bluuebunny/crossref_metadata_2025 huggingface dataset:
A mechanogated nonselective cation channel in proximal tubule that is ATP sensitive
Authors: Hurwitz Craig G. , Hu Vivian Y. , Segal Alan S.
Published: March 2015

Ion channels that are gated in response to membrane deformation or “stretch” are empirically designated stretch-activated channels. Here we describe a stretch-activated nonselective cation channel in the basolateral membrane (BLM) of the proximal tubule (PT) that is nucleotide sensitive. Single channels were studied in cell-intact and cell-free patches from the BLM of PT cells that maintain their epithelial polarity. The limiting inward Cs+ conductance is ∼28 pS, and channel activity persists after excision into a Ca2+- and ATP-free bath. The stretch-dose response is sigmoidal, with half-maximal activation of about −19 mmHg at −40 mV, and the channel is activated by depolarization. The inward conductance sequence is: NH[Formula: see text] ∼ Cs+ ∼ Rb+> K+ ∼ Na+ ∼ Li+ > Ca2+ ∼ Ba2+> N-methyl-d-glucamine ∼ tetraethylammonium. The venom of the common Chilean tarantula, Grammostola spatulata, completely blocks channel activity in cell-attached patches. Hypotonic swelling reversibly activates the channel. Intracellular ATP concentration ([ATP]i) reversibly blocks the channel (inhibitory constant ∼0.48 mM), suggesting that channel function is coupled to the metabolic state of the cell. We conclude that this channel may function as a Ca2+ entry pathway and/or be involved in regulation of cell volume. We speculate this channel may be important when [ATP]i is depleted, as occurs during periods of increased transepithelial transport or with ischemic injury.
Angiotensin II induces apoptosis in rat glomerular epithelial cells
Authors: Ding Guohua , Reddy Krishna , Kapasi Aditi A. , Franki Nicholas , Gibbons Nora , Kasinath Balakuntalam S. , Singhal Pravin C.
Published: March 2015

ANG II has been shown to modulate kidney cell growth and contribute to the pathobiology of glomerulosclerosis. Glomerular visceral epithelial cell (GEC) injury or loss is considered to play a pivotal role in the initiation and progression of glomerulosclerosis. In the present study, we investigated the effect of ANG II on GEC apoptosis. Rat GECs were incubated with increasing doses of ANG II for variable time periods. Apoptosis was evaluated by cell nucleus staining and DNA fragmentation assay. ANG II induced GEC apoptosis in a dose- and time-dependent manner. The proapoptotic effect was attenuated by the ANG II receptor type 1 antagonist losartan or the ANG II receptor type 2 antagonist PD-123319 and was completely blocked by incubation with the combined antagonists. Moreover, ANG II stimulated transforming growth factor (TGF)-β1 production as measured by ELISA. GECs exposed to TGF-β1 demonstrated a dose- and time-dependent increase in apoptosis. ANG II-induced apoptosis was significantly inhibited by addition of anti-TGF-β1 antibody. ANG II also upregulated the expression of Fas, FasL, and Bax and downregulated the expression of Bcl-2 in GECs. These studies suggest that ANG II induces GEC apoptosis by a mechanism involving TGF-β1 expression that may, importantly, contribute to the pathogenesis of glomerulosclerosis.
Osmolality and solute composition are strong regulators of AQP2 expression in renal principal cells
Authors: Storm R. , Klussmann E. , Geelhaar A. , Rosenthal W. , Maric K.
Published: March 2015

The water permeability of the renal collecting duct is regulated by the insertion of aquaporin-2 (AQP2) into the apical plasma membrane of epithelial (principal) cells. Using primary cultured epithelial cells from the inner medulla of rat kidney (IMCD cells), we show that osmolality and solute composition are potent regulators of AQP2 mRNA and protein synthesis, as well as the classical cAMP-dependent pathway, but do not affect the arginine vasopressin-induced AQP2 shuttle. In the presence of the cAMP analog dibutyryl cAMP (DBcAMP, 500 μM), NaCl and sorbitol, but not urea, evoked a robust increase of AQP2 expression in IMCD cells, with NaCl being far more potent than sorbitol. cAMP-responsive element-binding protein phosphorylation increased with DBcAMP concentrations but was not altered by changes in osmolality. In the rat and human AQP2 promoter, we identified a putative tonicity-responsive element. We conclude that, in addition to the arginine vasopressin/cAMP-signaling cascade, a further pathway activated by elevated effective osmolality (tonicity) is crucial for the expression of AQP2 in IMCD cells, and we suggest that the effect is mediated via the tonicity-responsive element.
Angiotensin II induces apoptosis in renal proximal tubular cells
Authors: Bhaskaran Madhu , Reddy Krishna , Radhakrishanan Neetu , Franki Nicholas , Ding Guohua , Singhal Pravin C.
Published: March 2015

