bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandins buffer ANG II-mediated increases in cytosolic calcium in preglomerular VSMC
Authors: Purdy Kit E. , Arendshorst William J.
Published: December 2017

In order to exert an appropriate biological effect, the action of the vasoconstrictive hormone angiotensin II (ANG II) is modulated by vasoactive factors such as prostaglandins PGE2and PGI2. The present study investigates whether prostaglandins alter ANG II-mediated increases in cytosolic calcium concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) isolated from rat renal preglomerular arterioles. [Ca2+]iwas assessed using the calcium-sensitive dye fura 2 and a microscope-based photometer system. ANG II (10−7M) caused a biphasic, time-dependent [Ca2+]iresponse: an initial peak increase from 52 ± 7 to 264 ± 25 nM, followed by a sustained plateau of 95 ± 9 nM in cultured VSMC. Coadministration of PGE2or PGI2or synthetic mimetics caused dose-dependent decreases in the peak [Ca2+]iresponse to ANG II, with attenuation of 40–50%. This degree of inhibition was even more pronounced in individual freshly isolated preglomerular VSMC. Increasing cAMP levels in cultured VSMC, by using either a cell-permeable analog or inhibiting phosphodiesterase activity, mirrored the antagonistic effects of prostaglandins on ANG II-stimulated increases in [Ca2+]i. Radioimmunoassays demonstrate that ANG II (10−7M) stimulates production of PGI2and PGE2; the stable prostacyclin metabolite 6-keto-PGF1αwas released in 10-fold greater concentrations than PGE2.Indomethacin blockade of prostaglandin production potentiated both the peak (264 to 337 ± 26 nM) and sustained [Ca2+]iresponses (95 to 181 ± 22 nM) to ANG II. When prostaglandin analogs were added during indomethacin treatment, the ANG II response was restored to the typical pattern. In conclusion, we demonstrate that modulation of intracellular calcium levels is one mechanism by which prostaglandins can buffer ANG II-mediated constriction in renal preglomerular VSMC. PGI2is more potent than PGE2in this regard.
Afferent arteriolar adenosine A2a receptors are coupled to KATP in in vitro perfused hydronephrotic rat kidney
Authors: Tang Lilong , Parker Michael , Fei Qing , Loutzenhiser Rodger
Published: December 2017

Adenosine is known to exert dual actions on the afferent arteriole, eliciting vasoconstriction, by activating A1 receptors, and vasodilation at higher concentrations, by activating lower-affinity A2 receptors. We could demonstrate both of these known adenosine responses in the in vitro perfused hydronephrotic rat kidney. Thus, 1.0 μM adenosine elicited a transient vasoconstriction blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and 10–30 μM adenosine reversed KCl-induced vasoconstriction. However, when we examined the effects of adenosine on pressure-induced afferent arteriolar vasoconstriction, we observed a third action. In this setting, a high-affinity adenosine vasodilatory response was observed at concentrations of 10–300 nM. This response was blocked by both 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM-241385) and glibenclamide and was mimicked by 2-phenylaminoadenosine (CV-1808) (IC50 of 100 nM), implicating adenosine A2a receptors coupled to ATP-sensitive K channels (KATP). Like adenosine, 5′- N-ethylcarboxamidoadenosine (NECA) elicited both glibenclamide-sensitive and glibenclamide-insensitive vasodilatory responses. The order of potency for the glibenclamide-sensitive component was NECA > adenosine = CV-1808. Our findings suggest that, in addition to the previously described adenosine A1 and low-affinity A2b receptors, the renal microvasculature is also capable of expressing high-affinity adenosine A2areceptors. This renal adenosine receptor elicits afferent arteriolar vasodilation at submicromolar adenosine levels by activating KATP.
Expression of peroxisomal proliferator-activated receptors and retinoid X receptors in the kidney
Authors: Yang Tianxin , Michele Daniel E. , Park John , Smart Ann M. , Lin Zhiwu , Brosius Frank C. , Schnermann Jurgen B. , Briggs Josephine P.
Published: December 2017

The discovery that 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2) is a ligand for the γ-isoform of peroxisome proliferator-activated receptor (PPAR) suggests nuclear signaling by prostaglandins. Studies were undertaken to determine the nephron localization of PPAR isoforms and their heterodimer partners, retinoid X receptors (RXR), and to evaluate the function of this system in the kidney. PPARα mRNA, determined by RT-PCR, was found predominately in cortex and further localized to proximal convoluted tubule (PCT); PPARγ was abundant in renal inner medulla, localized to inner medullary collecting duct (IMCD) and renal medullary interstitial cells (RMIC); PPARβ, the ubiquitous form of PPAR, was abundant in all nephron segments examined. RXRα was localized to PCT and IMCD, whereas RXRβ was expressed in almost all nephron segments examined. mRNA expression of acyl-CoA synthase (ACS), a known PPAR target gene, was stimulated in renal cortex of rats fed with fenofibrate, but the expression was not significantly altered in either cortex or inner medulla of rats fed with troglitazone. In cultured RMIC cells, both troglitazone and 15d-PGJ2significantly inhibited cell proliferation and dramatically altered cell shape by induction of cell process formation. We conclude that PPAR and RXR isoforms are expressed in a nephron segment-specific manner, suggesting distinct functions, with PPARα being involved in energy metabolism through regulating ACS in PCT and with PPARγ being involved in modulating RMIC growth and differentiation.
Regulation of renal epithelial cell affinity for calcium oxalate monohydrate crystals
Authors: Lieske John C. , Huang Erick , Toback F. Gary
Published: December 2017

