bluuebunny/crossref_metadata_2025 huggingface dataset:
Urinary tract infection in iNOS-deficient mice with focus on bacterial sensitivity to nitric oxide
Authors: Poljakovic Mirjana , Persson Katarina
Published: March 2015

Inducible nitric oxide synthase (iNOS)-deficient mice were used to examine the role of iNOS in Escherichia coli-induced urinary tract infection (UTI). The toxicity of nitric oxide (NO)/peroxynitrite to bacteria and host was also investigated. The nitrite levels in urine of iNOS+/+but not iNOS−/−mice increased after infection. No differences in bacterial clearance or persistence were noted between the genotypes. In vitro, the uropathogenic E. coli 1177 was sensitive to 3-morpholinosydnonimine, whereas the avirulent E. coli HB101 was sensitive to both NO and 3-morpholinosydnonimine. E. coli HB101 was statistically ( P < 0.05) more sensitive to peroxynitrite than E. coli 1177. Nitrotyrosine immunoreactivity was observed in infected bladders of both genotypes and in infected kidneys of iNOS+/+mice. Myeloperoxidase, neuronal (n)NOS, and endothelial (e)NOS immunoreactivity was observed in inflammatory cells of both genotypes. Our results indicate that iNOS−/−and iNOS+/+mice are equally susceptible to E. coli-induced UTI and that the toxicity of NO to E. colidepends on bacterial virulence. Furthermore, myeloperoxidase and nNOS/eNOS may contribute to nitrotyrosine formation in the absence of iNOS.
Interactions of MAP17 with the NaPi-IIa/PDZK1 protein complex in renal proximal tubular cells
Authors: Pribanic Sandra , Gisler Serge Mike , Bacic Desa , Madjdpour Caveh , Hernando Nati , Sorribas Victor , Gantenbein Andrea , Biber Jürg , Murer Heini
Published: March 2015

An essential role in phosphate homeostasis is played by Na/Pi cotransporter IIa that is localized in the brush borders of renal proximal tubular cells. Recent studies identified several PDZ proteins interacting with the COOH-terminal tail of NaPi-IIa, such as PDZK1 and NHERF-1. Here, by using yeast two-hybrid screen of mouse kidney cDNA library, we attempted to find proteins interacting with the NH2-terminal part of NaPi-IIa. We identified MAP17, a 17-kDa membrane protein that has been described to be associated with various human carcinomas, but it is also expressed in normal kidneys. Results obtained by various in vitro analyses suggested that MAP17 interacts with the fourth domain of PDZK1 but not with other PDZ proteins localized in proximal tubular brush borders. As revealed by immunofluorescence, MAP17 was abundant in S1 but almost absent in S3 segments. No alterations of the apical abundance of MAP17 were observed after maneuvers undertaken to change the content of NaPi-IIa (parathyroid hormone treatment, different phosphate diets). In agreement, no change in the amount of MAP17 mRNA was observed. Results obtained from transfection studies using opossum kidney cells indicated that the apical localization of MAP17 is independent of PDZK1 but that MAP17 is required for apical localization of PDZK1. In summary, we conclude that MAP17 1) interacts with PDZK1 only, 2) associates with the NH2 terminus of NaPi-IIa within the PDZK1/NaPi-IIa/MAP17 complex, and 3) acts as an apical anchoring site for PDZK1.
Expression of urea transporters in potassium-depleted mouse kidney
Authors: Jung Ju-Young , Madsen Kirsten M. , Han Ki-Hwan , Yang Chul-Woo , Knepper Mark A. , Sands Jeff M. , Kim Jin
Published: March 2015

Urea transport in the kidney is mediated by a family of transporter proteins that include the renal urea transporter (UT-A) and the erythrocyte urea transporter (UT-B). The purpose of this study was to determine the location of the urea transporter isoforms in the mouse kidney and to examine the effects of prolonged potassium depletion on the expression and distribution of these transporters by ultrastructural immunocytochemistry. C57BL6 mice were fed a low-potassium diet for 2 wk, and control animals received normal chow. After 2 wk on a low-potassium diet, urinary volume increased and urinary osmolality decreased (833 ± 30 vs. 1,919 ± 174 mosmol/kgH2O), as previously demonstrated. Kidneys were processed for immunocytochemistry with antibodies against UT-A1 (L446), UT-A1 and UT-A2 (L194), UT-A3 (Q2), and UT-B. In normal mice, UT-A1 and UT-A3 were expressed mainly in the cytoplasm of the terminal inner medullary collecting duct (IMCD). UT-A2 immunoreactivity was observed mainly on the basolateral membrane of the type 1 epithelium of the descending thin limb (DTL) of short-looped nephrons. The intensity of UT-A1 and UT-A3 immunoreactivity in the IMCD was markedly reduced in potassium-depleted mice. In contrast, there was a significant increase in UT-A2 immunoreactivity in the DTL. The intensity of UT-B immunoreactivity in the descending vasa recta (DVR) was reduced in potassium-depleted animals compared with controls. In control animals, UT-B immunoreactivity was predominantly observed in the plasma membrane, whereas in potassium-depleted mice, it was mainly observed in cytoplasmic granules in endothelial cells of the DVR. In summary, potassium depletion is associated with reduced expression of UT-A1, UT-A3, and UT-B but increased expression of UT-A2. We conclude that reduced expression of urea transporters may play a role in the impaired urine-concentrating ability associated with potassium deprivation.
Reversal of charge selectivity in cation or anion-selective epithelial lines by expression of different claudins
Authors: Van Itallie Christina M. , Fanning Alan S. , Anderson James M.
Published: March 2015

