bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of homocysteine on endothelial nitric oxide production
Authors: Zhang Xiaohui , Li Hong , Jin Haoli , Ebin Zachary , Brodsky Sergey , Goligorsky Michael S.
Published: December 2017

Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate. Controversy exists as to the effects of HHCy on nitric oxide (NO) production: it has been shown that HHCy both increases and suppresses it. We addressed this problem by using amperometric electrochemical NO detection with a porphyrinic microelectrode to study responses of endothelial cells incubated with homocysteine (Hcy) to the stimulation with bradykinin, calcium ionophore, or l-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 μM) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not affect the expression of endothelial nitric oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and immunoblotting of proteins modified by tyrosine nitration. Hcy did not directly affect the ability of recombinant eNOS to generate NO, but oxidation of sulfhydryl groups in eNOS reduced its NO-generating activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitrotyrosine-modified proteins. In addition, a scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by endothelial cells. In conclusion, electrochemical detection of NO release from cultured endothelial cells demonstrated that concentrations of Hcy >20 μM produce a significant indirect suppression of eNOS activity without any discernible effects on its expression. Folates, superoxide ions, and peroxynitrite scavengers restore the NO-generating activity to eNOS, collectively suggesting that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.
A hydrodynamic mechanosensory hypothesis for brush border microvilli
Authors: Guo P. , Weinstein A. M. , Weinbaum S.
Published: December 2017

In the proximal tubule of the kidney, Na+ and HCO3 − reabsorption vary proportionally with changes in axial flow rate. This feature is a critical component of glomerulotubular balance, but the basic mechanism by which the tubule epithelial cells sense axial flow remains unexplained. We propose that the microvilli, which constitute the brush border, are physically suitable to act as a mechanosensor of fluid flow. To examine this hypothesis quantitatively, we have developed an elastohydrodynamic model to predict the forces and torques along each microvillus and its resulting elastic bending deformation. This model indicates that: 1) the spacing of the microvilli is so dense that there is virtually no axial velocity within the brush border and that drag forces on the microvilli are at least 200 times greater than the shear force on the cell's apical membrane at the base of the microvilli; 2) of the total drag on a 2.5-μm microvillus, 74% appears within 0.2 μm from the tip; and 3) assuming that the structural strength of the microvillus derives from its axial actin filaments, then a luminal fluid flow of 30 nl/min produces a deflection of the microvillus tip which varies from about 1 to 5% of its 90-nm diameter, depending on the microvilli length. The microvilli thus appear as a set of stiff bristles, in a configuration in which changes in drag will produce maximal torque.
Multidrug resistance protein Mrp2 mediates ATP-dependent transport of classic renal organic anionp-aminohippurate
Authors: Van Aubel Rémon A. M. H. , Peters Janny G. P. , Masereeuw Rosalinde , Van Os Carel H. , Russel Frans G. M.
Published: December 2017

p-Aminohippurate (PAH) is widely used as a model substrate to characterize organic anion transport in kidney proximal tubules. The carrier responsible for uptake of PAH across the basolateral membrane has been cloned and well characterized, whereas transporters mediating PAH excretion across the brush-border (apical) membrane are yet unknown. In this study we investigated whether PAH is a substrate for the apical multidrug resistance protein 2 (Mrp2). Overexpression of recombinant rabbit Mrp2 in Sf9 cells significantly increased ATP-dependent [14C]PAH uptake into isolated membrane vesicles compared with endogenous ATP-dependent uptake. The Michaelis-Menten constant and maximal velocity for Mrp2-mediated ATP-dependent [14C]PAH transport were 1.9 ± 0.8 mM and 187 ± 29 pmol · mg−1· min−1, respectively. On the basis of the inhibitory profile, the endogenous ATP-dependent PAH transporter does not appear to be an ortholog of Mrp2. Together, our results show that Mrp2 is a low-affinity ATP-dependent PAH transporter, indicating that Mrp2 might contribute to urinary PAH excretion.
Expression characteristics and relevance of sodium glucose cotransporter-1 in mammalian renal tubulogenesis
Authors: Yang Qiwei , Tian Yufeng , Wada Jun , Kashihara Naoki , Wallner Elisabeth , Peterson Darryl , Kanwar Yashpal S.
Published: December 2017

Expression and role of sodium glucose cotransporter (SGLT-1) in tubulogenesis were investigated during renal development. A mouse SGLT-1 cDNA was cloned, and it had substantial homology with human and rat forms. Four mRNA transcripts were detected, which differed in size from other species. SGLT-1 transcripts were detected at day 13 of gestation, and their expression increased during later stages extending into the postnatal period. A high mRNA and protein expression of SGLT-1 was seen in tubular segments of the inner cortex and outer medulla at day 16, and it was developmentally regulated. Treatment with SGLT-1 antisense selectively decreased the population of tubules in the metanephric explants. Expression of glomerular mRNA and WGA binding were unchanged. SGLT-1 activity, as measured by [14C]methyl-α-d-glucopyranoside uptake, increased during gestation in the tubular segments where it is expressed. Glucose uptake was inhibited by the treatment with SGLT-1 antisense and d-galactose. The data suggest that SGLT-1 exhibits a restricted spatiotemporal expression with functional activity confined to the corresponding tubular segments, and it selectively maintains renal tubulogenesis during development.
Expression of aquaporins in the renal connecting tubule
Authors: Coleman Richard A. , Wu Daniel C. , Liu Jie , Wade James B.
Published: December 2017