The binding and internalization of calcium oxalate monohydrate (COM) crystals by tubular epithelial cells may be a critical step leading to kidney stone formation. Exposure of MDCK cells to arachidonic acid (AA) for 3 days, but not oleic or linoleic acid, decreased COM crystal adhesion by 55%. Exogenous prostaglandin PGE1or PGE2decreased crystal binding 96% within 8 h, as did other agents that raise intracellular cAMP. Actinomycin D, cycloheximide, or tunicamycin each blocked the action of PGE2, suggesting that gene transcription, protein synthesis, and N-glycosylation were required. Blockade of crystal binding by AA was not prevented by the cyclooxygenase inhibitor flurbiprofen, and was mimicked by the nonmetabolizable AA analog eicosatetryanoic acid (ETYA), suggesting that generation of PGE from AA is not the pathway by which AA exerts its effect. These studies provide new evidence that binding of COM crystals to renal cells is regulated by physiological signals that could modify exposure of cell surface molecules to which the crystals bind. Intrarenal AA, PGs, and/or other agents that raise the intracellular concentration of cAMP may serve a protective function by preventing crystal adhesion along the nephron, thereby defending the kidney against crystal retention and stone formation.
α1-Adrenoceptor subtypes in rat renal resistance vessels: in vivo and in vitro studies
Authors: Salomonsson Max , Brännström Kristina , Arendshorst William J.
Published: December 2017

This study provides new information about the relative importance of different α1-adrenoceptors during norepinephrine (NE) activation in rat renal resistance vessels. In Sprague-Dawley rats, we measured renal blood flow (RBF) using electromagnetic flowmetry in vivo and the intracellular free calcium concentration ([Ca2+]i) utilizing ratiometric photometry of fura 2 fluorescence in isolated afferent arterioles. Renal arterial bolus injection of NE produced a transient 46% decrease in RBF. In microdissected afferent arterioles, NE (1 μM) elicited an immediate square-shaped increase in [Ca2+]i, from 90 to 175 nM ( P < 0.001). Chloroethylclonidine (CEC) (50 μM) had no chronic irreversible alkylating effect in vitro but exerted acute reversible blockade on norepinephrine (NE) responses both on [Ca2+]iin vitro and on RBF in vivo. The RBF response was attenuated by ∼50% by the putative α1A-adrenoceptor and α1D-adrenoceptor antagonists 5-methylurapidil (5-MU), and 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) (12.5 and 62.5 μg/h), respectively. The in vitro [Ca2+]iresponse to NE was blocked ∼25% and 50% by 5-MU (100 nM and 1 μM). BMY-7378 (100 nM and 1 μM) attenuated the NE-induced response by ∼40% and 100%. The degree of inhibition in vitro was similar to the in vivo experiments. In conclusion, 5-MU and BMY-7378 attenuated the NE-induced responses, although relatively high concentrations were required, suggesting involvement of both the α1A-adrenoceptor and α1D-adrenoceptor. Participation of the α1B-adrenoceptor is less likely, as we found no evidence for CEC-induced alkylation.
Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment
Authors: Christensen Birgitte Mønster , Zelenina Marina , Aperia Anita , Nielsen Søren
Published: December 2017

Phosphorylation of Ser256, in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser256were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys1,d-Arg8]vasopressin (DDAVP) treatment or V2-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V2-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser256is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V2receptors by altering phosphorylation and/or dephosphorylation of Ser256in AQP2.
Abnormal water metabolism in mice lacking the type 1A receptor for ANG II
Authors: Oliverio Michael I. , Delnomdedieu Marielle , Best Christopher F. , Li Ping , Morris Mariana , Callahan Michael F. , Johnson G. Allan , Smithies Oliver , Coffman Thomas M.
Published: December 2017

Mice lacking AT1Areceptors for ANG II have a defect in urinary concentration manifested by an inability to increase urinary osmolality to levels seen in controls after thirsting. This defect results in extreme serum hypertonicity during water deprivation. In the basal state, plasma vasopressin levels are similar in wild-type controls and Agtr1a −/− mice. Plasma vasopressin levels increase normally in the AT1Areceptor-deficient mice after 24 h of water deprivation, suggesting that the defect in urine concentration is intrinsic to the kidney. Using magnetic resonance microscopy, we find that the absence of AT1Areceptors is associated with a modest reduction in the distance from the kidney surface to the tip of the papilla. However, this structural abnormality seems to play little role in the urinary concentrating defect in Agtr1a −/− mice since the impairment is largely reproduced in wild-type mice by treatment with an AT1-receptor antagonist. These studies demonstrate a critical role for the AT1Areceptor in maintaining inner medullary structures in the kidney and in regulating renal water excretion.
Dihydropyridine-sensitive Ca2+channels in human glomerular mesangial cells
Authors: Hall David A. , Carmines Pamela K. , Sansom Steven C.
Published: December 2017