ANG II has been demonstrated to play a role in the progression of tubulointerstial injury. We studied the direct effect of ANG II on apoptosis of cultured rat renal proximal tubular epithelial cells (RPTECs). ANG II promoted RPTEC apoptosis in a dose- and time-dependent manner. This effect of ANG II was attenuated by anti-transforming growth factor (TGF)-β antibody. Moreover, TGF-β triggered RPTEC apoptosis in a dose-dependent manner. ANG II also enhanced RPTEC expression of Fas and Fas ligand (FasL); furthermore, anti-FasL antibody attenuated ANG II-induced RPTEC apoptosis. In addition, ANG II increased RPTEC expression of Bax, a cell death protein. Both ANG II type 1 (AT1) and type 2 (AT2) receptor blockers inhibited ANG II-induced RPTEC apoptosis. SB-202190, an inhibitor of p38 MAPK phosphorylation, and caspase-3 inhibitor also attenuated ANG II-induced RPTEC apoptosis. ANG II enhanced RPTEC heme oxygenase (HO)-1 expression. Interestingly, pretreatment with hemin as well as curcumin (inducers of HO-1) inhibited the ANG II-induced tubular cell apoptosis; conversely, pretreatment with zinc protoporphyrin, an inhibitor of HO-1 expression, promoted the effect of ANG II. These results suggest that ANG II-induced apoptosis is mediated via both AT1and AT2receptors through the generation of TGF-β, followed by the transcription of cell death genes such as Fas, FasL, and Bax. Modulation of tubular cell expression of HO-1 has an inverse relationship with the ANG II-induced tubular cell apoptosis.
Regulated expression of pendrin in rat kidney in response to chronic NH4Cl or NaHCO3loading
Authors: Frische Sebastian , Kwon Tae-Hwan , Frøkiær Jørgen , Madsen Kirsten M. , Nielsen Søren
Published: March 2015

The anion exchanger pendrin is present in the apical plasma membrane of type B and non-A-non-B intercalated cells of the cortical collecting duct (CCD) and connecting tubule and is involved in HCO[Formula: see text]secretion. In this study, we investigated whether the abundance and subcellular localization of pendrin are regulated in response to experimental metabolic acidosis and alkalosis with maintained water and sodium intake. NH4Cl loading (0.033 mmol NH4Cl/g body wt for 7 days) dramatically reduced pendrin abundance to 22 ± 4% of control values ( n = 6, P < 0.005). Immunoperoxidase labeling for pendrin showed reduced intensity in NH4Cl-loaded animals compared with control animals. Moreover, double-label laser confocal microscopy revealed a reduction in the fraction of cells in the CCD exhibiting pendrin labeling to 65% of the control value ( n = 6, P < 0.005). Conversely, NaHCO3loading (0.033 mmol NaHCO3/g body wt for 7 days) induced a significant increase in pendrin expression to 153 ± 11% of control values ( n = 6, P < 0.01) with no change in the fraction of cells expressing pendrin. Immunoelectron microscopy revealed no major changes in the subcellular distribution, with abundant labeling in both the apical plasma membrane and the intracellular vesicles in all conditions. These results indicate that changes in pendrin protein expression play a key role in the well-established regulation of HCO[Formula: see text] secretion in the CCD in response to chronic changes in acid-base balance and suggest that regulation of pendrin expression may be clinically important in the correction of acid-base disturbances.
Aquaporin-2 localization in clathrin-coated pits: inhibition of endocytosis by dominant-negative dynamin
Authors: Sun Tian-Xiao , Van Hoek Alfred , Huang Yan , Bouley Richard , McLaughlin Margaret , Brown Dennis
Published: March 2015

Before the identification of aquaporin (AQP) proteins, vasopressin-regulated “water channels” were identified by freeze-fracture electron microscopy as aggregates or clusters of intramembraneous particles (IMPs) on hormonally stimulated target cell membranes. In the kidney collecting duct, these IMP clusters were subsequently identified as possible sites of clathrin-coated pit formation on the plasma membrane, and a clathrin-mediated mechanism for internalization of vasopressin-sensitive water channels was suggested. Using an antibody raised against the extracellular C loop of AQP2, we now provide direct evidence that AQP2 is concentrated in clathrin-coated pits on the apical surface of collecting duct principal cells. Furthermore, by using a fracture-label technique applied to LLC-PK1cells expressing an AQP2- c-myc construct, we show that AQP2 is located in IMP aggregates and is concentrated in shallow membrane invaginations on the surface of forskolin-stimulated cells. We also studied the functional role of clathrin-coated pits in AQP2 trafficking by using a GTPase-deficient dynamin mutation (K44A) to inhibit clathrin-mediated endocytosis. Immunofluorescence labeling and freeze-fracture electron microscopy showed that dominant-negative dynamin 1 and dynamin 2 mutants prevent the release of clathrin-coated pits from the plasma membrane and induce an accumulation of AQP2 on the plasma membrane of AQP2-transfected cells. These data provide the first direct evidence that AQP2 is located in clathrin-coated pits and show that AQP2 recycles between the plasma membrane and intracellular vesicles via a dynamin-dependent endocytotic pathway. We propose that the IMP clusters previously associated with vasopressin action represent sites of dynamin-dependent, clathrin-mediated endocytosis in which AQP2 is concentrated before internalization.
Synthesis of sulfated proteoglycans by bovine glomerular endothelial cells in culture
Authors: Sörensson Jenny , Björnson Anna , Ohlson Maria , Ballermann Barbara J. , Haraldsson Börje
Published: March 2015

