bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of inhibition of myo-inositol transport on MDCK cells under hypertonic environment
Authors: Kitamura H. , Yamauchi A. , Nakanishi T. , Takamitsu Y. , Sugiura T. , Akagi A. , Moriyama T. , Horio M. , Imai E.
Published: December 2017

To investigate the role of myo-inositol under hypertonic conditions, we examined the effects of inhibition of myo-inositol transport in Madin-Darby canine kidney (MDCK) cells using an analog of myo-inositol, 2-O,C-methylene-myo-inositol (MMI). We first characterized the inhibitory effects of MMI on myo-inositol transport in MDCK cells. The Na+-dependent component of [3H] myo-inositol uptake was inhibited by MMI in a concentration-dependent manner, although MMI did not inhibit the activities of the betaine transporter and system A neutral amino acid transporter. We found decreased affinity for myo-inositol in the presence of MMI, whereas the maximal velocity (Vmax) of the transporter did not change. Thus MMI behaves as a competitive inhibitor of myo-inositol transport with a relatively high inhibition constant (K(i)) value (1.6 mM). Myo-inositol content in hypertonic MDCK cells was markedly reduced in the presence of 5 mM MMI, but MMI itself did not accumulate in these cells. The hypertonic cells began to detach in the presence of MMI 3 days after increasing medium osmolality, whereas MMI did not affect the cells in isotonic medium. We also examined the effects of MMI on colony-forming efficiency of MDCK cells. MMI decreased colony-forming efficiency in a concentration-dependent manner, and addition of myo-inositol returned the efficiency to the value without MMI. Addition of betaine also increased colony-forming efficiency in the presence of MMI. These results indicate that myo-inositol plays an important role in survival and growth under hypertonic environment.
Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells
Authors: Berl T. , Siriwardana G. , Ao L. , Butterfield L. M. , Heasley L. E.
Published: December 2017

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.
Physiological role of nitric oxide in regulation of renal function in humans
Authors: Haynes W. G. , Hand M. F. , Dockrell M. E. , Eadington D. W. , Lee M. R. , Hussein Z. , Benjamin N. , Webb D. J.
Published: December 2017

The physiological role of endogenous nitric oxide in regulation of renal function in humans is unclear. Eight healthy men received an inhibitor of nitric oxide synthase, N(G)-monomethyl-L-arginine (L-NMMA, 3 mg/kg), and saline placebo intravenously on two occasions. L-NMMA significantly increased mean arterial pressure (+7%) and total peripheral resistance (+36%). However, because renal plasma flow did not decrease significantly, the increase in renal vascular resistance (+21%) was significantly less than the increase in total peripheral resistance. Glomerular filtration rate (-19%), filtration fraction (-10%), urine flow rate (-18%), sodium (-28%), and free water excretion (-25%) all decreased significantly. Fractional distal, but not proximal, sodium reabsorption increased. L-NMMA also significantly decreased plasma nitrate and urinary excretion of nitrate and dopamine. There were no significant changes in plasma renin activity, plasma endothelin, and aldosterone or in platelet number and ex vivo aggregation. L-NMMA had a plasma half-life of 75 min. Basal generation of nitric oxide appears to contribute less to vascular tone in the kidney than systemically but may alter afferent arteriolar tone. Decreased fractional sodium excretion supports an important physiological role for nitric oxide in inhibition of tubular sodium reabsorption, possibly mediated by the renal dopaminergic system.
Branching patterns and autoregulatory responses of juxtamedullary afferent arterioles
Authors: Casellas D. , Bouriquet N. , Moore L. C.
Published: December 2017