In mesangial cells (MC), the response of intracellular Ca2+concentration ([Ca2+]i) to a contractile agonist is biphasic with a large, transient increase in [Ca2+]ifollowed by a smaller but sustained elevation as Ca2+flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca2+channels in the plasmalemmal membrane. However, a role for voltage-gated Ca2+channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca2+entry pathway responsible for the sustained response of [Ca2+]iin human MC. We found that ANG II at 10 nM, 100 nM, and 1 μM increased [Ca2+]ito sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 μM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca2+. None of the peak responses (due to release of intracellular stores of Ca2+) were affected by removal of external Ca2+or addition of diltiazem. Upon elevating the extracellular [K+] from 5 mM to 75 mM, [Ca2+]ireached a sustained level of 48% greater than baseline. This effect of high K+was attenuated by either Ca2+removal or addition of diltiazem. In the presence of 75 or 140 mM K+, the dihydropyridine agonist BAY K 8644 (1 μM and 10 μM) initiated sustained [Ca2+]iresponses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca2+in the external solution, BAY K 8644 did not significantly affect [Ca2+]i. In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl2and 10 μM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between −30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca2+channels responsible for the voltage-regulated entry of Ca2+into the cell during an agonist-induced contraction.
Roles of the podocyte in glomerular function
Authors: Pavenstädt Hermann
Published: December 2017

The podocyte is the most differentiated cell type in the glomerulum, which forms a crucial component of the glomerular filtration barrier. It has been assumed that podocyte foot processes counteract the elastic force of the glomerular basement membrane and that vasoactive hormones may regulate the contractile state of their foot processes and thereby modulate the ultrafiltration coefficient Kf. Podocyte damage leads to proteinuria, and podocyte injury occurs in many glomerular diseases, which may progress to chronic renal failure. The understanding of the regulation of physiological properties of the podocyte and the mechanisms of its cellular response to injury may thus provide a clue to the understanding of the pathogenesis of proteinuria and glomerular diseases. In the past it was difficult to study cellular functions in this cell type, because of its unique anatomic location and the difficulty in characterizing podocytes in cell culture. However, recent advances in physiological, molecular biological, and cell culture techniques have increased the knowledge of the role of the podocyte in glomerular function. The present review attempts to outline new aspects of podocyte function in the glomerulum.
Upregulation of endothelin B receptors in kidneys of DOCA-salt hypertensive rats
Authors: Pollock David M. , Allcock Graham H. , Krishnan Arthi , Dayton Brian D. , Pollock Jennifer S.
Published: December 2017

Experiments were designed to elucidate the role of endothelin B receptors (ETB) on arterial pressure and renal function in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Male Sprague-Dawley rats underwent uninephrectomy and were treated with either DOCA and salt (0.9% NaCl to drink) or placebo. DOCA-salt rats given the ETB-selective antagonist, A-192621, for 1 wk (10 mg ⋅ kg−1⋅ day−1in the food) had significantly greater systolic arterial pressure compared with untreated DOCA-salt rats (208 ± 7 vs. 182 ± 4 mmHg) whereas pressure in placebo rats was unchanged. In DOCA-salt, but not placebo rats, A-192621 significantly decreased sodium and water excretion along with parallel decreases in food and water intake. To determine whether the response in DOCA-salt rats was due to increased expression of ETBreceptors, endothelin receptor binding was performed by using membranes from renal medulla. Maximum binding (Bmax) of [125I]ET-1, [125I]ET-3, and [125I]IRL-1620 increased from 227 ± 42, 146 ± 28, and 21 ± 1 fmol/mg protein, respectively, in placebo rats to 335 ± 27, 300 ± 38, and 61 ± 6 fmol/mg protein, respectively, in DOCA-salt hypertensive rats. The fraction of receptors that are the ETBsubtype was significantly increased in DOCA-salt (0.88 ± 0.07) compared with placebo (0.64 ± 0.01). The difference between [125I]ET-3 and [125I]IRL-1620 binding is consistent with possible ETBreceptor subtypes in the kidney. These results indicate that ETBreceptors in the renal medulla are up-regulated in the DOCA-salt hypertensive rat and may serve to maintain a lower arterial pressure by promoting salt and water excretion.
Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitivetrans-Golgi-associated compartment
Authors: Gustafson Corinne E. , Katsura Toshiya , McKee Mary , Bouley Richard , Casanova James E. , Brown Dennis
Published: December 2017

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK1cells even in the absence of hormonal stimulation. Incubating cells at 20°C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H+-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20°C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.
Substance P dependence of endosomal fusion during bladder inflammation
Authors: Hammond T. G. , Saban R. , Bost K. L. , Harris H. W. , Kaysen J. H. , Goda F. O. , Wang X.-C. , Lewis Fawn C. , Navar G. L. , Campbell W. C. , Bjorling D. E. , Saban M. , Zeidel M. L.
Published: December 2017