The renal connecting tubule (CNT) is a distinct segment that occurs between the distal convoluted tubule (DCT) and the cortical collecting duct. On the basis of its characterization in rabbit it is widely believed that connecting tubule cells have a low permeability to water and do not respond to vasopressin. Here we utilize segment-specific markers and specific aquaporin antibodies to characterize expression of water channels in CNT of the rat by immunocytochemistry. Colocalization of aquaporin 2 (AQP2), AQP3, and AQP4 with Na+, Ca2+exchanger (NCX), a transporter characteristic of the connecting tubule, gave heterogeneous labeling. There was aquaporin labeling in many but not all regions labeled by NCX. Colocalization of AQP2 with AQP3 and with AQP4 showed that AQP3 and AQP4 labeling were always accompanied by AQP2. Immunogold labeling and electron microscopy showed that NCX-labeled cells with AQP2 labeling had the morphology of CNT cells, whereas NCX-labeled cells without AQP2 labeling were DCT cells. The latter regions were identified as the late region of the DCT known as DCT2. Additionally, regions of CNT lacking AQP2 labeling could be identified in Brattleboro rats not treated with vasopressin but not in such animals chronically treated with deamino-Cys1,d-Arg8-vasopressin (dDAVP). Quantitative analysis of labeling was consistent with expression of AQP2 over a longer region of CNT after dDAVP exposure.
PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values
Authors: Leipziger Jens , MacGregor Gordon G. , Cooper Gordon J. , Xu Jason , Hebert Steven C. , Giebisch Gerhard
Published: December 2017

Close similarity between the rat native low-conductance K+channel in the apical membrane of renal cortical collecting duct principal cells and the cloned rat ROMK channel strongly suggest that the two are identical. Prominent features of ROMK regulation are a steep pH dependence and activation by protein kinase A (PKA)-dependent phosphorylation. In this study, we investigated the pH dependence of cloned renal K+channel (ROMK2), wild-type (R2-WT), and PKA site mutant channels (R2-S25A, R2-S200A, and R2-S294A). Ba2+-sensitive outward whole cell currents (holding voltage −50 mV) were measured in two-electrode voltage-clamp experiments in Xenopus laevisoocytes expressing either R2-WT or mutant channels. Intracellular pH (pHi) was measured with pH-sensitive microelectrodes in a different group of oocytes from the same batch on the same day. Resting pHiof R2-WT and PKA site mutants was the same: 7.32 ± 0.02 ( n = 22). The oocytes were acidified by adding 3 mM Na butyrate with external pH (pHo) adjusted to 7.4, 6.9, 6.4, or 5.4. At pHo7.4, butyrate led to a rapid (τ: 163 ± 14 s, where τ means time constant, n= 4) and stable acidification of the oocytes (ΔpHi0.13 ± 0.02 pH units, where Δ means change, n = 12). Intracellular acidification reversibly inhibited ROMK2-dependent whole cell current. The effective acidic dissociation constant (p Ka) value of R2-WT was 6.92 ± 0.03 ( n = 8). Similarly, the effective p Kavalue of the N-terminal PKA site mutant R2-S25A was 6.99 ± 0.02 ( n = 6). The effective p Kavalues of the two COOH-terminal PKA site mutant channels, however, were significantly shifted to alkaline values; i.e., 7.15 ± 0.06 ( n = 5) for R2-S200A and 7.16 ± 0.03 ( n = 8) for R2-S294A. The apparent ΔpH shift between the R2-WT and the R2-S294A mutant was 0.24 pH units. In excised inside-out patches, alkaline pH 8.5 activated R2-S294A channel current by 32 ± 6.7%, whereas in R2-WT channel patches alkalinzation only marginally increased current by 6.5 ± 1% ( n = 5). These results suggest that channel phosphorylation may substantially influence the pH sensitivity of ROMK2 channel. Our data are consistent with the hypothesis that in the native channel PKA activation involves a shift of the pKavalue of ROMK channels to more acidic values, thus relieving a H+-mediated inhibition of ROMK channels.
Interaction of angiotensin II and atrial natriuretic peptide on pHiregulation in MDCK cells
Authors: Oliveira-Souza M. , De Mello-Aires M.
Published: December 2017

The effect of ANG II and atrial natriuretic peptide (ANP) on intracellular pH (pHi) and cytosolic free calcium concentration ([Ca2+]i) was investigated in Madin-Darby canine kidney cells by using the fluorescent probes 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (AM) and fura 2-AM or fluo 4-AM. pHirecovery rate was examined in the first 2 min after the acidification of pHiwith a NH4Cl pulse. In the control situation, the pHirecovery rate was 0.088 ± 0.014 pH units/min ( n = 14); in the absence of external Na+, this value was decreased. ANG II (10−12or 10−9M) caused an increase in this value, but ANG II (10−7M) decreased it. ANP (10−6M) or dimethyl-1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM (50 μM) alone did not affect this value but impaired both stimulatory and inhibitory effects of ANG II. ANG II (10−12, 10−9, or 10−7M) increased [Ca2+]iprogressively from 99 ± 10 ( n = 20) to 234 ± 7 mM ( n = 10). ANP or dimethyl-BAPTA-AM decreases [Ca2+]i, and the subsequent addition of ANG II caused a recovery of [Ca2+]ibut without reaching ANG II values found in the absence of these agents. The results indicate a role for [Ca2+]iin regulating the process of pHirecovery mediated by the Na+/H+exchanger, stimulated/impaired by ANG II, and not affected by ANP or ANG II plus ANP. This hormonal interaction may represent physiologically relevant regulation in conditions of volume alterations in the intact animal.
Spontaneous shift in transcriptional profile of explanted glomeruli via activation of the MAP kinase family
Authors: Ishikawa Yoshihisa , Konta Tsuneo , Kitamura Masanori
Published: December 2017