Tight junctions (TJ) regulate paracellular ionic charge selectivity and conductance across epithelial tissues and cell lines. These properties vary among epithelia, and recent evidence implicates the claudins, a family of TJ transmembrane proteins, as important determinants of both characteristics. To test the hypothesis that each claudin contributes a characteristic charge discrimination to the TJ, we expressed claudins-2, -4, -11, and -15 in both cation-selective Madin-Darby canine kidney (MDCK) II cells and in anion-selective LLC-PK1cells and examined changes in transepithelial electrical resistance (TER) and paracellular charge selectivity. Regulated expression and localization were verified by immunoblot analysis and immunofluorescence microscopy, respectively. Expression of claudin-4 increased TER in both cell lines, whereas effects of the others on TER were variable. Claudin-4 and -11 decreased paracellular permeability for Na+in MDCK II cells, whereas neither claudin-2 nor -15 had an effect. Conversely, in LLC-PK1cells, claudin-2 and -15 increased the permeability for Na+, whereas claudin-4 and -11 were without effect. We conclude that the contribution of each claudin is most easily detectable when it reverses the direction of monolayer charge selectivity. These results are consistent with a model in which exogenous claudins add new charge-selective pores, leading to a physiological phenotype that combines endogenous and exogenous contributions. Additionally, it is possible to rationalize the direction of charge selectivity conferred by the individual claudins on the basis of electrostatic effects of the charged amino acids in their first extracellular loops.
Platelet-activating factor and solute transport processes in the kidney
Authors: Handa Rajash K. , Strandhoy Jack W. , Giammattei Carlos E. , Handa Shelly E.
Published: March 2015

We examined the hemodynamic and tubular transport mechanisms by which platelet-activating factor (PAF) regulates salt and water excretion. In anesthetized, renally denervated male Wistar rats, with raised systemic blood pressure and renal arterial blood pressure maintained at normal levels, intrarenal PAF infusion at 2.5 ng · min−1· kg−1resulted in a small fall in systemic blood pressure (no change in renal arterial blood pressure) and an increase in renal blood flow and urinary water, sodium, and potassium excretion rates. The PAF-induced changes in cardiovascular and renal hemodynamic function were abolished and renal excretory function greatly attenuated by treating rats with a nitric oxide synthase inhibitor. To determine whether a tubular site of action was involved in the natriuretic effect of PAF, cortical proximal tubules were enzymatically dissociated from male Wistar rat kidneys, and oxygen consumption rates (Qo2) were used as an integrated index of transcellular sodium transport. PAF at 1 nM maximally inhibited Qo2in both untreated and nystatin-stimulated (sodium entry into renal cell is not rate limiting) proximal tubules by ∼20%. Blockade of PAF receptors or Na+-K+-ATPase pump activity with BN-52021 or ouabain, respectively, abolished the effect of PAF on nystatin-stimulated proximal tubule Qo2. Inhibition of nitric oxide synthase or guanylate cyclase systems did not alter PAF-mediated inhibition of nystatin-stimulated proximal tubule Qo2, whereas phospholipase A2or cytochrome- P-450 monooxygenase inhibition resulted in a 40–60% reduction. These findings suggest that stimulation of PAF receptors on the proximal tubule decreases transcellular sodium transport by activating phospholipase A2and the cytochrome- P-450 monooxygenase pathways that lead to the inhibition of an ouabain-sensitive component of the basolateral Na+-K+-ATPase pump. Thus PAF can activate both an arachidonate pathway-mediated suppression of proximal tubule sodium transport and a nitric oxide pathway-mediated dilatory action on renal hemodynamics that likely contributes to the natriuresis and diuresis observed in vivo.
An update on renal peptide transporters
Authors: Daniel Hannelore , Rubio-Aliaga Isabel
Published: March 2015

The brush-border membrane of renal epithelial cells contains PEPT1 and PEPT2 proteins that are rheogenic carriers for short-chain peptides. The carrier proteins display a distinct surface expression pattern along the proximal tubule, suggesting that initially di- and tripeptides, either filtered or released by surface-bound hydrolases from larger oligopeptides, are taken up by the low-affinity but high-capacity PEPT1 transporter and then by PEPT2, which possesses a higher affinity but lower transport capacity. Both carriers transport essentially all possible di- and tripeptides and numerous structurally related drugs. A unique feature of the mammalian peptide transporters is the capability of proton-dependent electrogenic cotransport of all substrates, regardless of their charge, that is achieved by variable coupling in proton movement along with the substrate down the transmembrane potential difference. This review focuses on the postcloning research efforts to understand the molecular physiology of peptide transport processes in renal tubules and summarizes available data on the underlying genes, protein structures, and transporter function as derived from studies in heterologous expression systems.
Role of tyrosine kinase and p44/42 MAPK in D2-like receptor-mediated stimulation of Na+, K+-ATPase in kidney
Authors: Narkar Vihang , Hussain Tahir , Lokhandwala Mustafa
Published: March 2015