Urinary bladder instillation of ovalbumin into presensitized guinea pigs stimulates rapid development of local bladder inflammation. Substance P is an important mediator of this inflammatory response, as substance P antagonists largely reverse the process. Vacuolization of the subapical endosomal compartment of the transitional epithelial cells lining the bladder suggests that changes in endosomal trafficking and fusion are also part of the inflammatory response. To test directly for substance P mediation of changes in endosomal fusion, we reconstituted fusion of transitional cell endosomes in vitro using both cuvette-based and flow cytometry energy transfer assays. Bladders were loaded with fluorescent dyes by a hypotonic withdrawal protocol before endosomal isolation by gradient centrifugation. Endosomal fusion assayed by energy transfer during in vitro reconstitution was both cytosol and ATP dependent. Fusion was confirmed by the increase in vesicle size on electron micrographs of fused endosomal preparations compared with controls. In inflamed bladders, dye uptake was inhibited 20% and endosomal fusion was inhibited 50%. These changes are partly mediated by the neurokinin-1 (NK1) receptor (NK1R), as 4 mg/kg of CP-96,345, a highly selective NK1 antagonist, increased fusion in inflamed bladders but had no effect on control bladders. The receptor-mediated nature of this effect was demonstrated by the expression of substance P receptor mRNA in rat bladder lumen scrapings and by the detection of the NK1R message in guinea pig subapical endosomes by Western blot analysis. The NK1Rs were significantly upregulated following induction of an inflammatory response in the bladder. These results demonstrate that 1) in ovalbumin-induced inflammation in the guinea pig bladder, in vitro fusion of apical endosomes is inhibited, showing endocytotic processes are altered in inflammation; 2) pretreatment in vivo with an NK1R antagonist blocks this inhibition of in vitro fusion, demonstrating a role for NK1R in this process; and 3) the NK1R is present in higher amounts in apical endosomes of inflamed bladder, suggesting changes in translation or trafficking of the NK1R during the inflammatory process. This suggests that NK1R can change the fusion properties of membranes in which it resides.
Surviving rat distal tubule bicarbonate reabsorption: effects of chronic AT1blockade
Authors: Levine David Z. , Iacovitti Michelle , Luck Brian , Hincke Maxwell T. , Burns Kevin D. , Fryer James N. ,   (With the Technical Assistance of A. Slater)
Published: December 2017

To determine the in vivo effects of chronic ANG II type 1 (AT1)-receptor blockade by losartan (Los) on enhanced unidirectional bicarbonate reabsorption ( JHCO3) of surviving distal tubules, nephrectomized rats drank either water or a solution of Los, 7 days before microperfusion. JHCO3was suppressed by 50% after Los without further reduction by 5 nM concanamycin A (Conc), suggesting that Los suppresses all Conc-sensitive H+-ATPase pumping. Indeed, ultrastructural analysis of A-type intercalated cells revealed a 50% reduction of H+-ATPase immunogold labeling of the apical plasma membrane, whereas Western blotting showed that H+-ATPase protein levels were also reduced by one-half by Los treatment. To identify other transporters sustaining JHCO3, we perfused three inhibitors simultaneously [5-( N, N-dimethyl) amiloride hydrochloride, Conc, Schering 28080] with or without prior Los treatment: JHCO3was unchanged despite marked reduction of water reabsorption. We conclude enhanced distal tubule JHCO3of surviving nephrons is largely mediated by AT1receptor-dependent synthesis and insertion of apical H+-ATPase pumps in A-type intercalated cells.
Growth factor ultrafiltration in experimental diabetic nephropathy contributes to interstitial fibrosis
Authors: Wang Shi-Nong , Hirschberg Raimund
Published: December 2017

Glomerular proteinuria is a risk factor for progression of chronic renal failure and contributes to renal interstitial fibrosis. In experimental diabetic glomerular sclerosis, there is translocation of high-molecular-weight growth factors, namely, hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β, from plasma into tubular fluid, both of which act on tubular cells through apical membrane receptors. In the present studies, the hypothesis is examined that ultrafiltered HGF and TGF-β induce increased expression of extracellular matrix (ECM) proteins directly in tubular cells, or induce increased expression of cytokines that may act on interstitial myofibroblasts. Incubation of cultured tubular cells with recombinant human (rh) TGF-β modestly raises expression of collagen type III, but rhHGF dose dependently blocks expression of this ECM protein. Both growth factors raise fibronectin expression up to fourfold and increase expression of platelet-derived growth factor (PDGF)-BB up to sixfold, but not of fibroblast growth factor-2. Pooled, diluted glomerular ultrafiltrate that had been collected by nephron micropuncture from rats with diabetic nephropathy (24–30 wk) also raises expression of fibronectin as well as PDGF-BB in proximal tubular cells. In the presence of neutralizing antibodies that block actions of HGF and TGF-β, diabetic rat glomerular ultrafiltrate fails to increase tubular cell PDGF-BB expression. In NRK-49F renal interstitial myofibroblasts, rhPDGF-BB, in turn, raises the expression of collagen type III but not type I or fibronectin. The findings provide evidence for ultrafiltered HGF and TGF-β to contribute to interstitial accumulation of ECM proteins by direct effects on tubular cells as well as indirect mechanisms, via PDGF-BB and its action on myofibroblasts. These events may be important mechanisms of proteinuria-induced renal interstitial fibrosis and accelerated progression of chronic renal failure in diabetic nephropathy and perhaps other proteinuric glomerular diseases.
Effects of Ca2+channel activity on renal hemodynamics during acute attenuation of NO synthesis in the rat
Authors: Kramp R. A. , Fourmanoir P. , Ladrière L. , Joly E. , Gerbaux C. , el Hajjam A. , Caron N.
Published: December 2017