The spatial organization of autoregulatory responses (AR) was assessed in couples of afferent arterioles (AA), either grouped as anatomic pairs or branched sequentially along the same arcuate arterial branch (ArcB). With blood-perfused juxtamedullary nephron (JMN) preparations, AR were elicited by raising blood perfusion pressure from 60 to 120 mmHg and quantified by videomicroscopy as pressure-induced constrictions. Paired AA had unequal lengths (long-to-short ratio, 1.9 +/- 0.1; n = 36); however, no statistical difference in AR was found between long and short AA at juxtaglomerular or early AA (EAA) sites. Sequentially branched AA had the same length heterogeneity as paired AA (proximal-to-distal AA length ratio, 2.0 +/- 0.2; n = 30). However, AR exhibited a significant axial gradient, being higher in distal than in proximal AA or ArcB sites. In both AA branching patterns, EAA and nearby sites of the feed arteries had similar AR. Hence, our results are consistent with hemodynamic coupling in paired JMN. Around branching sites, AR are spatially organized in a way consistent with electrotonic vascular coupling.
Membrane polarity in epithelial cells: protein sorting and establishment of polarized domains
Authors: Caplan M. J.
Published: December 2017

Epithelial cells go to great trouble to organize the subdomains of their plasma membranes. The apical and basolateral surfaces of polarized epithelia are equipped with markedly distinct populations of channels, carriers, and pumps. This anisotropy is an absolute prerequisite for vectorial solute and fluid transport. The physiological properties of an individual epithelial cell type are determined not only by its census of transport proteins but also by the manner in which these proteins are segregated between the apical and basolateral portions of the plasmalemma (Curr. Top. Membr. 39: 37-86, 1991). To achieve this asymmetry, an epithelial cell must be able to establish distinct surface domains, to target newly synthesized transport proteins to their appropriate sites of functional residence, and to retain them there following their delivery. Studies of the cellular pathways involved in generating and maintaining the polarized state have begun to illuminate an elegant network of cell biological specializations that may be involved not only in establishing the distributions of transport proteins but in dynamically regulating their function as well.
Putting the actin cytoskeleton into perspective: pathophysiology of ischemic alterations
Authors: Molitoris B. A.
Published: December 2017

The actin cytoskeleton plays an ever-increasingly understood role in mediating a myriad of processes necessary for cellular structure and function. New and exciting information regarding the dynamic aspects of the actin cytoskeleton and its intracellular regulation are unfolding at a rapid rate. Actin cytoskeletal-surface membrane interactions mediating such diverse cellular events as cell polarity, endocytosis, exocytosis, cell division, cellular migration, cell adhesion, signal transduction, and ion channel activity are part of an ever-growing list of cellular processes dependent on precise actin polarization and regulation of assembly and disassembly. The purpose of this review is to highlight recent advances in the understanding of actin cytoskeleton-mediated cellular processes, to provide a framework that interrelates the complex protein-protein interactions necessary for localization, regulation, and mediation of these essential cellular functions, and to outline the role of actin effector proteins in the pathophysiology of ischemic cell injury.
Distribution and developmentally regulated expression of murine polycystin
Authors: Geng L. , Segal Y. , Pavlova A. , Barros E. J. , Lohning C. , Lu W. , Nigam S. K. , Frischauf A. M. , Reeders S. T. , Zhou J.
Published: December 2017

PKD1, the gene that is mutated in approximately 85% of autosomal dominant polycystic kidney disease (ADPKD) cases in humans, has recently been identified (Eur. PKD Consortium. Cell 77: 881-894, 1994; also, erratum in Cell 78: 1994). The longest open-reading frame of PKD1 encodes polycystin, a novel approximately 460-kDa protein that contains a series of NH2-terminal adhesive domains (J. Hughes, C. J. Ward, B. Peral, R. Aspinwall, K. Clark, J. San Millan, V. Gamble, and P. C. Harris. Nat. Genet. 10: 151-160, 1995; and Int. PKD Consortium. Cell 81: 289–298, 1995) and several putative transmembrane segments. To extend studies of polycystin to an experimentally accessible animal, we have isolated a cDNA clone encoding the 3' end of Pkd1, the mouse homologue of PKD1, and raised a specific antibody to recombinant murine polycystin. This antibody was used to determine the subcellular localization and tissue distribution of the protein by Western analysis and immunocytochemistry. In the mouse, polycystin is an approximately 400-kDa molecule that is predominantly found in membrane fractions of tissue and cell extracts. It is expressed in many tissues including kidney, liver, pancreas, heart, intestine, lung, and brain. Renal expression, which is confined to tubular epithelia, is highest in late fetal and early neonatal life and drops 20-fold by the third postnatal week, maintaining this level into adulthood. Thus the temporal profile of polycystin expression coincides with kidney tubule differentiation and maturation.
Evidence for the distinct nature of F2-isoprostane receptors from those of thromboxane A2
Authors: Fukunaga M. , Yura T. , Grygorczyk R. , Badr K. F.
Published: December 2017