Our laboratory has shown that dopamine D2-like receptor activation causes stimulation of Na+, K+-ATPase (NKA) activity in the proximal tubules of the rat kidney. The present study was designed to investigate the cellular signaling mechanisms mediating this response to D2-like receptor activation. We measured the stimulation of NKA activity by bromocriptine (D2-like receptor agonist) in the absence and presence of PD-98059 [p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor] and genistein (tyrosine kinase inhibitor) in renal proximal tubules. Both agents inhibited bromocriptine-mediated stimulation of NKA, suggesting the involvement of p44/42 MAPK and tyrosine kinase in this response. Additionally, we found that bromocriptine increased the phosphorylation of p44/42 MAPK in the proximal tubules, which was blocked by PD-98059 and genistein. These results show that D2-like receptor activation causes stimulation of NKA activity by means of a tyrosine kinase-p44/42 MAPK pathway in the proximal tubules of the kidney.
Role of oxidative stress and heme oxygenase activity in morphine-induced glomerular epithelial cell growth
Authors: Patel Jaimita , Manjappa Nagarathna , Bhat Rajani , Mehrotra Pavni , Bhaskaran Madhu , Singhal Pravin C.
Published: March 2015

Opiate addiction has been reported to contribute to the progression of renal injury. In addition, opiate addiction is a major risk factor for the development of human immunodeficiency virus-associated nephropathy. In the present study, we evaluated the effects of morphine, an active metabolite of heroin, on glomerular epithelial cell (GEC) growth and the involved molecular mechanism. At lower concentrations, morphine promoted GEC proliferation; however, at higher concentrations, morphine triggered apoptosis. Antioxidants inhibited morphine-induced proliferation as well as apoptosis. Similarly, free radical scavengers prevented morphine-induced GEC proliferation and apoptosis. Because proliferative and proapoptotic effects of morphine were inhibited by free radical scavengers as well as antioxidants, it appears that these effects of morphine are mediated through oxidative stress. Hemin, an inducer of heme oxygenase (HO) activity, inhibited GEC proliferation and promoted GEC apoptosis under basal and morphine-stimulated conditions. On the other hand, zinc protoporphyrin, an inhibitor of HO activity, promoted GEC proliferation and inhibited GEC apoptosis under basal as well as morphine-stimulated conditions. These findings suggest that HO activity is directly related to GEC apoptosis and inversely related to GEC proliferation. Morphine, de novo, had bimodal effects on HO activity: lower concentrations increased and higher concentrations decreased HO activity. It appears that HO activity may be modifying morphine-induced GEC growth.
Estrogen upregulates renal angiotensin II AT2receptors
Authors: Armando Ines , Jezova Miroslava , Juorio Augusto V. , Terrón José A. , Falcón-Neri Alicia , Semino-Mora Cristina , Imboden Hans , Saavedra Juan M.
Published: March 2015

AT2receptors may act in opposition to and in balance with AT1receptors, their stimulation having beneficial effects. We found renal AT2receptor expression in female mice higher than in male mice. We asked the question of whether such expression might be estrogen dependent. In male, female, ovariectomized, and estrogen-treated ovariectomized mice, we studied renal AT1and AT2receptors by immunocytochemistry and autoradiography, AT2receptor mRNA by RT-PCR, and cAMP, cGMP, and PGE2by RIA. AT1receptors predominated. AT2receptors were present in glomeruli, medullary rays, and inner medulla, and in female kidney capsule. AT1and AT2receptors colocalized in glomeruli. Female mice expressed fewer glomerular AT1receptors. Ovariectomy decreased AT1receptors in medullary rays and capsular AT2receptors. Estrogen administration normalized AT1receptors in medullary rays and increased AT2receptors predominantly in capsule and inner medulla, and also in glomeruli, medullary rays, and inner stripe of outer medulla. In medullas of estrogen-treated ovariectomized mice there was higher AT2receptor mRNA, decreased cGMP, and increased PGE2content. We propose that the protective effects of estrogen may be partially mediated through enhancement of AT2receptor stimulation.
Histopathological analysis of renal cystic epithelia in thePkd2WS25/-mouse model of ADPKD
Authors: Thomson R. Brent , Mentone SueAnn , Kim Robert , Earle Karen , Delpire Eric , Somlo Stefan , Aronson Peter S.
Published: March 2015

It has been proposed that autosomal dominant polycystic kidney disease (ADPKD)affected renal epithelial cells undergo a phenotypic transition from a highly differentiated absorptive state to a much less differentiated secretory state during cystogenesis and that this transition is accompanied by loss of epithelial cell polarity and mistargeting of specific membrane proteins. We conducted a detailed evaluation of this hypothesis in the Pkd2WS25/-mouse model of ADPKD. Ultrastructural analysis of Pkd2WS25/-cysts by electron microscopy confirmed that cystic epithelial cells progressively dedifferentiate with cyst enlargement. Immunocytochemical analysis of both early- and late-stage cysts with antibodies directed against Na+-K+-ATPase, Ksp-cadherin, and E-cadherin failed to detect evidence of altered cyst cell polarity. Na+-K+-ATPase and Ksp-cadherin were expressed exclusively on the basolateral membranes (BLM) of epithelial cells in all early cysts. Expression levels of both Na+-K+-ATPase and Ksp-cadherin decreased progressively with the degree of cyst cell dedifferentiation, but neither protein was ever mislocalized. Highly dedifferentiated cysts did not express immunodetectable levels of either Na+-K+-ATPase or Ksp-cadherin. E-cadherin was expressed prominently on the BLM of all cysts. Cysts were subsequently stained with an antibody directed against the secretory isoform of the Na+-K+-Cl-cotransporter NKCC1. NKCC1 expression was detected on the BLM of advanced cysts only. Our data are consistent with a model of progressive cystic epithelial cell dedifferentiation in which fluid accumulation in late-stage cysts is mediated by transepithelial secretion of chloride rather than secretion of sodium by apical Na+-K+-ATPase.
Characterization of PMCA isoforms and their contribution to transcellular Ca2+flux in MDCK cells
Authors: Kip Sertac N. , Strehler Emanuel E.
Published: March 2015