It has been suggested that proteinuria is caused by alterations of the charge selectivity of the basement membrane and/or the epithelial cell layer (podocytes). However, recent findings suggest that the endothelial luminal surface coat, consisting of proteoglycans with their connected glycosaminoglycan (GAG) branches and glycoproteins, may contribute to the permselectivity. Therefore, we wanted to investigate the effects on endothelial GAG synthesis during normal and pathological conditions. We treated glomerular endothelial cell cultures with puromycin aminonucleoside (PAN, a nephrosis-inducing agent) or interleukin-1β (IL-1β) for a total of 72 h and compared the metabolic turnover and incorporation of [35S]sulfate during the last 2 days. In control cultures, the GAG content in the media supernatants increased 66 ± 6% (mean ± SE) between 12 and 42 h of incubation with radioactivity ( P < 0.01, n = 8). The content of35S-labeled GAGs in the media was reduced by 31 ± 1% by PAN ( P < 0.001, n = 8) and increased by 141 ± 15% by 10 U/ml IL-1β ( P < 0.01, n = 8). Treatment with enzymes revealed a dominance of heparan, chondroitin, and dermatan sulfate GAGs. Thus the glomerular endothelial cell production of GAGs was increased by IL-1β and reduced by PAN. Therefore, it is conceivable that certain nephrotic conditions may be due to endothelial dysfunction, rather than other renal causes.
Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine
Authors: Zelenina Marina , Zelenin Sergey , Bondar Alexander A. , Brismar Hjalmar , Aperia Anita
Published: March 2015

Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability ( Pf) was measured in individual LLC-PK1cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH2terminus with green fluorescent protein. Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased Pfof cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the Pfin transfected cells. The effect was abolished by the PKC inhibitor Ro 31–8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser180, a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser180and that the effect is likely mediated by gating of the channel.
Determinants of GFR depression in early membranous nephropathy
Authors: Hladunewich M. A. , Lemley K. V. , Blouch K. L. , Myers B. D.
Published: March 2015

We evaluated the glomerular filtration rate (GFR) in 34 subjects with membranous nephropathy (MN) of new onset. We used physiological techniques to measure GFR, renal plasma flow, and oncotic pressure and computed a value for the two-kidney ultrafiltration coefficient ( Kf). A morphometric analysis of glomeruli in the diagnostic biopsy permitted computation of the single-nephron ultrafiltration coefficient (SN Kf). MN subjects were divided into two groups: moderate or severe, according to whether GFR was depressed by less or more than 50%. SN Kfwas subnormal but similar in moderate and severe MN. In contrast, two-kidney Kfwas significantly more depressed in severe than in moderate MN. We estimated the total number of functioning glomeruli ( Ng) by dividing two-kidney Kfby SN Kf. Whereas mean Ngwas similar in controls and moderate MN (1.5 and 1.4–1.7 × 106, respectively), it was significantly lower in severe MN (0.5 × 106). This degree of glomerulopenia was not reflected in the rate of global sclerosis. We conclude that a combination of depressed SN Kf(due to foot process broadening) and profound glomerulopenia accounts for GFR depression of >50% early in the course of MN. The cause of the glomerulopenia remains to be elucidated.
Identification of functionally important sites in the first intracellular loop of the NaPi-IIa cotransporter
Authors: Köhler Katja , Forster Ian C. , Stange Gerti , Biber Jürg , Murer Heini
Published: March 2015

Intrasequence comparison of the type IIa Na+-Picotransport protein revealed two regions with high similarity in the first intracellular (ICL-1) and third extracellular (ECL-3) loops. Because the ECL-3 loop contains functionally important sites that have been identified by cysteine scanning, we applied this method to corresponding sites in the ICL-1 loop. The accessibility of novel cysteines by methanethiosulfonate reagents was assayed electrophysiologically. Mutants N199C and V202C were fully inhibited after methanethiosulfonate ethylammonium exposure, whereas other mutants showed marginal reductions in cotransport function. None showed significant functional loss after exposure to impermeant methanethiosulfonate ethyltrimethylammonium, which suggested a sidedness of Cys modification. Compared with the wild-type (WT), mutant A203C showed altered Na+leak kinetics, whereas N199C exhibited decreased apparent substrate affinities. To delineate the role of residue N199 in conferring substrate affinity, other mutations at this site were made. Only two mutants yielded significant32Piuptake and inward Pi-induced currents with decreased Piaffinity; for the others, Piapplication suppressed only the Na+leak. We suggest that ICL-1 and ECL-3 sites contribute to the transport pathway and that site N199 is implicated in defining the transport mode.
Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter
Authors: Vázquez Norma , Monroy Adriana , Dorantes Elisa , Muñoz-Clares Rosario A. , Gamba Gerardo
Published: March 2015

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161–F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na+than for Cl−, with apparent Michaelis-Menten constant ( Km) values of 58.2 ± 7.1 and 22.1 ± 4.2 mM, respectively. These Kmvalues are significantly higher than those observed in rTSC. The Na+and Cl−affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10−9to 10−7M) resulted in activation of the cotransporter, whereas higher concentrations (10−6to 10−4M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na+or the Cl−concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg2+, with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.
1,25-Dihydroxyvitamin D-stimulated calmodulin binding proteins: a sustained effect on distal tubules
Authors: Siaw Emmanuel K. O. , Walters Marian R.
Published: March 2015

The tubular localization of 1,25-dihydroxyvitamin D[1,25(OH)2D3]-stimulated calmodulin binding proteins (CaMBP-Ds) in the rat kidney and the specificity of their induction were characterized to better understand renal responses to protracted 1,25(OH)2D3treatment in vivo. None of the other hormones tested (parathyroid hormone, calcitonin, estradiol-17β, testosterone, progesterone, hydrocortisone, or dexamethasone) stimulated the CaMBP-Ds, whereas maximal 1,25(OH)2D3stimulation occurred after a 5- to 7-day treatment with 100 ng/day 1,25(OH)2D3. With the exception of the more ubiquitously distributed CaMBP-D150, the CaMBP-Ds were localized in distal, but not proximal, tubule preparations. 1,25(OH)2D3induction of vitamin D receptors and the CaMBP-Ds was similar with respect to dose-response and time course. Finally, the CaMBP-Ds remained elevated for at least 4 wk after 1,25(OH)2D3withdrawal. Because the vitamin D-stimulated renal CaMBP-Ds are principally proteins of the distal tubule, they may be associated with renal regulation of Ca2+homeostasis. The sustained induction of CaMBP-Ds is important in addressing the question of whether their induction is a function of normal Ca2+homeostasis or a pathophysiological consequence of hypervitaminosis D and hypercalcemia.
Transport of cimetidine by flounder and human renal organic anion transporter 1
Authors: Burckhardt Birgitta C. , Brai Stefan , Wallis Sönke , Krick Wolfgang , Wolff Natascha A. , Burckhardt Gerhard
Published: March 2015