In rat glomeruli and mesangial cells, the thromboxane A2 (TxA2) mimetic, U-46,619, but not 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), reduced glomerular inulin space and increased inositol 1,4,5-trisphosphate production, effects abolished by SQ-29,548. In competitive binding studies using 8-iso-[3H]PGF2alpha or [3H]SQ-29,548, mesangial cells displayed TxA2 binding sites but not ones for 8-iso-PGF2alpha. In contrast, rat aortic smooth muscle cells possessed specific binding sites for both TxA2 and 8-iso-PGF2alpha and displayed functional responses to both agonists, such as time- and dose-dependent activation of mitogen-activated protein kinases. In these cells, the mean dissociation constant value for the isoprostane receptor was 31.8 +/- 5.7 nM. When human TxA2 receptor cDNA was expressed in Xenopus oocytes injected with the Ca2+-specific photoprotein, aequorin, 8-iso-PGF2alpha gave much weaker responses than U-46,619. These studies provide the first radioligand binding characteristics of the F2-isoprostane receptor and demonstrate its specific and heterologous cellular localization. These studies support the distinct nature and biological significance of isoprostane receptors and provide a tool for their further molecular characterization.
Angiotensin II responses in AT1A receptor-deficient mice: a role for AT1B receptors in blood pressure regulation
Authors: Oliverio M. I. , Best C. F. , Kim H. S. , Arendshorst W. J. , Smithies O. , Coffman T. M.
Published: December 2017

Most of the classic functions of the renin-angiotensin system are mediated by type 1 (AT1) angiotensin receptors, of which two subtypes, AT1A and AT1B, have been identified. However, distinct functions for these two AT1 receptors have been difficult to separate. We examined the pressor effects of angiotensin II in Agtr1A -/- mice, which lack AT1A receptors. In enalapril-pretreated Agtr1A -/- mice, angiotensin II caused significant and dose-proportional increases in mean arterial pressure. This pressor response was not blocked by pretreatment with sympatholytic agents but was completely inhibited by the AT1-receptor antagonists, losartan and candesartan, suggesting that it is directly mediated by AT1B receptors. Chronic treatment of Agtr1A -/- mice with losartan reduced systolic blood pressure from 80 +/- 5 to 72 +/- 4 mmHg (P < 0.04), suggesting a role for AT1B receptors in chronic blood pressure regulation. These studies provide the first demonstration of in vivo pressor effects mediated by AT1B receptors and demonstrate that, when AT1A receptors are absent, the AT1B receptor contributes to the regulation of resting blood pressure.
Expression of carbonic anhydrase IV mRNA in rabbit kidney: stimulation by metabolic acidosis
Authors: Winkler C. A. , Kittelberger A. M. , Schwartz G. J.
Published: December 2017

The renal carbonic anhydrases, CA II (cytosolic) and CA IV (membrane bound), are believed to facilitate renal acid secretion. We have recently shown that renal cortical sodium dodecyl sulfate (SDS)-resistant hydratase (presumably CA IV) activity was stimulated 241% during chronic metabolic acidosis (CMA). In the present study, we examined the expression and regulation of CA IV mRNA in kidneys from control and acidotic rabbits. To obtain a CA IV probe, we reverse transcribed rabbit kidney total RNA and amplified a approximately 780-base pair (bp) DNA product using primers derived from the human CA IV sequence. Using this product, we screened one-half of a kidney cortex cDNA library and sequenced a 1,194-bp cDNA, which contained the entire open-reading frame of rabbit CA IV. The cDNA was 78% identical to human and 71% to rat CA IV. The deduced amino acid sequence projected an active zinc binding site and two glycosylation sites. Northern analysis yielded a single transcript of approximately 1,600 bp in size expressed more abundantly in cortex and inner medulla than in outer medulla. CA IV mRNA was also expressed abundantly in lung but not in liver or spleen. The high abundance of CA IV mRNA in inner medulla was localized by in situ hybridization to medullary collecting duct cells. Rabbits exposed to CMA showed significant upregulation of CA IV mRNA expression in kidney cortex and outer medulla. Despite a numerical increase, excessive variability precluded statistical significance in the inner medulla. Thus CA IV mRNA was expressed abundantly in kidney and stimulated by CMA, similar to what has been previously observed for SDS-resistant hydratase (presumed CA IV) activity. It is likely that the regulation of CA IV mRNA and activity is relevant to the kidney's adaptation to CMA.
Agmatine affects glomerular filtration via a nitric oxide synthase-dependent mechanism
Authors: Schwartz D. , Peterson O. W. , Mendonca M. , Satriano J. , Lortie M. , Blantz R. C.
Published: December 2017