In cultured vascular muscle cells, nitric oxide (NO) has been shown to inhibit voltage-dependent Ca2+channels, which are involved in renal blood flow (RBF) autoregulation. Therefore, our purpose was to specify in vivo the effects of this interaction on RBF autoregulation. To do so, hemodynamics were investigated in anesthetized rats during Ca2+channel blockade before or after acute NO synthesis inhibition. Rats were treated intravenously with vehicle ( n = 10), 0.3 mg/kg body wt NG-nitro-l-arginine-methyl ester (l-NAME; n = 7), 4.5 μg ⋅ kg body wt−1⋅ min−1nifedipine ( n = 8) alone, or with nifedipine infused before ( n = 8), after ( n = 8), or coadministered with l-NAME ( n = 10). Baseline renal vascular resistance (RVR) averaged 14.0 ± 1.2 resistance units and did not change after vehicle. RVR increased or decreased significantly by 27 and 29% afterl-NAME or nifedipine, respectively. Nifedipine reversed, but did not prevent, RVR increase after or coadministered withl-NAME. RBF autoregulation was maintained afterl-NAME, but the autoregulatory pressure limit (PA) was significantly lowered by 15 mmHg. Nifedipine pretreatment or coadministration with l-NAME limited PAresetting or suppressed autoregulation at higher doses. Results were similar with verapamil. Intrarenal blockade of Ca2+-activated K+channels also prevented autoregulatory resetting by l-NAME ( n = 8). These findings suggest NO inhibits voltage-dependent Ca2+channels and thereby modulates RBF autoregulatory efficiency.
Developmentally regulated expression of organic ion transporters NKT (OAT1), OCT1, NLT (OAT2), and Roct
Authors: Pavlova Anna , Sakurai Hiroyuki , Leclercq Baudouin , Beier David R. , Yu Alan S. L. , Nigam Sanjay K.
Published: December 2017

Several xenobiotic (organic cation and anion) transporters have recently been identified, although their endogenous substrates, if such exist, remain unknown. When we initially identified NKT, also known as OAT1, the first member of the organic anion transporter (OAT) family (Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, and Nigam SK. J Biol Chem 272: 6471–6478, 1997), we noted its expression in the embryonic kidney. We have now demonstrated its transporter function and more fully examined the spatiotemporal expression patterns of representative organic ion transporters, [NKT (OAT1), Roct, OCT1, and NLT, also known as OAT2] during murine development. In the kidney, NKT (OAT1), OCT1, and Roct transcripts appeared at midgestation, coinciding with proximal tubule differentiation, and gradually increased during nephron maturation. A similar pattern was observed for NLT (OAT2) in the liver and kidney, although, in the kidney, NLT (OAT2) transcription did not increase as dramatically. The roughly cotemporal expression of these related transporters in the developing proximal tubule may indicate common transcriptional regulation. Expression during embryogenesis in extrarenal sites could suggest a role in the formation and maintenance of nonrenal tissues. Importantly, all four genes were expressed in unexpected places during nonrenal organogenesis: Roct in the fetal liver (temporally coinciding with the onset of hematopoiesis) and neural tissue; NKT (OAT1) in the fetal brain; OCT1 in the ascending aorta and atrium; and NLT (OAT2) in the fetal lung, intestine, skin, and developing bone. Because these gene products mediate the transport of a broad range of metabolites and toxins, it seems likely that, apart from their known functions, these transporters play a role in transport of organic molecules, perhaps including those with morphogenetic activity. These genes could also play important developmental roles independent of transport function.
Localization of protein inhibitor of neuronal nitric oxide synthase in rat kidney
Authors: Roczniak Agnes , Levine David Z. , Burns Kevin D.
Published: December 2017

We have recently demonstrated that in rats with 5/6 nephrectomy (5/6 Nx), renal cortical and inner medullary neuronal NOS (nNOS) expression is downregulated, associated with decreased urinary excretion of nitric oxide (NO) products. Recently, a novel 89-amino acid protein [protein inhibitor of nNOS (PIN)] was isolated from rat brain and shown to inhibit nNOS activity. The present studies localized PIN in the rat kidney and determined the effect of 5/6 Nx on PIN expression. By RT-PCR, PIN mRNA was detected in the kidney cortex and inner medulla. Immunohistochemistry revealed staining for PIN in glomerular and vasa rectae endothelial cells. PIN was also localized to the apical membranes of inner medullary collecting duct (IMCD) cells. Two weeks after 5/6 Nx, inner medullary PIN expression was significantly upregulated (sham, 0.18 ± 0.07 vs. 5/6 Nx, 0.58 ± 0.13 arbitrary units; n = 6, P < 0.02), as determined by Western blotting. In summary, our data show that PIN, a specific inhibitor of nNOS activity, is expressed in the IMCD, a site of high nNOS expression in the kidney. PIN expression is upregulated in the inner medulla of 5/6 Nx rats. Inhibition of nNOS activity by PIN may have important implications for the regulation of NO synthesis in the IMCD of normal and remnant kidneys.
Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract
Authors: Breton Sylvie , Nsumu Ndona N. , Galli Thierry , Sabolic Ivan , Smith Peter J. S. , Brown Dennis
Published: December 2017