The H2-receptor antagonist cimetidine is efficiently excreted by the kidneys. In vivo studies indicated an interaction of cimetidine not only with transporters for basolateral uptake of organic cations but also with those involved in excretion of organic anions. We therefore tested cimetidine as a possible substrate of the organic anion transporters cloned from winter flounder (fROAT) and from human kidney (hOAT1). Uptake of [3H]cimetidine into fROAT-expressing Xenopus laevis oocytes exceeded uptake into control oocytes. At −60-mV clamp potential, 1 mM cimetidine induced an inward current, which was smaller than that elicited by 0.1 mM PAH. Cimetidine concentrations exceeding 0.1 mM decreased PAH-induced inward currents, indicating interaction with the same transporter. At pH 6.6, no current was seen with 0.1 mM cimetidine, whereas at pH 8.6 a current was readily detectable, suggesting preferential translocation of uncharged cimetidine by fROAT. Oocytes expressing hOAT1 also showed [3H]cimetidine uptake. These data reveal cimetidine as a substrate for fROAT/hOAT1 and suggest that organic anion transporters contribute to cimetidine excretion in proximal tubules.
Expression of isoforms of the Na+/H+exchanger in M-1 mouse cortical collecting duct cells
Authors: Hill C. , Giesberts A. N. , White S. J.
Published: March 2015

Na+/H+exchanger (NHE) proteins perform a variety of functions in the kidney and are differentially distributed among nephron segments. The purpose of this study was to identify NHE isoforms in murine M-1 cells as a model of cortical collecting duct principal cells. It was found that mRNAs corresponding to NHE1, NHE2, and NHE4 are expressed in M-1 cells. NHE-dependent regulation of intracellular pH (pHi) was investigated in the absence of extracellular HCO[Formula: see text]. Application of a 20 mM NH4Cl pulse resulted in a reversible intracellular acidification from which recovery was partially inhibited by application of 1 mM amiloride to either the apical or the basolateral membranes and was abolished when amiloride was applied to both sides of the monolayers, which suggests that NHEs are expressed in both the apical and the basolateral cell membranes of M-1 cells. The purinergic agonists ATP and benzoylbenzoyl-ATP caused a reduction of pHiwhen applied to the apical membrane, which suggests pHimay be influenced by extracellular nucleotides in the luminal fluid of the cortical collecting duct.
Isolation of mouse THP gene promoter and demonstration of its kidney-specific activity in transgenic mice
Authors: Zhu Xinhua , Cheng Jin , Gao Jing , Lepor Herbert , Zhang Zhong-Ting , Pak Joanne , Wu Xue-Ru
Published: March 2015

Tamm-Horsfall protein (THP), the most abundant urinary protein synthesized by the kidney epithelial cells, is believed to play important and diverse roles in the urinary system, including renal water balance, immunosuppression, urinary stone formation, and inhibition of bacterial adhesion. In the present study, we describe the isolation of a 9.3-kb, 5′-region of the mouse THP gene and show the highly conserved nature of its proximal 589-bp, 5′-flanking sequence with that in rats, cattle, and humans. We also demonstrate using the transgenic mouse approach that a 3.0-kb, proximal 5′-flanking sequence is sufficient to drive the kidney-specific expression of a heterologous reporter gene. Within the kidney, transgene expression was confined to the renal tubules that endogenously expressed the THP protein, which suggests specific transgene activity in the thick ascending limb of the loop of Henle and early distal convoluted tubules. Our results establish the kidney- and nephron-segment-specific expression of the mouse THP gene. The availability of the mouse THP gene promoter that functions in vivo should facilitate additional studies of the molecular mechanisms of kidney-specific gene regulation and should provide new molecular tools for better understanding renal physiology and disease through nephron-specific gene targeting.
Structural determinants and specificities for ROMK1-phosphoinositide interaction
Authors: Zeng Wei-Zhong , Liou Horng-Huei , Krishna U. Murali , Falck J. R. , Huang Chou-Long
Published: March 2015

We have recently reported that direct interaction between phosphatidylinositol bisphosphate (PIP2) and the COOH-terminal cytoplasmic domain of ROMK1 is important for opening of the channel. We identified arginine-188 of ROMK1 as a critical residue for this interaction. Here, we further report that substitution of a neutral amino acid for lysine-181, arginine-217, or lysine-218 decreases single-channel open probability for the full-conductance state and increases the frequency of opening at a subconductance state. Compared with wild-type ROMK1 channels, these substitution mutants also display an increased sensitivity to the block by anti-PIP2 antibodies and to inhibition by intracellular protons. These results indicate that, like arginine-188, lysine-181, arginine-217, and lysine-218 are also involved in interactions with PIP2 and are critical for ROMK1 to open at full conductance. Using synthetic phosphoinositides containing phosphates at different positions in the head group, we also examined the specificities of phosphoinositides in the regulation of ROMK1 channels. We found that phosphoinositides containing phosphate at both positions 4 and 5 of the inositol head group have the highest efficacy in activating ROMK1 channels. These results suggest that phosphatidylinositol 4,5-bisphosphate is likely the important phosphoinositide in the regulation of ROMK1 channels in a physiological membrane milieu.
Physiology of the renal medullary microcirculation
Authors: Pallone Thomas L. , Zhang Zhong , Rhinehart Kristie
Published: March 2015