To understand how isolation and explantation of glomeruli affect the function of resident cells, the present study investigated the transcriptional profile of explanted normal glomeruli. We found that ex vivo incubation of glomeruli spontaneously expressed monocyte chemoattractant protein-1 (MCP-1) and stromelysin, the genes regulated by activator protein-1 (AP-1). The expression was suppressed by heparin and quercetin, the drugs with anti-AP-1 activities. The gene expression was preceded by 1) induction of AP-1 components c- fos and c- jun and 2) phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein (MAP) kinase, and c-Jun NH2-terminal kinase (JNK), the upstream inducers/activators of AP-1. Suppression of ERK by PD098059 abrogated induction of c- fos and c- jun, and the p38 MAP kinase inhibitor SB203580 attenuated c- fos expression. Furthermore, treatment with either PD098059, SB203580, or the JNK-AP-1 inhibitor curcumin diminished the expression of MCP-1 and stromelysin. The transcriptional profile of glomerular cells thus alters dramatically after explantation of glomeruli. It is, at least in part, due to activation of multiple MAP kinases that lead to induction of AP-1-dependent gene expression.
Regulation of AQP6 mRNA and protein expression in rats in response to altered acid-base or water balance
Authors: Promeneur Dominique , Kwon Tae-Hwan , Yasui Masato , Kim Gheun-Ho , Frøkiær Jørgen , Knepper Mark A. , Agre Peter , Nielsen Søren
Published: December 2017

In the rat, aquaporin-6 (AQP6) is mainly localized in intercalated cells (ICs) in collecting ducts, where it is exclusively associated with intracellular vesicles. In this study, we examined whether AQP6 protein and mRNA expression were regulated in the inner medulla or inner stripe of the outer medulla. Rats treated with dietary alkali or acid load for 7 days with a fixed daily water intake revealed appropriate changes in urine pH but unchanged urine output. AQP6 protein and mRNA abundance were increased in alkali-loaded rats (187 ± 18 and 151 ± 17% of control, respectively), whereas no changes were observed in acid-loaded rats. Immunohistochemistry revealed increased IC AQP6 labeling in alkali-loaded rats but not in acid-loaded rats. In contrast, administration of NH4Cl in the drinking water for 2 wk (free access to water) revealed a significant increase in AQP6 protein abundance (194 ± 9% of control), but this was associated with increased water intake. Combined, this suggests that AQP6 expression was not affected by acid loading per se but rather was in response to changes in water intake. Consistent with this, water loading for 48 h was associated with increased AQP6 protein abundance, compared with thirsted rats. Moreover, rats with lithium-induced nephrogenic diabetes insipidus had a threefold increase in both AQP6 protein and mRNA expression. Overall, these results suggest that AQP6 expression in collecting duct ICs is regulated by altered acid/alkali load or water balance. Thus AQP6 may contribute to maintenance of acid-base homeostasis and water balance.
Cellular origin and hormonal regulation of K+-ATPase activities sensitive to Sch-28080 in rat collecting duct
Authors: Laroche-Joubert Nicolas , Marsy Sophie , Doucet Alain
Published: December 2017

Rat collecting ducts exhibit type I or type III K+-ATPase activities when animals are fed a normal (NK) or a K+-depleted diet (LK). This study aimed at determining functionally the cell origin of these two K+-ATPases. For this purpose, we searched for an effect on K+-ATPases of hormones that trigger cAMP production in a cell-specific fashion. The effects of 1-deamino-8-d-arginine vasopressin (dD-AVP), calcitonin, and isoproterenol in principal cells, α-intercalated cells, and β-intercalated cells of cortical collecting duct (CCD), respectively, and of dD-AVP and glucagon in principal and α-intercalated cells of outer medullary collecting duct (OMCD), respectively, were examined. In CCDs, K+-ATPase was stimulated by calcitonin and isoproterenol in NK rats (type I K+-ATPase) and by dD-AVP in LK rats (type III K+-ATPase). In OMCDs, dD-AVP and glucagon stimulated type III but not type I K+-ATPase. These hormone effects were mimicked by the cAMP-permeant analog dibutyryl-cAMP. In conclusion, in NK rats, cAMP stimulates type I K+-ATPase activity in α- and β-intercalated CCD cells, whereas in LK rats it stimulates type III K+-ATPase in principal cells of both CCD and OMCD and in OMCD intercalated cells.
Mathematical model of an avian urine concentrating mechanism
Authors: Layton H. E. , Davies John M. , Casotti Giovanni , Braun Eldon J.
Published: December 2017

A mathematical model was used to investigate how concentrated urine is produced within the medullary cones of the quail kidney. Model simulations were consistent with a concentrating mechanism based on single-solute countercurrent multiplication and on NaCl cycling from ascending to descending limbs of loops of Henle. The model predicted a urine-to-plasma (U/P) osmolality ratio of ∼2.26, a value consistent with maximum avian U/P osmolality ratios. Active NaCl transport from descending limb prebend thick segments contributed 70% of concentrating capability. NaCl entry and water extraction provided 80 and 20%, respectively, of the concentrating effect in descending limb flow. Parameter studies indicated that urine osmolality is sensitive to the rate of fluid entry into descending limbs and collecting ducts at the cone base. Parameter studies also indicated that the energetic cost of concentrating urine is sensitive to loop of Henle population as a function of medullary depth: as the fraction of loops reaching the cone tip increased above anatomic values, urine osmolality increased only marginally, and, ultimately, urine osmolality decreased.
Renal Na+-glucose cotransporters
Authors: Wright Ernest M.
Published: December 2017