Plasma membrane Ca2+ATPases (PMCAs) are ubiquitous in Ca2+-transporting organs, including the kidney. Using RT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na+/Ca2+exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring45Ca2+flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca2+transport was provided by Na+/Ca2+exchanger and one-third by PMCAs, as determined in Na+-free media and using various PMCA inhibitors (La3+, vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca2+efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca2+transport to ∼140%, whereas antisense treatment reduced Ca2+flux ∼45% compared with controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca2+reabsorption in the distal kidney.
A mathematical model of rat collecting duct III. Paradigms for distal acidification defects
Authors: Weinstein Alan M.
Published: March 2015

The present clinical taxonomy of distal renal tubular acidoses includes “gradient,” “secretory,” and “voltage” defects. These categories refer to presumed collecting duct defects in which the epithelium may be abnormally permeable and unable to sustain an ion gradient, in which luminal proton ATPases are defective, or in which electrogenic Na+reabsorption is impaired and luminal electronegativity is reduced. Classification of affected individuals is based on urinary pH and ion concentrations during spontaneous acidosis and during SO[Formula: see text] infusion, as well as urinary Pco2during HCO[Formula: see text] loading. A model of rat CD has been developed that has been used to examine determinants of urinary acidification (Weinstein AM. Am J Physiol Renal Physiol 283: F1252–F1266, 2002) and the interplay of HCO[Formula: see text] and PO[Formula: see text] loads to generate a disequlibrium pH and equilibrium Pco2. In this paper, pure forms of gradient, voltage, and secretory defects are simulated, with attention to variability in the locus of the defect in the cortical (CCD), outer medullary (OMCD), or inner medullary collecting duct (IMCD). The objective of these calculations is to discover whether the intuitive description of these defects is sustained quantitatively. The most important positive finding is that the locus of the transport defect along the CD plays a critical role in the apparent severity of the lesion, with more proximal defects being less severe and more easily correctable. In particular, model calculations suggest that for gradient or secretory defects to be clinically detectable they need to involve the OMCD and/or IMCD. Additionally, the calculations reveal a possible mechanism for CD K+wasting, which does not involve failure of H+-K+-ATPase but derives from a paracellular anion leak and thereby a more electronegative lumen. The most important negative finding is the lack of support for the category of renal tubular acidosis associated with a voltage defect. Although CD lesions that present with both K+and H+secretory defects suggest mediation by transepithelial electrical potential difference (PD), both PD changes and proton pump PD sensitivity appear too small to account for the observed abnormalities.
Theoretical effects of UTB urea transporters in the renal medullary microcirculation
Authors: Zhang Wensheng , Edwards Aurélie
Published: March 2015

A mathematical model of transport in the renal medullary microcirculation was used to investigate the role of the UTB urea transporter expressed in descending vasa recta (DVR) endothelia and red blood cell (RBC) membranes. Our simulations suggest that UTB raises RBC and plasma and interstitial urea concentrations by facilitating radial diffusion of the solute and therefore serves to increase the contribution of urea to the corticomedullary osmolality gradient, assuming no secondary effects on tubular transport. However, by lowering transmural urea concentration gradients, UTB reduces water efflux from DVR through aquaporin-1 (AQP1) water channels, thereby decreasing plasma sodium concentration. The net result of these competing effects on the osmolality gradient depends on the fraction of filtered urea that is reabsorbed by vasa recta. We also found that the contribution of UTB to water transport across DVR and RBCs is negligible, even in the absence of AQP1. Our model predicts that UTB plays a significant role, however, in reducing the shrinking and swelling of RBCs as blood flows along the medulla.
Induction of a laminin isoform and α3β1-integrin in renal ischemic injury and repair in vivo
Authors: Zuk Anna , Matlin Karl S.
Published: March 2015

Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of ∼220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the α3-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and α3β1-integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium.
The interrelationship between TGF-β1and nitric oxide is altered in salt-sensitive hypertension
Authors: Ying Wei-Zhong , Sanders Paul W.
Published: March 2015