Perfusion of the renal medulla plays an important role in salt and water balance. Pericytes are smooth muscle-like cells that impart contractile function to descending vasa recta (DVR), the arteriolar segments that supply the medulla with blood flow. DVR contraction by ANG II is mediated by depolarization resulting from an increase in plasma membrane Cl−conductance that secondarily gates voltage-activated Ca2+entry. In this respect, DVR may differ from other parts of the efferent microcirculation of the kidney. Elevation of extracellular K+constricts DVR to a lesser degree than ANG II or endothelin-1, implying that other events, in addition to membrane depolarization, are needed to maximize vasoconstriction. DVR endothelial cytoplasmic Ca2+is increased by bradykinin, a response that is inhibited by ANG II. ANG II inhibition of endothelial Ca2+signaling might serve to regulate the site of origin of vasodilatory paracrine agents generated in the vicinity of outer medullary vascular bundles. In the hydropenic kidney, DVR plasma equilibrates with the interstitium both by diffusion and through water efflux across aquaporin-1. That process is predicted to optimize urinary concentration by lowering blood flow to the inner medulla. To optimize urea trapping, DVR endothelia express the UT-B facilitated urea transporter. These and other features show that vasa recta have physiological mechanisms specific to their role in the renal medulla.
Control of descending vasa recta pericyte membrane potential by angiotensin II
Authors: Pallone Thomas L. , Huang James M.-C.
Published: March 2015

Using nystatin perforated-patch whole cell recording, we investigated the role of Cl−conductance in the modulation of outer medullary descending vasa recta (OMDVR) pericyte membrane potential (Ψm) by ANG II. ANG II (10−11 to 10−7 M) consistently depolarized OMDVR and induced Ψm oscillations at lower concentrations. The Cl− channel blockers anthracene-9-decarboxylate (1 mM) and niflumic acid (10 μM) hyperpolarized resting pericytes and repolarized ANG II-treated pericytes. In voltage-clamp experiments, ANG II-treated pericytes exhibited slowly activating currents that were nearly eliminated by treatment with niflumic acid (10 μM) or removal of extracellular Ca2+. Those currents reversed at −31 and −10 mV when extracellular Cl− concentration was 152 and 34 mM, respectively. In pericytes held at −70 mV, oscillating inward currents were sometimes observed; the reversal potential also shifted with extracellular Cl− concentration. We conclude that ANG II activates a Ca2+-dependent Cl− conductance in OMDVR pericytes to induce membrane depolarization and Ψm oscillations.
Regulation of sodium transporters in the thick ascending limb of rat kidney: response to angiotensin II
Authors: Kwon Tae-Hwan , Nielsen Jakob , Kim Young-Hee , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na+transporters. Rats were on a fixed intake of Na+and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng·min-1·kg-1ANG II (sc). Semiquantitative immunoblotting revealed that Na+/H+exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 ± 28 (ANG II-50, n = 5), 166 ± 23 (ANG II-100, n = 7), and 167 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, P < 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na+-K+-2Cl-cotransporter (BSC-1) in the ISOM was also increased to 187 ± 28 (ANG II-50), 162 ± 23 (ANG II-100), and 166 ± 19% (ANG II-200) of control levels ( P < 0.05), but there were no changes in the abundance of Na+-K+-ATPase and the electroneutral Na+-HCO3cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng·min-1·kg-1for 5 days, n = 6) had a higher urinary pH compared with controls. NH4Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na+and HCO3reabsorption in response to ANG II.
Tetanus toxin abolishes exocytosis of ROMK1 induced by inhibition of protein tyrosine kinase
Authors: Sterling Hyacinth , Lin Dao-Hong , Wei Yuan , Wang Wen-Hui
Published: March 2015

We used confocal microscopy, patch-clamp, and biotin-labeling techniques to examine the role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in mediating the effect of inhibition of PTK on ROMK1 trafficking in HEK-293 cells transfected with c-Src and green fluorescent protein (GFP)-ROMK1. Inhibition of c-Src with herbimycin A significantly decreased the tyrosine phosphorylation level of ROMK1. Patch-clamp studies demonstrated that addition of herbimycin A increased the activity of ROMK1 in cell-attached patches. Confocal microscopic imaging showed that herbimycin A decreased the intracellular intensity of GFP-ROMK1. The biotin-labeling technique demonstrated that the inhibition of c-Src increased surface ROMK1 by 110%. In contrast, inhibition of c-Src did not increase the K channel number in HEK cells transfected with R1Y337A, a ROMK1 mutant in which tyrosine residue 337 was mutated to alanine. This suggests that tyrosine residue 337 is essential for the herbimycin A-induced increase in surface ROMK1 channels. To determine whether SNARE proteins are involved in mediating exocytosis of ROMK1 induced by the inhibition of c-Src, we examined the effect of herbimycin A on ROMK1 trafficking in cells treated with tetanus toxin. The incubation of cells in a medium containing tetanus toxin abolished the herbimycin A-induced increase in the number of surface ROMK1. In contrast, inhibition of c-Src still increased the numbers of surface ROMK1 in cells treated with boiled tetanus toxin. We conclude that tyrosine dephosphorylation enhances the exocytosis of ROMK1 and that SNARE proteins are required for exocytosis induced by inhibition of PTK.
Intravital videomicroscopy of peritubular capillaries in renal ischemia
Authors: Yamamoto Tokunori , Tada Tetsuhiro , Brodsky Sergey V. , Tanaka Hiroyoshi , Noiri Eisei , Kajiya Fumihiko , Goligorsky Michael S.
Published: March 2015