In humans, the kidneys filter ∼180 g of d-glucose from plasma each day, and this is normally reabsorbed in the proximal tubules. Although the mechanism of reabsorption is well understood, Na+-glucose cotransport across the brush-border membrane and facilitated diffusion across the basolateral membrane, questions remain about the identity of the genes responsible for cotransport across the brush border. Genetic studies suggest that two different genes regulate Na+-glucose cotransport, and there is evidence from animal studies to suggest that the major bulk of sugar is reabsorbed in the convoluted proximal tubule by a low-affinity, high-capacity transporter and that the remainder is absorbed in the straight proximal tubule by a high-affinity, low-capacity transporter. There are at least three different candidates for these human renal Na+-glucose cotransporters. This review will focus on the structure-function relationships of these three transporters, SGLT1, SGLT2, and SGLT3.
Temporary effects of circulating Amadori products on glomerular filtration properties in the normal mouse
Authors: Londoño I. , Bendayan M.
Published: December 2017

Previous studies have established a preferential glomerular filtration of glycated BSA (gBSA), as well as a facilitated filtration of BSA in the presence of gBSA. We intend to determine whether these modifications are permanent or transitory. gBSA was intravenously injected into anesthetized normal mice and maintained in circulation for 30 min, 1, 2, 24, and 48 h. Five minutes before death, FITC-BSA was injected. On immunocytochemical evaluations, increased glomerular filtration of FITC-BSA was found at all circulating time points. Changes at 24 and 48 h were less pronounced. Glomerular basement membrane (GBM)-to-lumen gBSA labeling ratios were similar at all time points suggesting no accumulation of gBSA in the GBM. Seventy percent of the gBSA was cleared from the circulation and the GBM after 24 h, and 95% after 48 h. This was confirmed in experiments with radiolabeled tracers. These results suggest that the alteration in GBM permeability to BSA in the normal mouse are due to the presence of gBSA and are gradually overcome along with its clearance from circulation. In early diabetes, increasing concentrations of circulating glycated proteins could be responsible for changes in glomerular permselectivity and probably for the alteration in glomerular filtration properties leading to diabetic nephropathy.
Acute upregulation of COX-2 by renal artery stenosis
Authors: Mann Bianca , Hartner Andrea , Jensen Boye L. , Hilgers Karl F. , Höcherl Klaus , Krämer Bernhard K. , Kurtz Armin
Published: December 2017

This study aimed to characterize the influence of acute renal artery stenosis on cyclooxygenase-2 (COX-2) and renin expression in the juxtaglomerular apparatus. For this purpose, male Sprague-Dawley rats received a left renal artery clip, and COX-2 mRNA, COX-2 immunoreactivity, plasma renin activity, and renin mRNA levels were determined. COX-2 mRNA and COX-2 immunoreactivity in the macula densa region in the clipped kidneys increased as early as 6 h after clipping and reached a maximal expression 1–2 days after clipping. Although values for plasma renin activity were elevated markedly at all time points examined, remaining renin mRNA levels were unchanged after 6 h and then increased to reach a maximum value 1–2 days after clipping. In the contralateral intact kidney, renin mRNA and COX-2 immunoreactivity decreased to ∼50% of their normal values. To investigate a possible causal relationship between the changes of COX-2 and of renin expression, clipped rats were treated with the COX-2 blocker celecoxib (40 mg · kg−1 · day−1). This treatment, however, did not change renin mRNA either in the clipped or in the contralateral intact kidney. Our findings indicate that renal artery stenosis causes ipsilaterally an acute upregulation and contralaterally a downregulation of juxtaglomerular COX-2 expression. The lacking effect of celecoxib on renin gene expression does not support the concept of a direct mediator function of COX-2-derived prostaglandins in the control of renin expression during renal hypoperfusion.
BMP7 controls collecting tubule cell proliferation and apoptosis via Smad1-dependent and -independent pathways
Authors: Piscione Tino D. , Phan Tien , Rosenblum Norman D.
Published: December 2017

Bone morphogenetic protein-7 (BMP7) controls ureteric bud and collecting duct morphogenesis in a dose-dependent manner (Piscione TD, Yager TD, Gupta IR, Grinfeld B, Pei Y, Attisono L, Wrana JL, and Rosenblum ND. Am J Physiol Renal Physiol 273: F961–F975, 1997). We defined cellular and molecular mechanisms underlying these effects in embryonic kidney explants and in the mIMCD-3 cell model of collecting tubule morphogenesis. Low-dose (0.25 nM) BMP7 significantly increased tubule number and cell proliferation. Similar to BMP2, high-dose (10 nM) BMP7 inhibited cell proliferation and stimulated apoptosis. To define molecular mechanisms, we identified signaling events downstream of BMP7. High-dose BMP7, but not low-dose BMP7, activated Smad1 in mIMCD-3 cells. Moreover, the inhibitory effects of high-dose BMP7 and BMP2, but not the stimulatory effects of low-dose BMP7, on tubulogenesis and cell proliferation were significantly reduced in mIMCD-3 cells stably expressing Smad1(Δ458), a dominant negative mutant form of Smad1, but not in cells stably expressing wild-type Smad1. We conclude that BMP7 exerts dose-dependent effects on ureteric bud or collecting duct cell proliferation and apoptosis by signaling via Smad1-dependent and Smad1-independent pathways.
Proximal tubule cell response to radiographic contrast media
Authors: Hardiek Kathy , Katholi Richard E. , Ramkumar Vickram , Deitrick Cynthia
Published: December 2017