The study of salt-sensitive hypertension has been facilitated by development of genetic models, especially the Dahl/Rapp salt-sensitive (S) rat. S rats rapidly become hypertensive after initiation of a diet containing 8.0% NaCl and subsequently develop arteriolonephrosclerosis and renal failure, whereas the salt-resistant (R) strain remains normotensive on the same diet. The purpose of the present study was to use these strains to demonstrate the interactions between transforming growth factor-β1(TGF-β1) and nitric oxide (NO). Young, male S and R rats were fed for 4 days diets that contained either 0.3 or 8.0% NaCl. An increase in dietary salt increased kinase activities of both p38 MAPK and p42/44 MAPK in cytoplasmic extracts from aortic rings and isolated glomeruli from both strains. Inhibition of either pathway with PD-098059 or SB-203580 decreased production of TGF-β1and nitrate plus nitrite (NOx). In both strains, production of active TGF-β1and NOxlinearly correlated. Incubation of aortic rings and isolated glomeruli with the NO donor NOR3 decreased TGF-β1levels, whereas the NO synthase inhibitor Nω-nitro-l-arginine methyl ester increased production. The inhibitory effect of NO on production of TGF-β1was reduced in preparations from S rats. Although a close interrelationship existed between TGF-β1and NO in both strains, production of TGF-β1was increased in prehypertensive S rats and was further exaggerated with the increase in dietary salt intake. Augmented vascular and glomerular production of TGF-β1and diminished NO may contribute to the development of hypertensive nephrosclerosis in S rats.
Role of nitric oxide in the natriuretic and diuretic responses in pregnant rats
Authors: Khraibi Ali A. , Yu Tianzheng , Tang Daiyi
Published: March 2015

Normal pregnancy is characterized by sodium conservation and increase in plasma volume, yet the natriuretic response to acute saline volume expansion (VE) is intact in pregnant rats. Nitric oxide (NO) has been suggested to play a role in renal and cardiovascular adaptations to normal pregnancy. The objective of this study was to determine the role of NO in the natriuretic and diuretic responses to VE during pregnancy. Infusion of NG-monomethyl-l-arginine (l-NMMA) was used to inhibit NO synthesis. Nine groups of Sprague-Dawley (SD) rats were studied: nonpregnant (NP-VE, n = 7), midterm pregnant (MP-VE, n = 8), and late-term pregnant (LP-VE, n = 7) SD groups that underwent VE alone after a control period; NP-l-NMMA ( n = 7), MP-l-NMMA ( n = 8), and LP-l-NMMA ( n = 7) SD groups that were infused with l-NMMA after a control period; and another three groups of SD rats (NP-VE-l-NMMA, n = 8; MP-VE-l-NMMA, n = 7; and LP-VE-l-NMMA, n = 12) that underwent simultaneous VE and l-NMMA infusion after a control period. The change in fractional excretion of sodium was 7.22 ± 1.03% for NPVE, 9.89 ± 1.85% for NP-l-NMMA, and 17.66 ± 1.85% for NP-VE-l-NMMA ( P < 0.05 vs. NP-VE and NP-l-NMMA); 6.61 ± 1.07% for MP-VE, 7.99 ± 1.92% for MP-l-NMMA, and 10.24 ± 1.91% for MP-VE-l-NMMA [not significant (NS) vs. MP-VE and MP-l-NMMA]; 8.20 ± 1.92% for LP-VE, 8.09 ± 0.70% for LP-l-NMMA, and 7.57 ± 1.11% for LP-VE-l-NMMA (both NS vs. LP-VE and LP-l-NMMA). The increase in renal interstitial hydrostatic pressure was significantly greater in all NP compared with pregnant groups with similar experimental intervention (i.e., VE, l-NMMA, or VE-l-NMMA). In conclusion, the natriuretic and diuretic responses to VE and l-NMMA infusion were additive in NP but not in pregnant rats, indicating a possible lower ability of pregnant rats to respond to combined significant natriuretic and diuretic stimuli.
Interaction of MAPK and 12-lipoxygenase pathways in growth and matrix protein expression in mesangial cells
Authors: Reddy Marpadga A. , Adler Sharon G. , Kim Young-Sook , Lanting Linda , Rossi John , Kang Shin-Wook , Nadler Jerry L. , Shahed Asha , Natarajan Rama
Published: March 2015

The lipoxygenase (LO) pathway of arachidonate metabolism and mitogen-activated protein kinases (MAPKs) can mediate cellular growth and ANG II effects in vascular smooth muscle cells. However, their role in renal mesangial cells (MC) is not very clear. ANG II treatment of rat MC significantly increased 12-LO mRNA expression and formation of the 12-LO product 12( S)-hydroxyeicosatetraenoic acid [12( S)-HETE; P < 0.03]. ANG II-induced [3H]leucine incorporation was blocked by an LO inhibitor, cinnamyl-3,4-dihydroxy-α-cyanocinnamate ( P < 0.02). 12( S)-HETE and ANG II directly induced cellular hypertrophy and fibronectin (FN) expression ( P < 0.01) to a similar extent. ANG II and 12( S)-HETE led to activation of p38MAPKand its target transcription factor cAMP-responsive element-binding protein (CREB). ANG II- and 12( S)-HETE-induced CREB activation and [3H]leucine incorporation were blocked by the p38MAPKinhibitor SB-202190. A specific molecular inhibitor of rat 12-LO mRNA, namely, a novel ribozyme, could attenuate ANG II-induced FN mRNA. Thus p38MAPK-dependent CREB activation may mediate ANG II- and LO product-induced FN expression and cellular growth in rat MC. ANG II effects may be mediated by the LO pathway. These results suggest a novel interaction between LO and p38MAPKactivation in MC matrix synthesis associated with renal complications.
SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes
Authors: Li Xin , Low Seng Hui , Miura Masumi , Weimbs Thomas
Published: March 2015