The recent refinement and computerization of intravital microscopy have permitted us to monitor microcirculation in vivo with minimal invasion. Here, we report on the first findings made with the use of a pencil-lens intravital microscope as applied to the ischemic rat kidney. Peritubular capillary and glomerular blood flow were monitored under basal conditions, during renal artery occlusion, and immediately after release of the clamp. Erythrocyte velocity was calculated as an angle in consecutive spatiotemporal images. Intravital videomicroscopy during the reperfusion period showed intermittent cessation and partial recovery of blood flow in both peritubular and glomerular capillaries. Blood flow was uniformly orthograde under control conditions; however, the retrograde flow occurred on reperfusion. The patency of peritubular capillaries was partially compromised during the early reperfusion period but rapidly recovered. The recovery of glomerular microcirculation occurred faster than that of peritubular capillaries. We suggest that a functional vasculopathy develops very early in the course of ischemia-reperfusion in superficial cortical microvasculature and is more pronounced in peritubular capillaries, thus accounting for the development of patchy injury of tubular epithelia.
Effects of luminal flow and nucleotides on [Ca2+]iin rabbit cortical collecting duct
Authors: Woda Craig B. , Leite Maurilo , Rohatgi Rajeev , Satlin Lisa M.
Published: March 2015

Nucleotide binding to purinergic P2 receptors contributes to the regulation of a variety of physiological functions in renal epithelial cells. Whereas P2 receptors have been functionally identified at the basolateral membrane of the cortical collecting duct (CCD), a final regulatory site of urinary Na+, K+, and acid-base excretion, controversy exists as to whether apical purinoceptors exist in this segment. Nor has the distribution of receptor subtypes present on the unique cell populations that constitute Ca2+the CCD been established. To examine this, we measured nucleotide-induced changes in intracellular Ca2+concentration ([Ca2+]i) in fura 2-loaded rabbit CCDs microperfused in vitro. Resting [Ca2+]idid not differ between principal and intercalated cells, averaging ∼120 nM. An acute increase in tubular fluid flow rate, associated with a 20% increase in tubular diameter, led to increases in [Ca2+]iin both cell types. Luminal perfusion of 100 μM UTP or ATP-γ-S, in the absence of change in flow rate, caused a rapid and transient approximately fourfold increase in [Ca2+]iin both cell types ( P< 0.05). Luminal suramin, a nonspecific P2 receptor antagonist, blocked the nucleotide- but not flow-induced [Ca2+]itransients. Luminal perfusion with a P2X (α,β-methylene-ATP), P2X7(benzoyl-benzoyl-ATP), P2Y1(2-methylthio-ATP), or P2Y4/P2Y6(UDP) receptor agonist had no effect on [Ca2+]i. The nucleotide-induced [Ca2+]itransients were inhibited by the inositol-1,4,5-triphosphate receptor blocker 2-aminoethoxydiphenyl borate, thapsigargin, which depletes internal Ca2+stores, luminal perfusion with a Ca2+-free perfusate, or the L-type Ca2+channel blocker nifedipine. These results suggest that luminal nucleotides activate apical P2Y2receptors in the CCD via pathways that require both internal Ca2+mobilization and extracellular Ca2+entry. The flow-induced rise in [Ca2+]iis apparently not mediated by apical P2 purinergic receptor signaling.
Glomerular filtration rate dependence of sieving of albumin and some neutral proteins in rat kidneys
Authors: Lund Ulla , Rippe Anna , Venturoli Daniele , Tenstad Olav , Grubb Anders , Rippe Bengt
Published: March 2015

The size and charge-selective properties of the glomerular barrier are partly controversial. Glomerular sieving coefficients (θ) for proteins have rarely been determined noninvasively before in vivo. Therefore, θ was assessed vs. glomerular filtration rate (GFR;51Cr-EDTA clearance) in intact rats for radiolabeled myoglobin, κ-dimer, neutral horseradish peroxidase (nHRP), neutral human serum albumin (nHSA), and native albumin (HSA). To obtain θ, glomerular tracer clearance, assessed from the 7- to 8-min kidney uptake of protein, was divided by the GFR. The data were fitted with a two-pore model of glomerular permeability, where the small-pore radius was 37.35 ± 1.11 (SE) Å, and the “unrestricted pore area over diffusion path length” ( A0/Δ X) 1.84 ± 0.43 · 106cm. Although seemingly horizontal for nHRP and nHSA, the log θ vs. GFR curves showed slightly negative slopes for the proteins investigated in the GFR interval of 2–4.5 ml/min. Strong negative (linear) correlations between (log) θ and GFR were obtained for myoglobin ( P = 0.002) and HSA ( P = 0.006), whereas they were relatively weak for nHRP and nHSA and nonsignificant for κ-dimer. θ for nHSA was markedly higher than that for HSA. In conclusion, there were no indications of increases in θ vs. GFR, as indicative of concentration polarization, for the proteins investigated at high GFRs. Furthermore, the glomerular small-pore radius assessed from endogenous (neutral) protein sieving data was found to be smaller than previously determined using dextran or Ficoll as test molecules.
Chronic renal injury-induced hypertension alters renal NHE3 distribution and abundance
Authors: Yang Li E. , Zhong Huiqin , Leong Patrick K. K. , Perianayagam Anjana , Campese Vito M. , McDonough Alicia A.
Published: March 2015