Renal dysfunction associated with contrast media (CM) administration is generally attributed to reduced renal blood flow. Studies, however, also suggest direct tubular effects of CM, whose mechanisms remain unclear. This study was conducted to assess the chemotoxic effects of iopamidol, a prototypic CM, on a porcine proximal tubule (PT) cell line, LLC-PK1 cells. Results indicate that iopamidol did not affect cell viability (determined by trypan blue exclusion and fluorescein staining), but did reduce cell proliferation. Moreover, iopamidol altered mitochondrial function, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and mitochondrial membrane potential. Decreased MTT reduction was evident with all CM tested, and its rapid recovery after CM removal suggests that inhibition of mitochondrial function is reversible. Injury to PT cells by iopamidol is supported by the fact that CM increase extracellular adenosine, an indicator of cellular stress. This study provides greater insight into the mechanism underlying the nephrotoxicity induced by contrast in patients and explains the reversibility of this toxicity.
Impaired solute accumulation in inner medulla ofClcnk1−/− mice kidney
Authors: Akizuki Norikazu , Uchida Shinichi , Sasaki Sei , Marumo Fumiaki
Published: December 2017

The CLC-K1 chloride channel is a kidney-specific CLC chloride channel expressed in the thin ascending limb of Henle's loop (tAL). Recently, we determined that Clcnk1−/− mice show nephrogenic diabetes insipidus (NDI). To investigate the pathogenesis of impaired urinary concentrating ability, we analyzed renal functions of Clcnk1−/− mice in more detail. The osmolar clearance-to-creatinine clearance ratio was not significantly different between Clcnk1+/− and Clcnk1+/+ mice. Fractional excretion of sodium, chloride, and urea was also not significantly affected in Clcnk1−/− mice. These results indicate that the polyuria observed in Clcnk1−/− mice was water diuresis and not osmotic diuresis. The papillary osmolarity in Clcnk1−/− mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1−/− mice were at about one-half the levels observed in Clcnk1+/+ mice. Furthermore, the accumulation of urea was also impaired in Clcnk1−/− mice, suggesting that the overall countercurrent system was impaired by a defect of its single component, chloride transport in the tAL. The aldose reductase mRNA abundance in Clcnk1−/− mice was decreased, further evincing that inner medullary tonicity is decreased in Clcnk1−/− mice. We concluded that NDI in Clcnk1−/− mice resulted from an impairment in the generation of inner medullary hypertonicity by a dysfunction of the countercurrent systems.
Short-term K+deprivation provokes insulin resistance of cellular K+uptake revealed with the K+clamp
Authors: Choi Cheol S. , Thompson Curtis B. , Leong Patrick K. K. , McDonough Alicia A. , Youn Jang H.
Published: December 2017

We aimed to test the feasibility of quantifying insulin action on cellular K+uptake in vivo in the conscious rat by measuring the exogenous K+infusion rate needed to maintain constant plasma K+concentration ([K+]) during insulin infusion. In this “K+clamp” the K+infusion rate required to clamp plasma [K+] is a measure of insulin action to increase net plasma K+disappearance. K+infusion rate required to clamp plasma [K+] was insulin dose dependent. Renal K+excretion was not significantly affected by insulin at a physiological concentration (∼90 μU/ml, P > 0.05), indicating that most of insulin-mediated plasma K+disappearance was due to K+uptake by extrarenal tissues. In rats deprived of K+for 2 days, plasma [K+] fell from 4.2 to 3.8 mM, insulin-mediated plasma glucose clearance was normal, but insulin-mediated plasma K+disappearance decreased to 20% of control, even though there was no change in muscle Na-K-ATPase activity or expression, which is believed to be the main K+uptake route. After 10 days K+deprivation, plasma [K+] fell to 2.9 mM, insulin-mediated K+disappearance decreased to 6% of control (glucose clearance normal), and there were 50% decreases in Na-K-ATPase activity and α2-subunit levels. In conclusion, the present study proves the feasibility of the K+clamp technique and demonstrates that short-term K+deprivation leads to a near complete insulin resistance of cellular K+uptake that precedes changes in muscle sodium pump expression.
Molecular and biochemical characterization of prostacyclin receptors in rat kidney
Authors: Nasrallah Rania , Zimpelmann Joseph , Singh Sonia , Hébert Richard L.
Published: December 2017

The prostacyclin (IP) message was detected by RT-PCR in the renal cortex, outer (OM) and inner medulla (IM), and in freshly isolated (IMCD-f) and cultured inner medullary collecting duct (IMCD-c), and also the E-prostanoid (EP)1,3,4receptor subtypes, but not EP2. Digoxigenin in situ hybridization localized IP mRNA in the tubules of the OM and IM, and the vasculature, and also in the glomeruli, arteries, and tubules of the cortex. IP splice variants or subtypes could not be detected by RT-PCR followed by TA cloning, though several nonfunctional point mutations or single base pair deletions were observed. Iloprost (ILP), cicaprost (CCP), PGE2, and arginine vasopressin (AVP) stimulated cAMP in both IMCD preparations. In addition, AVP-stimulated cAMP in IMCD-f was inhibited by all three prostanoids, but not in IMCD-c. Calcium experiments were performed on IMCD-c or microdissected IMCD (IMCD-m). CCP, ILP, and PGE2did not alter intracellular calcium concentration ([Ca2+]i) in IMCD-c. However, on IMCD-m, both PGE2and ILP increased [Ca2+]ilevels equipotently and CCP had no effect. Pretreatment with the EP1antagonist AH-6809 indicates that the response to ILP and PGE2is mediated via EP1. These results suggest that IP receptors in the rat IMCD mediate the cAMP but not calcium signaling linked to PGI2; to date no subtypes or splice variants have been identified.
Na+-to-sugar stoichiometry of SGLT3
Authors: Díez-Sampedro Ana , Eskandari Sepehr , Wright Ernest M. , Hirayama Bruce A.
Published: December 2017