Our laboratory has previously shown that the vacuolar H+-ATPase, located in a subpopulation of specialized cells establishes a luminal acidic environment in the epididymis and proximal part of the vas deferens (Breton S, Smith PJS, Lui B, and Brown D. Nat Med 2: 470–472, 1996). Low luminal pH is critical for sperm maturation and maintenance of sperm in a quiescent state during storage in these organs. In the present study we examined the regulation of proton secretion in the epididymis and vas deferens. In vivo microtubule disruption by colchicine induced an almost complete loss of H+-ATPase apical polarity. Endocytotic vesicles, visualized by Texas red-dextran internalization, contain H+-ATPase, indicating active endocytosis of the pump. Cellubrevin, an analog of the vesicle soluble N-ethyl malemide-sensitive factor attachment protein (SNAP) receptor (v-SNARE) synaptobrevin, is highly enriched in H+-ATPase-rich cells of the epididymis and vas deferens, and tetanus toxin treatment markedly inhibited bafilomycin-sensitive proton secretion by 64.3 ± 9.0% in the proximal vas deferens. Western blotting showed effective cleavage of cellubrevin by tetanus toxin in intact vas deferens, demonstrating that the toxin gained access to cellubrevin. These results suggest that H+-ATPase is actively endocytosed and exocytosed in proton-secreting cells of the epididymis and vas deferens and that net proton secretion requires the participation of the v-SNARE cellubrevin.
C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis
Authors: Hughes Jeremy , Nangaku Masaomi , Alpers Charles E. , Shankland Stuart J. , Couser William G. , Johnson Richard J.
Published: December 2017

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6−), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6− rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.
Water transport by the renal Na+-dicarboxylate cotransporter
Authors: Meinild Anne-Kristine , Loo Donald D. F. , Pajor Ana M. , Zeuthen Thomas , Wright Ernest M.
Published: December 2017

This study investigated the ability of the renal Na+-dicarboxylate cotransporter, NaDC-1, to transport water. Rabbit NaDC-1 was expressed in Xenopus laevis oocytes, cotransporter activity was measured as the inward current generated by substrate (citrate or succinate), and water transport was monitored by the changes in oocyte volume. In the absence of substrates, oocytes expressing NaDC-1 showed an increase in osmotic water permeability, which was directly correlated with the expression level of NaDC-1. When NaDC-1 was transporting substrates, there was a concomitant increase in oocyte volume. This solute-coupled influx of water took place in the absence of, and even against, osmotic gradients. There was a strict stoichiometric relationship between Na+, substrate, and water transport of 3 Na+, 1 dicarboxylate, and 176 water molecules/transport cycle. These results indicate that the renal Na+-dicarboxylate cotransporter mediates water transport and, under physiological conditions, may contribute to fluid reabsorption across the proximal tubule.
Stimulation of TGF-β type II receptor by high glucose in mouse mesangial cells and in diabetic kidney
Authors: Isono Motohide , Mogyorósi András , Han Dong Cheol , Hoffman Brenda B. , Ziyadeh Fuad N.
Published: December 2017

Transforming growth factor-β (TGF-β) is important in the pathogenesis of diabetic nephropathy, but little is known about the regulation of the ligand-binding TGF-β type II signaling receptor (TβIIR). There were significant increases in TβIIR protein and mRNA levels in kidney cortex after 1–6 wk of streptozotocin-induced diabetes. Mouse mesangial cells cultured in high glucose demonstrated significantly increased TβIIR protein and mRNA levels compared with normal glucose. This effect was independent of stimulation of TGF-β bioactivity by high glucose. Consistent with transcriptional activation by high glucose, the half-life (∼4 h) of TβIIR mRNA was not affected by glucose concentration. Moreover, mouse mesangial cells transiently transfected with reporter constructs containing the first 47- or 274-bp promoter fragments of TβIIR demonstrated significantly increased reporter activity in high glucose. Cells grown in high glucose demonstrated increased responsiveness to a relatively small dose of exogenous TGF-β1(0.5 ng/ml): [3H]proline incorporation and α1(IV) collagen mRNA were significantly greater in cells cultured in high than in normal glucose. Hence, the expression of TβIIR is increased in the diabetic kidney and in mesangial cells cultured in high glucose, primarily because of stimulation of gene transcription. TβIIR upregulation by high ambient glucose may contribute to the increased sensitivity of mesangial cells to the profibrogenic action of TGF-β1.
Onset of glomerular hypertension with aging precedes injury in the spontaneously hypertensive rat
Authors: Tolbert Evelyn M. , Weisstuch Joseph , Feiner Helen D. , Dworkin Lance D.
Published: December 2017

