater Na+reabsorption. Recently, Nedd4 and Nedd4–2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. To further understand the role of human Nedd4–2 (hNedd4–2), we cloned its cDNAs and determined its genomic organization using a bioinformatic approach. The gene is present as a single copy, spans at least 400 kb, and contains >40 exons. Multiple 5′-exons were identified by 5′-rapid amplification of cDNA ends, and tissue-specific expression of these transcripts was noted by RT-PCR and RNase protection assay. Alternate polyadenylation signal sequences led to varying lengths of the 3′-untranslated region. Alternate splicing events within internal exons were also noted. Open reading frame analysis indicates that hNedd4–2 encode multiple protein variants with and without a C2 domain, and with a variable number of WW domains. Coexpression, in Fischer rat thyroid epithelia, of ENaC and Nedd4–2 cDNAs leads to a significant reduction in amiloride-sensitive currents, confirming a role in Na+transport regulation. In vitro binding studies demonstrated that individual PY motifs of α-, β-, and γ-ENaC have strong affinity for WW domains 3 and 4 but not 1 and 2. These studies indicate that alternate transcripts of Nedd4–2 may interact with ENaC differently. Understanding the function of variant proteins will increase our knowledge of the role of hNedd4–2 in the regulation of ENaC and define protein domains important for Nedd4–2 function.
Epithelial sodium channel activity in detergent-resistant membrane microdomains
Authors: Shlyonsky Vadim G. , Mies Frédérique , Sariban-Sohraby Sarah
Published: March 2015

The activity of epithelial Na+selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na+channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergent-soluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na+channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, high- and low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na+current, single-Na+channel activity, or the expression of Na+channel protein. This is the first reconstitution study of native epithelial Na+channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane.
Production of superoxide through NADH oxidase in thick ascending limb of Henle's loop in rat kidney
Authors: Li Ningjun , Yi Fu-Xian , Spurrier Jamie L. , Bobrowitz Carol A. , Zou Ai-Ping
Published: March 2015

We recently reported that NADH oxidase is one of the major enzymes responsible for superoxide (O[Formula: see text]·) production in the rat kidney. However, the functional significance of NADH oxidase-mediated O[Formula: see text]· production and the mechanisms regulating this enzyme activity are poorly understood. Using fluorescence microscopic imaging analysis, the present study demonstrated that thick ascending limbs of Henle's loop (TALHs) exhibited red fluorescence when incubated with dihydroethidium (DHE), suggesting that O[Formula: see text]· is produced in this tubular segment. Compared with other nephron segments, TALHs from both renal cortex and medulla showed the highest fluorescence intensity. By incubating cortical TALHs (cTALHs) with the substrates of NADH oxidase, xanthine oxidase, nitric oxide synthase, arachidonic acid-metabolizing enzymes, and intramitochondrial oxidases, NADH oxidase was found to be one of the most important enzymes for O[Formula: see text]· production in this tubular segment. The NADH oxidase inhibitor diphenyleneiodonium (DPI; 100 μM) completely blocked NADH-induced O[Formula: see text]· production in cTALHs. Exposure of cTALHs to low Po2(5–10 Torr) significantly increased O[Formula: see text]· production regardless of the absence or presence of NADH. Furthermore, angiotensin II (100 nM) increased NADH oxidase activity by 32%, which was completely blocked by DPI. These results suggest that NADH oxidase is a major enzyme responsible for O[Formula: see text]· production in the TALHs and that the production of O[Formula: see text]· via NADH oxidase may be regulated by renal tissue oxygenation and circulating hormones.
Dynamic alterations of glomerular charge density in fixed rat kidneys suggest involvement of endothelial cell coat
Authors: Ciarimboli Giuliano , Hjalmarsson Clara , Bökenkamp Arend , Schurek Hans-Joachim , Haraldsson Börje
Published: March 2015

In a previous paper, we found that low ionic strength (I) reversibly reduced the glomerular charge density, suggesting increased volume of the charge-selective barrier. Because glutaraldehyde makes most structures rigid, we considered the isolated, perfusion-fixed rat kidney to be an ideal model for further analysis. The fixed kidneys were perfused with albumin solutions containing FITC-Ficoll at two different Is (I = 151 and 34 mM). At normal I, the fractional clearance (θ) for albumin was 0.0049 (SE –0.0017, +0.0027, n = 6), whereas θ for neutral Ficoll35.5Åof similar size was significantly higher 0.104 (SE 0.010, n = 5, P < 0.001). At low I, θ for albumin was 0.0030 (SE –0.0011, +0.0018, n = 6, not significant from θalbuminat normal I) and θ for Ficoll35.5Åwas identical to that at normal I, 0.104 (SE 0.015, n = 6, P < 0.01 compared with θalbuminat low I). According to a heterogeneous charged fiber model, low I reduced the fiber density from 0.056 to 0.0315, suggesting a 78% gel volume expansion. We conclude that 1) there is a significant glomerular charge barrier. 2) Solutions with low I increase the volume of the charge barrier even in kidneys fixed with glutaraldehyde. Our findings suggest that polysaccharide-rich structures, such as the endothelial cell coat, are key components in the glomerular barrier.
CFTR null mutation altered cAMP-sensitive and swelling-activated Cl−currents in primary cultures of mouse nephron
Authors: Barrière Hervé , Belfodil Radia , Rubera Isabelle , Tauc Michel , Poujeol Chantal , Bidet Michel , Poujeol Philippe
Published: March 2015

The role of cystic fibrosis transmembrane conductance regulator (CFTR) in the control of Cl−currents was studied in mouse kidney. Whole cell clamp was used to analyze Cl−currents in primary cultures of proximal and distal convoluted and cortical collecting tubules from wild-type (WT) and cftr knockout (KO) mice. In WT mice, forskolin activated a linear Cl−current only in distal convoluted and cortical collecting tubule cells. This current was not recorded in KO mice. In both mice, Ca2+-dependent Cl−currents were recorded in all segments. In WT mice, volume-sensitive Cl−currents were implicated in regulatory volume decrease during hypotonicity. In KO mice, regulatory volume decrease and swelling-activated Cl−current were impaired but were restored by adenosine perfusion. Extracellular ATP also restored swelling-activated Cl−currents. The effect of ATP or adenosine was blocked by 8-cyclopentyl-1,3-diproxylxanthine. The ecto-ATPase inhibitor ARL-67156 inhibited the effect of hypotonicity and ATP. Finally, in KO mice, volume-sensitive Cl−currents are potentially functional, but the absence of CFTR precludes their activation by extracellular nucleosides. This observation strengthens the hypothesis that CFTR is a modulator of ATP release in epithelia.