Sodium-glucose cotransporters (SGLTs) mediate active transport of sugar across cell membranes coupled to Na+, by using the electrochemical gradient as a driving force. In the kidney, there is evidence for two kinds of cotransporters, a high-affinity, low-capacity system, and a low-affinity, high-capacity system, with differences in substrate specificity and kinetics. Three renal SGLT clones have been identified: SGLT1 corresponding to the high-affinity system, and SGLT2 and SGLT3 with properties reminiscent of the low-affinity system. We have determined the stoichiometry of pig SGLT3 (pSGLT3) by using a direct method, comparing the substrate-induced inward charge to22Na or [14C]α-methyl-d-glucopyranoside uptake in the same oocyte. pSGLT3 stoichiometry is 2 Na+:1 sugar, the same as that for SGLT1, but different from SGLT2 (1:1). The Na+Hill coefficient for SGLT3 is ∼1.5, suggesting low cooperativity between Na+binding sites. Thus SGLT3 has functional characteristics intermediate between SGLT1 and SGLT2, so, whereas SGLT3 stoichiometry is the same as that for SGLT1 (2:1), sugar affinity and specificity are similar to SGLT2.
NHE3 activity and trafficking depend on the state of actin organization in proximal tubule
Authors: Chalumeau C. , Du Cheyron D. , Defontaine N. , Kellermann O. , Paillard M. , Poggioli J.
Published: December 2017

The present study was addressed to define the contribution of cytoskeleton elements in the kidney proximal tubule Na+/H+exchanger 3 (NHE3) activity under basal conditions. We used luminal membrane vesicles (LMV) isolated from suspensions of rat cortical tubules pretreated with either colchicine (Colch) or cytochalasin D (Cyto D). Colch pretreatment of suspensions (200 μM for 60 min) moderately decreased LMV NHE3 activity. Cyto D pretreatment (1 μM for 60 min) elicited an increase in LMV NHE3 transport activity but did not increase Na-glucose cotransport activity. Cyto D pretreatment of suspensions did not change the apparent affinity of NHE3 for internal H+. In contrast, after Cyto D pretreatment of the suspensions, NHE3 protein abundance was increased in LMV and remained unchanged in cortical cell homogenates. The effect of Cyto D on NHE3 was further assessed with cultures of murine cortical cells. The amount of surface biotinylated NHE3 increased on Cyto D treatment, whereas NHE3 protein abundance was unchanged in cell homogenates. In conclusion, under basal conditions NHE3 activity depends on the state of actin organization possibly involved in trafficking processes between luminal membrane and intracellular compartment.
Mechanisms of chloride transport in thymic lymphocytes
Authors: Stakisaitis Donatas , Lapointe Michael S. , Batlle Daniel
Published: December 2017

This study examined mechanisms of Cl−transport in rat lymphocytes under a variety of conditions. Basal intracellular Cl−concentration ([Cl−]i) was not different between cells assayed in the presence of HCO3−or its absence (HEPES). Removal of external Cl−resulted in a fall in [Cl−]iand a rapid rise in intracellular pH (pHi). Both Cl−efflux and the rise in pHiwere blocked by DIDS or removal of external Na+but were unaffected by furosemide. The mechanisms governing Cl−influx were assessed in cells that had been Cl−depleted for 1 h. Reexposure to Cl−resulted in a rapid rise in [Cl−]ithat was partially inhibited by pretreatment with DIDS (57%) and partially inhibited by pretreatment with furosemide (45%). Pretreatment with both compounds together completely blocked Cl−influx. Cl−depletion caused a marked increase in pHithat rapidly declined toward normal when the cells were reexposed to Cl−. Preincubation with DIDS completely blocked this decrease in pHi. In contrast, neither removal of Na+nor preincubation with furosemide affected the decline in pHiwhen the cells were reexposed to Cl−. We conclude that, in thymic lymphocytes, Cl−/HCO3−(or Cl−/base exchange) regulates both Cl−influx and efflux. Cl−efflux is totally inhibited by DIDS and is mediated by a Na+-dependent Cl−/HCO3−exchanger. Cl−influx is partially DIDS sensitive and partially furosemide sensitive and is mediated by both a Na+-independent Cl−/HCO3−exchanger and by a Na+-K+-2Cl−cotransporter.
UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD
Authors: Terris James M. , Knepper Mark A. , Wade James B.
Published: December 2017

UT-A3 has recently been identified as a splicing variant transcript of the UT-A gene present in the kidney. To study the cellular and subcellular localization of UT-A3, we raised a new polyclonal antibody to its COOH-terminal end. Immunoblots identified bands at 44 and 67 kDa predominately in the inner medulla and showed that the antibody does not recognize UT-A1. Deglycosylation with PNGase decreased the molecular mass of both forms to 40 kDa. UT-A3 is most abundant in the inner third of the inner medulla and is present in membrane fractions. Cell fractionation studies showed that UT-A3 is only detectable in inner medullary collecting duct (IMCD) cells. These observations were confirmed with immunolocalization studies demonstrating an exclusive labeling of IMCD cells. Double-labeling studies with anti-Na-K-ATPase demonstrated UT-A3 in intracellular membranes and in the apical region but were incompatible with a basolateral site for UT-A3. Although the function of this isoform in the inner medulla is unknown, the large abundance suggests that it may be important in the renal handling of urea.
NHERF: targeting and trafficking membrane proteins
Authors: Shenolikar Shirish , Weinman Edward. J.
Published: December 2017