The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H+-ATPase in α- but not β-intercalated cells, suggesting its involvement in H+-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.
Bone morphogenic protein-7 inhibits progression of chronic renal fibrosis associated with two genetic mouse models
Authors: Zeisberg Michael , Bottiglio Cindy , Kumar Navin , Maeshima Yohei , Strutz Frank , Müller Gerhard A. , Kalluri Raghu
Published: March 2015

Tubulointerstitial fibrosis is a hallmark feature of chronic renal injury. Specific therapies to control the progression of renal fibrosis toward end-stage renal failure are limited. Previous studies have demonstrated that expression of endogenous bone morphogenic protein-7 (BMP-7) is reduced in the kidneys of several inducible mouse models of acute and chronic renal disease and that administration of exogenous recombinant human BMP-7 (rhBMP-7) has a beneficial effect on kidney function. Here we report that treatment with rhBMP-7 leads to improved renal function, histology, and survival in mice deficient in the α3-chain of type IV collagen and MRL/MpJlpr/lprlupus mice, two genetic models for chronic renal injury and fibrosis. Such therapeutic benefit is also associated with a significant decrease in the expression of profibrotic molecules, such as type I collagen and fibronectin, in renal fibroblasts. Additionally, rhBMP-7 induces expression of active matrix metalloproteinase-2, which is potentially important for removal of fibrotic matrix. Collectively, these studies provide further evidence for rhBMP-7 as an important bone-associated protein with protective function against renal pathology.
Claudin-2 is selectively expressed in proximal nephron in mouse kidney
Authors: Enck Alissa H. , Berger Urs V. , Yu Alan S. L.
Published: March 2015

First published August 15, 2001; 10.1152/ajprenal.00021.2001.—The proximal nephron possesses a leaky epithelium with unique paracellular permeability properties that underlie its high rate of passive NaCl and water reabsorption, but the molecular basis is unknown. The claudins are a large family of transmembrane proteins that are part of the tight junction complex and likely form structural components of a paracellular pore. To localize claudin-2 in the mouse kidney, we performed in situ hybridization using an isoform-specific riboprobe and immunohistochemistry using a polyclonal antibody directed against a COOH-terminal peptide. Claudin-2 mRNA and protein were found throughout the proximal tubule and in the contiguous early segment of the thin descending limb of long-looped nephrons. The level of expression demonstrated an axial increase from proximal to distal segments. In confocal images, the subcellular localization of claudin-2 protein coincided with that of the tight junction protein ZO-1. Our findings suggest that claudin-2 is a component of the paracellular pathway of the most proximal segments of the nephron and that it may be responsible for their uniquely leaky permeability properties.
ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion
Authors: Ashworth Sharon L. , Southgate Erica L. , Sandoval Ruben M. , Meberg Peter J. , Bamburg James R. , Molitoris Bruce A.
Published: March 2015

Ischemic injury induces actin cytoskeleton disruption and aggregation, but mechanisms affecting these changes remain unclear. To determine the role of actin-depolymerizing factor (ADF)/ cofilin participation in ischemic-induced actin cytoskeletal breakdown, we utilized porcine kidney cultured cells, LLC-PKA4.8, and adenovirus containing wild-type (wt), constitutively active, and inactive Xenopus ADF/cofilin linked to green fluorescence protein [XAC(wt)-GFP] in an ATP depletion model. High adenoviral infectivity (70%) in LLC-PKA4.8cells resulted in linearly increasing XAC(wt)-GFP and phosphorylated (p)XAC(wt)-GFP (inactive) expression. ATP depletion rapidly induced dephosphorylation, and, therefore, activation, of endogenous pcofilin as well as pXAC(wt)-GFP in conjunction with the formation of fluorescent XAC(wt)-GFP/actin aggregates and rods. No significant actin cytoskeletal alterations occurred with short-term ATP depletion of LLC-PKA4.8cells expressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, but cells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal and ATP-depleted LLC-PKA4.8cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC(wt)-GFP apical and basal signal in addition to XAC-GFP/actin aggregate formation. These data demonstrate XAC(wt)-GFP participates in ischemia-induced actin cytoskeletal alterations and determines the rate and extent of these ATP depletion-induced cellular alterations.
Stimulation of protein kinase C pathway mediates endocytosis of human nongastric H+-K+-ATPase, ATP1AL1
Authors: Reinhardt J. , Kosch M. , Lerner M. , Bertram H. , Lemke D. , Oberleithner H.
Published: March 2015