The changes in renal hemodynamics that develop with aging in spontaneously hypertensive rats (SHR) were examined. Micropuncture studies revealed that glomerular capillary pressure was elevated in SHR at 9 mo of age compared with 3-mo-old SHR and 9-mo-old normotensive Wistar-Kyoto rats. Glomerular hypertension developed because of a small increase in systemic blood pressure and a decline in preglomerular vascular resistance, allowing transmission of elevated systemic pressure to the glomerular capillaries. The hemodynamic alterations were not a compensatory response to injury, inasmuch as vascular and glomerular morphology were normal in 9-mo-old SHR. To determine the mechanism of these changes, the activity of several vasoactive systems was examined. Similar changes in renal hemodynamics were observed in young and old SHR after blockade of nitric oxide production and after intravenous administration of endothelin. However, ANG II produced a proportionally greater reduction in glomerular filtration rate than renal blood flow in older SHR. These data suggest that reduced endogenous activity of the renin-angiotensin system leads to glomerular hypertension in aging SHR. Late development of glomerular hypertension may contribute to the subsequent appearance of glomerular sclerosis and progressive renal failure in these rats.
Luminal hypotonicity in proximal tubules of aquaporin-1-knockout mice
Authors: Vallon V. , Verkman A. S. , Schnermann J.
Published: December 2017

To examine the role of aquaporin-1 (AQP1) in near-isosmolar fluid reabsorption in the proximal tubule, we compared osmolalities in micropuncture samples of late proximal tubular fluid and plasma in wild-type (+/+) and AQP1-knockout (−/−) mice. Compared with matched wild-type mice, the −/− animals produce a relatively hypotonic urine (607 ± 42 vs. 1,856 ± 101 mosmol/kgH2O) and have a higher plasma osmolality under micropuncture conditions (346 ± 11 vs. 318 ± 5 mosmol/kgH2O; P < 0.05). Measurements of tubular fluid osmolality were done in three groups of mice, +/+, −/−, and hydrated −/− mice in which plasma osmolality was reduced to 323 ± 1 mosmol/kgH2O. Late proximal tubular fluid osmolalities were 309 ± 5 (+/+, n= 21), 309 ± 4 (−/−, n = 24), and 284 ± 3 mosmol/kgH2O (hydrated −/−, n = 19). Tubular fluid chloride concentration averaged 152 ± 1 (+/+), 154 ± 1 (−/−), and 140 ± 1 mM (hydrated −/−). Transtubular osmotic gradients in untreated and hydrated AQP1 −/− mice were 39 ± 4 ( n = 25) and 39 ± 3 mosmol/kgH2O ( n = 19), values significantly higher than in +/+ mice (12 ± 2 mosmol/kgH2O; n = 24; both P < 0.001). AQP1 deficiency in mice generates marked luminal hypotonicity in proximal tubules, resulting from the retrieval of a hypertonic absorbate and indicating that near-isosmolar fluid absorption requires functional AQP1.
Potassium restriction downregulates ROMK expression in rat kidney
Authors: Mennitt Patricia A. , Frindt Gustavo , Silver Randi B. , Palmer Lawrence G.
Published: December 2017

The ROMK family of proteins has biophysical properties and distribution within the kidney similar to those of secretory potassium channels of the distal nephron. To study the regulation of ROMK during variations in dietary potassium, we measured the abundance of ROMK protein in rat kidney by immunoblotting. Neither 2 nor 5 days of a high-potassium diet had an effect on protein abundance in the cortex or medulla. Potassium deprivation (2 or 5 days) decreased ROMK protein content in both cortical and medullary fractions, to 51 and 40% of controls, respectively. To see whether the Na-K-2Cl cotransporter is similarly affected by potassium restriction, we analyzed immunoblots by using an antibody for the rat type 1 bumetanide-sensitive cotransporter (BSC-1). Like ROMK, BSC-1 protein content was found to decrease significantly in the renal medulla of potassium-deprived rats. In the thick ascending limb of Henle's loop, a decrease in ROMK and BSC-1 could result in decreased reabsorption of NaCl, a finding associated with hypokalemia.
Store-operated Ca2+channels in human glomerular mesangial cells
Authors: Ma Rong , Smith Sonja , Child Angie , Carmines Pamela K. , Sansom Steven C.
Published: December 2017