Vectorial ion transport initiated by Na+/H+exchanger isoform 3 (NHE3) mediates the reabsorption of NaCl and NaHCO3in renal proximal tubule cells. NHE3 activity is modulated by numerous physiological stimuli. Biochemical and cellular experiments identified Na+/H+exchanger regulatory factor (NHERF) as a protein cofactor essential for cAMP-mediated inhibition of NHE3 activity. Identification of numerous NHERF targets, including several transmembrane receptors and ion transporters, has broadened the role of this PSD-95/Dlg-1, Drososphila disk large/ZO-1 domain-containing adapter protein in membrane physiology. NHERF also associates with members of the ezrin/radixin/moesin family of actin-binding proteins and thus links NHE3 to the actin cytoskeleton. Formation of this multiprotein complex facilitates NHE3 phosphorylation and hormonal control of Na+/H+exchange. NHERF also plays a critical role in targeting transport proteins to apical membranes. Moreover, the NHERF signaling complex functions as a regulatory unit to control endocytosis and internal trafficking of membrane proteins. This article reviews the new evidence that implicates NHERF in wider aspects of epithelial membrane biology.
A gel-membrane model of glomerular charge and size selectivity in series
Authors: Ohlson Maria , Sörensson Jenny , Haraldsson Börje
Published: December 2017

We have analyzed glomerular sieving data from humans, rats in vivo, and from isolated perfused rat kidneys (IPK) and present a unifying hypothesis that seems to resolve most of the conflicting results that exist in the literature. Particularly important are the data obtained in the cooled IPK, because they allow a variety of experimental conditions for careful analysis of the glomerular barrier; conditions that never can be obtained in vivo. The data strongly support the classic concept of a negative charge barrier, but separate components seem to be responsible for charge and size selectivity. The new model is composed of a dynamic gel and a more static membrane layer. First, the charged gel structure close to the blood compartment has a charge density of 35–45 meq/l, reducing the concentration of albumin to 5–10% of that in plasma, due to ion-ion interactions. Second, the size-selective structure has numerous functional small pores (radius 45–50 Å) and far less frequent large pores (radius 75–115 Å), the latter accounting for 1% of the total hydraulic conductance. Both structures are required for the maintenance of an intact glomerular barrier.
Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis
Authors: Bagnis Corinne , Marsolais Mireille , Biemesderfer Daniel , Laprade Raynald , Breton Sylvie
Published: December 2017

An acidic luminal pH in the epididymis and vas deferens (VD) helps maintain mature sperm in an immotile state during storage. We have previously shown that the majority of proton secretion in the VD is due to the activity of the vacuolar H+-ATPase. Acidification is dependent on luminal sodium in more proximal regions of the epididymis, and we examined the distribution of the Na+/H+exchanger, NHE3, by immunofluorescence and measured Na+/H+exchange (NHE) activity in isolated epididymal tubules. NHE3 was detected in the apical pole of nonciliated cells of the efferent ducts and principal cells (PC) of the epididymis. No staining was seen in the distal cauda epididymidis and the VD. Isolated tubules from the distal initial segment (DIS) and proximal cauda epididymidis were perfused in vitro and loaded with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6′)-carboxyfluorescein. Ethylisopropyl amiloride (EIPA) (50 μM) reduced the initial rate of intracellular pH recovery (dpHi/d t), in response to an acute acid load, by 51% and 45% in the DIS and cauda epididymidis, respectively. In the DIS, removal of luminal sodium reduced dpHi/d t by 52%. HOE694 (50 μM) inhibited all EIPA-sensitive dpHi/d t in the DIS, despite the previously reported absence of NHE2 in this region (Cheng Chew SB, Leung GPH, Leung PY, Tse CM, and Wong PYD, Biol Reprod 62: 755–758, 2000). These data indicate that HOE694- and EIPA-sensitive Na+/H+exchange may participate, together with the H+-ATPase, in luminal acidification in the male excurrent duct.
Membrane topology and immunolocalization of CHIF in kidney and intestine
Authors: Shi Haikun , Levy-Holzman Rivi , Cluzeaud Francoise , Farman Nicolette , Garty Haim
Published: December 2017

Corticosteroid hormone-induced factor (CHIF) is an aldosterone-induced gene, the function of which is yet unknown. It is specifically expressed in kidney collecting duct (CD) and distal colon and is upregulated by either Na+deprivation or K+loading. Hence, it may play a role in epithelial electrolyte transport. Previous studies have characterized regulation and tissue distribution of CHIF mRNA but provided no information on the protein itself. The present paper addresses this issue by using Western blotting, immunochemistry, and in vitro translation. CHIF is an ∼8-kDa membranal protein, and protease digestion experiments suggest that its COOH tail faces the cell interior. The protein is abundant in distal colon, kidney medulla, and papilla but cannot be detected in a variety of other tissues. Confocal immunocytochemistry demonstrates that CHIF is present in the basolateral membrane of CD principal cells and distal colon surface cells, with occasional intracellular staining. Dexamethasone and low Na+intake increase the abundance of CHIF. Unlike previous Northern data, induction of CHIF protein by low-Na+intake was apparent not only in the distal colon but also in the kidney.
A quantitative analysis of the glomerular charge barrier in the rat
Authors: Sörensson Jenny , Ohlson Maria , Haraldsson Börje
Published: December 2017