The human nongastric H+-K+-ATPase, ATP1AL1, shown to reabsorb K+in exchange for H+or Na+, is localized in the luminal plasma membrane of renal epithelial cells. It is presumed that renal H+-K+-ATPases can be regulated by endocytosis. However, little is known about the molecular mechanisms that control plasma membrane expression of renal H+-K+-ATPases. In our study, activation of protein kinase C (PKC) using phorbol esters (phorbol 12-myristate 13-acetate) leads to clathrin-dependent internalization and intracellular accumulation of the ion pump in stably transfected Madin-Darby canine kidney cells. Functional inactivation of the H+-K+-ATPase by PKC activation is shown by intracellular pH measurements. Proton extrusion capacity of ATP1AL1-transfected cells is drastically reduced after phorbol 12-myristate 13-acetate incubation and can be prevented with the PKC blocker bisindolylmaleimide. Ion pump internalization and inactivation are specifically mediated by the PKC pathway, whereas activation of the protein kinase A pathway has no influence. Our results show that the nongastric H+-K+-ATPase is a specific target for the PKC pathway. Therefore, PKC-mediated phosphorylation is a potential regulatory mechanism for apical nongastric H+-K+-ATPase plasma membrane expression.
Role of luminal anion and pH in distal tubule potassium secretion
Authors: Amorim J. B. O. , Bailey M. A. , Musa-Aziz R. , Giebisch G. , Malnic G.
Published: March 2015

Potassium secretory flux ( JK) by the distal nephron is regulated by systemic and luminal factors. In the present investigation, JKwas measured with a double-barreled K+electrode during paired microperfusion of superficial segments of the rat distal nephron. We used control solutions (100 mM NaCl, pH 7.0) and experimental solutions in which Cl−had been replaced with a less permeant anion and/or pH had been increased to 8.0. JKincreased when Cl−was replaced by either acetate (∼37%), sulfate (∼32%), or bicarbonate (∼62%), and also when the pH of the control perfusate was increased (∼26%). The majority (80%) of acetate-stimulated JKwas Ba2+sensitive, but furosemide (1 mM) further reduced secretion (∼10% of total), suggesting that K+-Cl−cotransport was operative. Progressive reduction in luminal Cl−concentration from 100 to 20 to 2 mM caused increments in JKthat were abolished by inhibitors of K+-Cl−cortransport, i.e., furosemide and [(dihydroindenyl)oxy]alkanoic acid. Increasing the pH of the luminal perfusion fluid also increased JKeven in the presence of Ba2+, suggesting that this effect cannot be accounted for only by K+channel modulation of K+secretion in the distal nephron of the rat. Collectively, these data suggest a role for K+-Cl−cotransport in distal nephron K+secretion.
Renal arterial 20-hydroxyeicosatetraenoic acid levels: regulation by cyclooxygenase
Authors: Cheng Monica K. , McGiff John C. , Carroll Mairead A.
Published: March 2015

20-HETE, a potent vasoconstrictor, is generated by cytochrome P-450 ω-hydroxylases and is the principal eicosanoid produced by preglomerular microvessels. It is released from preglomerular microvessels by ANG II and is subject to metabolism by cyclooxygenase (COX). Because low-salt (LS) intake stimulates the renin-angiotensin system and induces renal cortical COX-2 expression, we examined 20-HETE release from renal arteries (interlobar and arcuate and interlobular arteries) obtained from 6- to 7-wk-old male Sprague-Dawley rats fed either normal salt (0.4% NaCl) or LS (0.05% NaCl) diets for 10 days. With normal salt intake, the levels of 20-HETE recovered were similar in arcuate and interlobular arteries and interlobar arteries: 30.1 ± 8.5 vs. 24.6 ± 5.3 ng · mg protein−1· 30 min−1, respectively. An LS diet increased 20-HETE levels in the incubate of either arcuate and interlobular or interlobar renal arteries only when COX was inhibited. Addition of indomethacin (10 μM) to the incubate of arteries obtained from rats fed an LS diet resulted in a two- to threefold increase in 20-HETE release from arcuate and interlobular arteries, from 39.1 ± 13.2 to 101.8 ± 42.6 ng · mg protein−1· 30 min−1( P < 0.03), and interlobar arteries, from 31.7 ± 15.1 to 61.9 ± 29.4 ng · mg protein−1· 30 min−1( P < 0.05) compared with release of 20-HETE when COX was not inhibited. An LS diet enhanced vascular expression of cytochrome P-4504A and COX-2 in arcuate and interlobular arteries; COX-1 was unaffected. Metabolism of 20-HETE by COX is proposed to represent an important regulatory mechanism in setting preglomerular microvascular tone.
Enhanced ammonia secretion by proximal tubules from mice receiving NH4Cl: role of angiotensin II
Authors: Nagami Glenn T. ,   (With the Technical Assistance of Evelyn M. Warech)
Published: March 2015

Acidosis and angiotensin II (ANG II) stimulate ammonia production and transport by the proximal tubule. We examined the effect of short-term (18 h) in vivo acid loading with NH4Cl on ammonia production and secretion rates by mouse S2 proximal tubule segments microperfused in vitro with or without ANG II in the luminal microperfusion solution. S2 tubules from NH4Cl-treated mice displayed higher rates of luminal ammonia secretion compared with those from control mice. The adaptive increase in ammonia secretion in NH4Cl-treated mice was eliminated when losartan was coadministered in vivo with NH4Cl. Ammonia secretion rates from both NH4Cl-treated and control mice were largely inhibited by amiloride. Addition of ANG II to the microperfusion solution enhanced ammonia secretion and production rates to a greater extent in tubules from NH4Cl-treated mice compared with those from controls, and the stimulatory effects of ANG II were blocked by losartan. These results demonstrate that a short-term acid challenge induces an adaptive increase in ammonia secretion by the proximal tubule and suggest that ANG II plays an important role in the adaptive enhancement of ammonia secretion that is observed with short-term acid challenges.
Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases
Authors: Çetinkaya Ibrahim , Ciarimboli Giuliano , Yalçinkaya Gülay , Mehrens Thomas , Velic Ana , Hirsch Jochen R. , Gorboulev Valentin , Koepsell Hermann , Schlatter Eberhard
Published: March 2015