Experiments were performed to identify the biophysical properties of store-operated Ca2+channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([Ca2+]i) evoked by elevating external [Ca2+] from 10 nM to 1 mM (Δ[Ca2+]). Under control conditions, Δ[Ca2+] averaged 6 nM and was unaffected by elevating bath [K+]. After treatment with 1 μM thapsigargin to deplete the intracellular Ca2+store, the change in [Ca2+]i(Δ[Ca2+]th) averaged 147 ± 16 nM. In thapsigargin-treated MC studied under depolarizing conditions (75 mM bath K+), Δ[Ca2+]thwas 45 ± 7 nM. The Δ[Ca2+]thresponse of thapsigargin-treated cells was inhibited by La3+(IC50= 335 nM) but was unaffected by 5 μM Cd2+. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM Ba2+or Ca2+in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with Ba2+and 0.7 pS with Ca2+. The estimated selectivities were Ca2+> Ba2+>> K+. These channels were sensitive to 2 μM La3+, insensitive to 5 μM Cd2+, and voltage independent, with an average channel activity ( NPo) of 1.02 at command potential (− Vp) ranging from 0 to −80 mV. In summary, MC exhibited an electrogenic Ca2+influx pathway that is suggestive of Ca2+entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell Ca2+influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative Ca2+entry in response to depletion of the intracellular store.
Extracellular ATP increases [Ca2+]i in distal tubule cells. II. Activation of a Ca2+-dependent Cl− conductance
Authors: Rubera Isabelle , Tauc Michel , Bidet Michel , Verheecke-Mauze Catherine , De Renzis Guy , Poujeol Chantal , Cuiller Béatrice , Poujeol Philippe
Published: December 2017

We characterized Cl− conductance activated by extracellular ATP in an immortalized cell line derived from rabbit distal bright convoluted tubule (DC1). 125I− efflux experiments showed that ATP increased 125I− loss with an EC50 = 3 μM. Diphenylamine-2-carboxylate (10−3 M) and NPPB (10−4 M) abolished the125I− efflux. Preincubation with 10 μM 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-acetoxymethyl ester or 10−7 M thapsigargin inhibited the effect of ATP. Ionomycin (2 μM) increased125I− efflux with a time course similar to that of extracellular ATP, suggesting that the response is dependent on the intracellular Ca2+ concentration ([Ca2+]i). The ATP agonist potency order was ATP ≥ UTP > ATPγS. Suramin (500 μM) inhibited the ATP-induced 125I− efflux, consistent with P2 purinoceptors. 125I− effluxes from cells grown on permeable filters suggest that ATP induced an apical efflux that was mediated via apical P2 receptors. Whole cell experiments showed that ATP (100 μM) activated outwardly rectifying Cl− currents in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the involvement of P1 receptors. Ionomycin activated Cl−currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical P2Y2 receptors, and Ca2+ mobilization for apical Cl− conductance in a distal tubule cell line.
Prostaglandin E receptors and the kidney
Authors: Breyer Matthew D. , Breyer Richard M.
Published: December 2017

Prostaglandin E2is a major renal cyclooxygenase metabolite of arachidonate and interacts with four G protein-coupled E-prostanoid receptors designated EP1, EP2, EP3, and EP4. Through these receptors, PGE2modulates renal hemodynamics and salt and water excretion. The intrarenal distribution and function of EP receptors have been partially characterized, and each receptor has a distinct role. EP1expression predominates in the collecting duct where it inhibits Na+absorption, contributing to natriuresis. The EP2receptor regulates vascular reactivity, and EP2receptor-knockout mice have salt-sensitive hypertension. The EP3receptor is also expressed in vessels as well as in the thick ascending limb and collecting duct, where it antagonizes vasopressin-stimulated salt and water transport. EP4mRNA is expressed in the glomerulus and collecting duct and may regulate glomerular tone and renal renin release. The capacity of PGE2to bidirectionally modulate vascular tone and epithelial transport via constrictor EP1and EP3receptors vs. dilator EP2and EP4receptors allows PGE2to serve as a buffer, preventing excessive responses to physiological perturbations.
Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction
Authors: Hruska Keith A. , Guo Guangjie , Wozniak Magdalena , Martin Daniel , Miller Steven , Liapis Helen , Loveday Kenneth , Klahr Saulo , Sampath T. Kuber , Morrissey Jeremiah
Published: December 2017

Unilateral ureteral obstruction (UUO) is a model of renal injury characterized by progressive tubulointerstitial fibrosis and renal damage, while relatively sparing the glomerulus and not producing hypertension or abnormalities in lipid metabolism. Tubulointerstitial fibrosis is a major component of several kidney diseases associated with the progression to end-stage renal failure. Here we report that when a critical renal developmental morphogen, osteogenic protein-1 (OP-1; 100 or 300 μg/kg body wt), is administered at the time of UUO and every other day thereafter, interstitial inflammation and fibrogenesis are prevented, leading to preservation of renal function during the first 5 days after obstruction. Compared with angiotensin-converting enzyme inhibition with enalapril treatment, OP-1 was more effective in preventing tubulointerstitial fibrosis and in preserving renal function. The mechanism of OP-1- induced renal protection was associated with prevention of tubular atrophy, an effect not shared with enalapril, and was related to preservation of tubular epithelial integrity. OP-1 blocked the stimulation of epithelial cell apoptosis produced by UUO, which promoted maintenance of tubular epithelial integrity. OP-1 preserved renal blood flow (RBF) during UUO, but enalapril also stimulated RBF. Thus OP-1 treatment inhibited tubular epithelial disruption stimulated by the renal injury of UUO, preventing tubular atrophy and diminishing the activation of tubulointerstitial inflammation and fibrosis and preserving renal function.