Modifying the ionic strength (I) is a gentle way to alter charge interactions, but it cannot be done for studies of the glomerular sieving of proteins in vivo. We therefore perfused 18 isolated rat kidneys with albumin solutions of different ionic strengths at a low temperature (cIPK) to inhibit tubular uptake and protease activity. Four anionic proteins were studied, namely albumin (Alb), orosomucoid (Oro), ovalbumin (Ova), and anionic horseradish peroxidase (aHRP), together with the neutral polymer Ficoll. With normal ionic strength of the perfusate (152 mM), the fractional clearance (θ) was 0.0018 ± 0.0003 for Alb, 0.0033 ± 0.0003 for Oro, 0.090 ± 0.008 for Ova, and 0.062 ± 0.002 for aHRP. These θ values were all lower than for Ficoll of similar hydrodynamic size; e.g., θFicoll 36 Åwas >20 times higher than θ for albumin. Low ionic strength (34 mM) increased size selectivity as θ for anionic proteins and Ficoll fell, suggesting a reduction in small-pore radius from 44 ± 0.4 to 41 ± 0.5 Å, P < 0.01. In contrast, low I reduced the charge density of the membrane, ω, to one-quarter of the 20–50 meq/l estimated at normal I. These dynamic changes in ω seem to be due to volume alterations of the charged gel, fluid shifts that easily are accounted for by the changes in electroosmotic pressures. The finding that low ionic strength induces inverse effects on size selectivity and charge density strongly suggests that separate structures of the glomerular wall are responsible for the two properties.
Hypoxia and high glucose cause exaggerated mesangial cell growth and collagen synthesis: role of osteopontin
Authors: Sodhi Chhinder P. , Phadke Sarojini A. , Batlle Daniel , Sahai Atul
Published: December 2017

The effect of hypoxia on the proliferation and collagen synthesis of cultured rat mesangial cells was examined under normal-glucose (NG, 5 mM) and high-glucose (HG, 25 mM)-media conditions. In addition, a role for osteopontin (OPN) in mediating these processes was assessed. Quiescent cultures were exposed to hypoxia (3% O2) and normoxia (18% O2) in a serum-free medium with NG or HG, and cell proliferation, collagen synthesis, and OPN expression were assessed. Cells exposed to hypoxia in NG medium resulted in significant increases in [3H]thymidine incorporation, cell number, and [3H]proline incorporation, respectively. HG incubations also produced significant stimulation of these parameters under normoxic conditions, which were markedly enhanced in cells exposed to hypoxia in HG medium. In addition, hypoxia and HG stimulated the mRNA levels of type IV collagen, and the combination of hypoxia and HG resulted in additive increases in type IV collagen expression. Hypoxia and HG also stimulated OPN mRNA and protein levels in an additive fashion. A neutralizing antibody to OPN or its β3-integrin receptor significantly blocked the effect of hypoxia and HG on proliferation and collagen synthesis. In conclusion, these results demonstrate for the first time that hypoxia in HG medium produces exaggerated mesangial cell growth and type IV collagen synthesis. In addition, OPN appears to play a role in mediating the accelerated mesangial cell growth and collagen synthesis found in a hyperglycemic and hypoxic environment.
Function and spatial distribution of ion channels and transporters in cell migration
Authors: Schwab Albrecht
Published: December 2017

Cell migration plays a central role in many physiological and pathophysiological processes, such as embryogenesis, immune defense, wound healing, or the formation of tumor metastases. Detailed models have been developed that describe cytoskeletal mechanisms of cell migration. However, evidence is emerging that ion channels and transporters also play an important role in cell migration. The purpose of this review is to examine the function and subcellular distribution of ion channels and transporters in cell migration. Topics covered will be a brief overview of cytoskeletal mechanisms of migration, the role of ion channels and transporters involved in cell migration, and ways by which a polarized distribution of ion channels and transporters can be achieved in migrating cells. Moreover, a model is proposed that combines ion transport with cytoskeletal mechanisms of migration.
Long-term adaptation of renal cells to hypertonicity: role of MAP kinases and Na-K-ATPase
Authors: Capasso J. M. , Rivard C. J. , Berl T.
Published: December 2017

Renal cells in culture have low viability when exposed to hypertonicity. We developed cell lines of inner medullary collecting duct cells adapted to live at 600 and 900 mosmol/kgH2O. We studied the three modules of the mitogen-activated protein (MAP) kinase family in the adapted cells. These cells had no increase in either extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, or p38 MAP kinase protein or basal activity. When acutely challenged with further increments in tonicity, they had blunted activation of these kinases, which was not due to enhanced phosphatase activity. In contrast, the cells adapted to the hypertonicity displayed a marked increment in Na-K-ATPase expression (5-fold) and ouabain-sensitive Na-K-ATPase activity (10-fold). The changes were reversible on return to isotonic conditions. Replacement of 300 mosmol/kgH2O of NaCl by urea in cells adapted to 600 mosmol/kgH2O resulted in marked decrement in Na-K-ATPase and failure to maintain the cell line. Replacement of NaCl for urea in cells adapted to 900 mosmol/kgH2O did not alter either Na-K-ATPase expression, or the viability of the cells. The in vivo modulation of Na-K-ATPase was studied in the renal papilla of water-deprived mice (urinary osmolality 2,900 mosmol/kgH2O), compared with that of mice drinking dextrose in water (550 mosmol/kgH2O). Increased water intake was associated with a ∼30% decrement in Na-K-ATPase expression ( P < 0.02, n = 6), suggesting that this enzyme is osmoregulated in vivo. We conclude that whereas MAP kinases play a role in the response to acute changes in tonicity, they are not central to the chronic adaptive response. Rather, in this setting there is upregulation of other osmoprotective proteins, among which Na-K-ATPase appears to be an important component of the adaptive process.