Renal cortical phenol injection provokes acute sympathetic nervous system-dependent hypertension and a shift of proximal tubule Na+/H+exchanger isoform 3 (NHE3) and Na+-Picotransporter type 2 (NaPi2) to apical microvilli. This study aimed to determine whether proximal tubule (PT) Na+transporter redistribution persists chronically and whether the pool sizes of renal Na+transporters are altered. At 5 wk after a 50-μl 10% phenol injection, blood pressure is elevated: 154 ± 8 vs. 113 ± 11 mmHg after saline injection. Cortical membranes were fractionated into three “windows” enriched in apical brush border ( WI), mixed apical and intermicrovillar cleft ( WII), and intracellular membranes ( WIII). NHE3 relative distribution in these windows, assessed by immunoblots and expressed as %total, remained shifted to apical from intracellular membranes ( WI: 25.3 ± 3 in phenol vs.12.7 ± 3% in saline and WIII: 9.1 ± 1.3 in phenol vs. 18.9 ± 3% in saline). NaPi2 and dipeptidyl-peptidase IV also remained shifted to WI, and alkaline phosphatase activity increased 100.9 ± 29.7 ( WI) and 51.4 ± 17.5% ( WII) in phenol-injected membranes. Na+transporter total abundance [NHE3, NaPi2, thiazide-sensitive Na-Cl cotransporter, bumetanide-sensitive Na-K-2Cl cotransporter, Na-K-ATPase α1- and β1-subunits, and epithelial Na+channel (ENaC) α- and β-subunits] was profiled by immunoblotting. Only cortical NHE3 abundance was altered, decreasing to 0.56 ± 0.06. The results demonstrate that phenol injury provokes a persistant shift of PT NHE3 and NaPi2 to the apical microvilli, along with a 44% decrease in total NHE3, evidence for an escape mechanism that would counteract the redistribution of a larger fraction of NHE3 to the apical surface by normalizing the total amount of NHE3 in apical membranes.
Expression of SSAT, a novel biomarker of tubular cell damage, increases in kidney ischemia-reperfusion injury
Authors: Zahedi Kamyar , Wang Zhaohui , Barone Sharon , Prada Anne E. , Kelly Caitlin N. , Casero Robert A. , Yokota Naoko , Porter Carl W. , Rabb Hamid , Soleimani Manoocher
Published: March 2015

Ischemia-reperfusion injury (IRI) is the major cause of acute renal failure in native and allograft kidneys. Identifying the molecules and pathways involved in the pathophysiology of renal IRI will yield valuable new diagnostic and therapeutic information. To identify differentially regulated genes in renal IRI, RNA from rat kidneys subjected to an established renal IRI protocol (bilateral occlusion of renal pedicles for 30 min followed by reperfusion) and time-matched kidneys from sham-operated animals was subjected to suppression subtractive hybridization. The level of spermidine/spermine N1-acetyltransferase (SSAT) mRNA, an essential enzyme for the catabolism of polyamines, increased in renal IRI. SSAT expression was found throughout normal kidney tubules, as detected by nephron segment RT-PCR. Northern blots demonstrated that the mRNA levels of SSAT are increased by greater than threefold in the renal cortex and by fivefold in the renal medulla at 12 h and returned to baseline at 48 h after ischemia. The increase in SSAT mRNA was paralleled by an increase in SSAT protein levels as determined by Western blot analysis. The concentration of putrescine in the kidney increased by ∼4- and ∼7.5-fold at 12 and 24 h of reperfusion, respectively, consistent with increased functional activity of SSAT. To assess the specificity of SSAT for tubular injury, a model of acute renal failure from Na+depletion (without tubular injury) was studied; SSAT mRNA levels remained unchanged in rats subjected to Na+depletion. To distinguish SSAT increases from the effects of tubular injury vs. uremic toxins, SSAT was increased in cis-platinum-treated animals before the onset of renal failure. The expression of SSAT mRNA and protein increased by ∼3.5- and >10-fold, respectively, in renal tubule epithelial cells subjected to ATP depletion and metabolic poisoning (an in vitro model of kidney IRI). Our results suggest that SSAT is likely a new marker of tubular cell injury that distinguishes acute prerenal from intrarenal failure.
Effects of dietary salt changes on renal renin-angiotensin system in rats
Authors: Ingert Catherine , Grima Michèle , Coquard Catherine , Barthelmebs Mariette , Imbs Jean-Louis
Published: March 2015