Arginine decarboxylase is present in the kidney and metabolizes the amino acid, arginine, to agmatine. Agmatine increases filtration rate in single nephrons (J. J. Lortie, W. F. Novotny, O. W. Peterson, V. Vallon, K. Malvey, M. Mendonca, J. Satriano, P. Insel, S. C. Thomson, and R. C. Blantz. J. Clin. Invest. 97:413-420, 1996). Experiments were conducted to determine whether exogenously administered agmatine exerts these effects via interaction with nitric oxide synthase (NOS) and whether this interaction depends upon alpha 2-adrenergic receptors. Agmatine microperfused (1 microM) into the urinary space of surface glomeruli of the rat increased nephron filtration rate from 33 +/- 4 to 40 +/- 5 nl/min with complete recovery within 10 min. When NG-monomethyl-L-arginine (L-NMMA), a nonselective NOS inhibitor, was systemically infused, agmatine no longer increased single-nephron glomerular filtration rate (SNGFR). BHT-933, an alpha 2-adrenergic agonist, did not increase SNGFR and was unaffected by concurrent L-NMMA. In vitro incubation of freshly harvested glomeruli with agmatine resulted in significant increases in the generation of cGMP, effects similar to carbachol, and blocked by nitro-L-arginine methyl ester (L-NAME) but not yohimbine, an alpha 2-adrenergic antagonist. Agmatine exerts effects on glomerular ultrafiltration via a constitutive NOS-dependent mechanism, and this does not require the participation of alpha 2-adrenoreceptors.
Polycystin expression is temporally and spatially regulated during renal development
Authors: Van Adelsberg J. , Chamberlain S. , D'Agati V.
Published: December 2017

Mutations in PKD1 cause autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease in which cysts form from kidney tubules. The predicted product of this gene is a novel protein with cell-adhesive and membrane-spanning domains. To test the hypothesis that polycystin, the product of the PKD1 gene, is a cell adhesion molecule, we raised antibodies against peptides derived from the unduplicated, membrane-spanning portion of the predicted amino acid sequence. These antibodies recognized membrane-associated polypeptides of 485 and 245 kDa in human fetal kidney homogenates. Expression was greater in fetal than adult kidney by both Western blot analysis and immunofluorescence. In fetal kidney, polycystin was localized to the plasma membranes of ureteric bud and comma and S-shaped bodies. However, in more mature tubules in fetal kidney, in adult kidney, and in polycystic kidney, the majority of polycystin staining was intracellular. The temporal and spatial regulation of polycystin expression during renal development lead us to speculate that polycystin may play a role in nephrogenesis.
Apical and basal uptake of L-dopa and L-5-HTP and their corresponding amines, dopamine and 5-HT, in OK cells
Authors: Vieira-Coelho M. A. , Soares-da-Silva P.
Published: December 2017