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]- N-methylpyridinium (ASP+). ASP+uptake was electrogenic and inhibited by TPA+(EC50= 2.7 μM), tetraethylammonium (EC50= 35 μM), cimetidine (EC50= 36 μM), or quinine (EC50= 6.7 μM). Stimulation with carbachol or ATP decreased initial uptake by 44 ± 3 ( n = 14) and 34 ± 4% ( n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 ± 4% ( n = 40). Inhibition of calmodulin (CaM), Ca2+/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 ± 2 ( n = 15), 40 ± 4 ( n = 30), and 31 ± 4% ( n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC50value for the inhibition of ASP+uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.
Glucocorticoids inhibit transcription and expression of the UT-A urea transporter gene
Authors: Peng Tao , Sands Jeff M. , Bagnasco Serena M.
Published: March 2015

Dexamethasone treatment increases urea excretion and decreases urea permeability and urea transporter UT-A1 protein abundance in the inner medullary collecting duct (IMCD) of adrenalectomized rats. We examined the effect of dexamethasone treatment for 3 days on the abundance of several UT-A mRNA transcripts in rat renal medulla. By Northern blot analysis, a significant decrease in mRNA expression was observed in the inner medulla of dexamethasone-treated rats compared with controls for UT-A1 (71%), UT-A3 (75%), and UT-A3b (75%), but not for UT-A2. We then tested the effect of 100 nM dexamethasone on the activity of promoter I in the UT-A gene, using LLC-PK1-GR101 cells that express the glucocorticoid receptor. Dexamethasone significantly decreased the activity of rat UT-A promoter I (72%) but did not affect UT-A promoter II. Deletion analysis and site-directed mutagenesis demonstrated that sequences between −423 and −244 are important for this inhibition and that a 10-bp sequence at −363, which binds a nuclear protein in a gel shift assay, is necessary for basal promoter activity. The specific factors involved in repression of UT-A promoter I activity by glucocorticoids remain to be determined.
Preserved macula densa-dependent renin secretion in A1adenosine receptor knockout mice
Authors: Schweda Frank , Wagner Charlotte , Krämer Bernhard K. , Schnermann Jürgen , Kurtz Armin
Published: March 2015

Recent studies demonstrated that the influence of the macula densa on glomerular filtration is abolished in adenosine A1receptor (A1AR) knockout mice. Inasmuch as the macula densa not only regulates glomerular filtration but also controls the activity of the renin system, the present study aimed to determine the role of the A1AR in macula densa control of renin synthesis and secretion. Although a high-salt diet over 1 wk suppressed renin mRNA expression and renal renin content to similar degrees in A1AR+/+, A1AR+/−, and A1AR−/−mice, stimulation of Ren-1 mRNA expression and renal renin content by salt restriction was markedly enhanced in A1AR−/−compared with wild-type mice. Pharmacological blockade of macula densa salt transport with loop diuretics stimulated renin expression in vivo (treatment with furosemide at 1.2 mg/day for 6 days) and renin secretion in isolated perfused mouse kidneys (treatment with 100 μM bumetanide) in all three genotypes to the same extent. Taken together, our data are consistent with the concept of a tonic inhibitory role of the A1AR in the renin system, whereas they indicate that the A1AR is not indispensable in macula densa control of the renin system.
PTH stimulates a Cl−-dependent and EIPA-sensitive current in chick proximal tubule cells in culture
Authors: Laverty Gary , McWilliams Colleen , Sheldon Amanda , Árnason Sighvatur S.
Published: March 2015

The electrophysiological effects of parathyroid hormone (PTH) were studied in a primary cell culture model of the chick ( Gallus domesticus) proximal tubule. In this model, confluent monolayers are grown on permeable filters and exhibit vectorial transport, including glucose-stimulated current. Under short-circuit conditions, PTH, at 10−9M, induced a positive current [short-circuit current ( Isc)] response, with an average 2-min peak response of 14.30 ± 1.58 μA/cm2over the baseline Isc,followed by a slow decay. The PTH response was dose dependent, with a half-maximal response at 5 × 10−9M and maximal response at 5 × 10−8M. Forskolin and dibutyryl-cAMP also stimulated Isc, as did the phosphodiesterase inhibitor IBMX. In contrast, the phorbol ester PMA inhibited baseline Isc. The PTH response was nearly abolished by apical addition of 100 μM EIPA, an inhibitor of Na+/H+exchangers, and partially blocked by the Cl−channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 μM) and glibenclamide (300 μM). Higher doses of EIPA or NPPB alone (500 μM) were almost fully effective, with no or slight additional effects of NPPB or EIPA, respectively. The anion exchange inhibitor DIDS (100 μM) and the Na+channel blocker amiloride (10 μM) had no effect. Bilateral reduction of Cl−in the buffer, from 137 to 2.6 mM, abolished the PTH response; increasing Cl−concentration restored the Iscresponse, with a half-maximal effect at 50 mM. These data suggest that, in the chick proximal tubule, PTH activates both an Na+/H+exchanger and a Cl−channel that may be functionally linked.