Renin (RA) and angiotensin-converting enzyme (ACE) activities and angiotensinogen, ANG I, and ANG II levels were measured in the kidney (cortex and medulla) and plasma of Wistar-Kyoto rats on a low-sodium (LS; 0.025% NaCl; n= 8), normal-sodium (NS; 1% NaCl; n = 7), or high-sodium (HS; 8% NaCl; n = 7) diet for 21 days. RA, ANG I, and ANG II levels increased in a manner inversely related to sodium content of the diet in both plasma and renal tissues. The LS diet resulted in a 16-, 2.8-, and 1.8-fold increase in plasma RA, ANG I, and ANG II levels, respectively, compared with those in HS rats. In the renal cortex and medulla, RA, ANG I, and ANG II levels were also increased by diminution of dietary salt content but, in contrast to plasma, ANG II levels increased much more than RA or ANG I levels [5.4 (cortex)- and 4.7 (medulla)-fold compared with HS rats]. In summary, we demonstrated variations of ANG II levels in the kidney during dietary salt modifications. Our results confirm that RA and ACE activity are not the steps limiting intrarenal ANG II levels. Nevertheless, despite RA and ACE activity differences between renal cortex and medulla, ANG I and ANG II levels are equivalent in these two tissues; these results argue against a compartmentalization of RAS in these two intrarenal areas.
The electrical resistance breakdown assay determines the role of proteinases in tumor cell invasion
Authors: Ludwig Thomas , Ossig Rainer , Graessel Susanne , Wilhelmi Marianne , Oberleithner Hans , Schneider Stefan W.
Published: March 2015

The electrical resistance breakdown of the Madin-Darby canine kidney (MDCK) cell monolayer provides a continuous assay system for cancer invasion that detects functional changes before morphological alterations. In this study, we address the question of whether physical contact between tumor cell and epithelial monolayer is a prerequisite for tumor cell invasion. When human melanoma cells were seeded directly (i.e., physical contact) on top of an electrically tight epithelial cell layer (5,800 ± 106 Ω · cm2), electrical monolayer leakage led to an 18 ± 3% reduction of transepithelial electrical resistance within 24 h. However, when melanoma cells were seeded close to the basolateral surface of the epithelial cell monolayer but separated by a filter membrane (i.e., no physical contact), electrical leakage occurred even more quickly (42 ± 3% reduction in 24 h). Atomic force microscopy detected discrete structural changes between cells. Electrical leakage was effectively blocked by α2-macroglobulin or ilomastat, inhibitors of matrix metalloproteinases. We conclude that exocytosis of soluble proteases causes electrical breakdown of the MDCK monolayer, independently of physical contact between tumor cells and the monolayer.
Moderate alcohol intake has no impact on acute and chronic progressive anti-thy1 glomerulonephritis
Authors: Peters Harm , Martini Sebastian , Woydt Raiko , Rückert Matthias , Shimizu Fuijo , Kawachi Hiroshi , Liefeldt Lutz , Krämer Stephanie , Neumayer Hans-H.
Published: March 2015

Moderate alcohol consumption has shown beneficial effects in experimental and human cardiovascular disease. With the use of rat models of acute and chronic progressive anti-thy1 glomerulonephritis (GN), we tested the hypothesis that moderate alcohol intake is protective in renal fibrotic disease. In acute anti-thy1 GN, untreated nephritic rats showed marked mesangial cell lysis and induced nitric oxide production at day 1 and high proteinuria, glomerular matrix accumulation, and transforming growth factor (TGF)-β1, fibronectin, and plasminogen activator inhibitor (PAI)-1 expression at day 7 after disease induction, respectively. In animals 15 wk after induction of chronic progressive anti-thy1 GN, disease was characterized by significantly reduced renal function, persisting albuminuria as well as increased glomerular and tubulointerstitial matrix expansion, TGF-β1, fibronectin, and PAI-1 protein expression. In both anti-thy1 GN models, an ethanol intake of ∼2 ml per day and animal was achieved, however, disease severity was not significantly altered by moderate alcohol consumption in any of the protocols. In conclusion, moderate alcohol intake does not influence renal matrix protein production and accumulation in acute and chronic progressive anti-thy1 glomerulofibrosis. The study suggests that, in contrast to cardiovascular disorders, moderate alcohol consumption might not provide specific protection in renal fibrotic disease.
A dual-pathway ultrastructural model for the tight junction of rat proximal tubule epithelium
Authors: Guo Peng , Weinstein Alan M. , Weinbaum Sheldon
Published: March 2015

A dual-pathway model is proposed for transport across the tight junction (TJ) in rat proximal tubule: large slit breaks formed by infrequent discontinuities in the TJ complex and numerous small circular pores, with spacing similar to that of claudin-2. This dual-pathway model is developed in the context of a proximal tubule model (Weinstein AM. Am J Physiol Renal Fluid Electrolyte Physiol 247: F848–F862, 1984) to provide an ultrastructural view of solute and water fluxes. Tubule model paramters (TJ reflection coefficient and water permeability), plus the measured epithelial NaCl and sucrose permeabilities, provide constraints for the dual-pathway model, which yields the small-pore radius and spacing and large slit height and area. For a small-pore spacing of 20.2 nm, comparable to the distance between adjacent particle pairs in apposing TJ strands, the small-pore radius is 0.668 nm and the large slit breaks have a height of 19.6 nm, occupying 0.04% of the total TJ length. This pore/slit geometry also satisfies the measured permeability for mannitol. The numerous small circular pores account for 91.25% of TJ NaCl permeability but only 5.0% of TJ water permeability. The infrequent large slit breaks in the TJ account for 95.0% of TJ water permeability but only 8.7% of TJ NaCl permeability. Sucrose and mannitol (4.6- and 3.6-Å radius) can pass through both the large slit breaks and the small pores. For sucrose, 78.3% of the flux is via the slits and 21.7% via the pores; for mannitol, the flux is split nearly evenly between the two pathways, 50.8 and 49.2%. In this ultrastructural model, the TJ water permeability is 21.2% of the entire transepithelial water permeability and thus an order of magnitude greater than that predicted by the single-pore/slit theory (Preisig PA and Berry CA. Am J Physiol Renal Fluid Electrolyte Physiol 249: F124–F131, 1985).