The present study defined the kinetic characteristics of L-3,4-dihydroxyphenylalanine (L-dopa) and L-5-hydroxytryptophan (L-5-HTP) transport and their decarboxylation products, dopamine and 5-hydroxytryptamine (5-HT), respectively, in OK cells. The apical uptake of both L-dopa and 5-HTP was temperature dependent and stereoselective. Nonlinear analysis of the saturation curves revealed for L-dopa and L-5-HTP Michaelis constants of 13.8 and 29.1 microM, respectively, and maximal velocities of 21 and 29 nmol.mg protein-1.6 min-1, respectively, L-5-HTP inhibited the apical uptake of L-dopa with a half-maximal inhibitory constant of 29.1 microM. The accumulation of L-dopa and L-5-HTP from the basolateral cell border was dependent on the concentration used and saturable at nearly 50 microM. The accumulation of dopamine and 5-HT was found to be saturable only when the substrates were applied from the basolateral cell border. These results demonstrate that the L-dopa and L-5-HTP transporters in OK cells are located in both the apical and basolateral cell borders, whereas the uptake of dopamine and 5-HT is a saturable process only when the substrates are applied from the basolateral cell border.
Molecular and functional identification of beta-adrenergic receptors in distal convoluted tubule cells
Authors: Gesek F. A. , White K. E.
Published: December 2017

Renal nerve stimulation or circulating catecholamines activate the beta-adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of beta-adrenergic receptors have been characterized: beta 1, beta 2, and beta 3. beta-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of beta-adrenergic receptors in DCT cells and 2) examine functional responses to beta-receptor activation on adenosine 3',5'-cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of beta-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for beta 1-, beta 2-, and beta 3-receptor subtypes. Products of the appropriate sizes were obtained with beta 1- and beta 2-primers. No product was observed with primers to the beta 3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse beta 1- and beta 2-receptor sequence. Receptor binding of[3H]dihydroalprenolol was 123 +/- 13 fmol/mg protein, and a 3:1 ratio of beta 1- to beta 2-receptors was observed with DCT cell membranes. Isoproterenol, a beta-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased 22Na+ uptake to 345 +/- 23 compared with a basal rate of 256 +/- 12 nmol.min-1.mg protein-1 and was blocked with propranolol and beta 1- and beta 2-selective antagonists. Isoproterenol had no effect on 45Ca2+ entry into DCT cells. In summary, DCT cells express three times more beta 1- than beta 2-receptors and express no detectable beta 3-adrenergic receptors. beta-Receptors couple to adenylyl cyclase, and activation of beta-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.
Effect of hypokalemia on the abundance of HK alpha 1 and HK alpha 2 protein in the rat kidney
Authors: Kraut J. A. , Hiura J. , Besancon M. , Smolka A. , Sachs G. , Scott D.
Published: December 2017

An H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) contributes to potassium reabsorption by the collecting ducts of the rat kidney. mRNAs for two isoforms of the H(+)-K(+)-ATPase, HK alpha 1 and HK alpha 2, have been found in the rat kidney. To evaluate whether the HK alpha 1 and HK alpha 2 proteins are present in the rat kidney, microsomes enriched in HK alpha 1 or HK alpha 2 were isolated using the MiniMac magnetic separation system with antibodies directed against either HK alpha 1 (HK 12.18) or HK alpha 2 (AS 31.7). Immunoblots of rat kidney microsomal protein isolated with HK 12.18 revealed a band approximately 94 kDa in size that comigrated with the G1 fraction of the stomach. Immunoblots of rat kidney microsomal protein isolated with AS 31.7 revealed a band slightly greater than 94 kDa that comigrated with a band obtained from rat colonic microsomal protein. To examine the effect of perturbations in potassium metabolism, the abundance of the HK alpha 1 and HK alpha 2 isoforms was compared in rats fed a normal or potassium-deficient diet. A low-potassium diet increased the abundance of HK alpha 2, whereas that of HK alpha 1 was not altered. These data suggest that HK alpha 2 might be the isoform responsible for potassium conservation by the kidney.
Acid-base changes alter Mg2+ uptake in mouse distal convoluted tubule cells
Authors: Dai L. J. , Friedman P. A. , Quamme G. A.
Published: December 2017

Metabolic alkalosis leads to renal magnesium conservation, whereas metabolic acidosis is associated with urinary magnesium wasting. Micropuncture studies suggest that these actions affect magnesium transport in the distal tubule. The cellular mechanisms of acid-base changes were investigated in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined by microfluorescence using the Mg(2+)-responsive dye, mag-fura 2. Mg2+ transport was assessed as a function of change in [Mg2+]i with time following placement of Mg(2+)-depleted cells into a buffer containing 1.5 mM magnesium. The uptake rate of Mg2+, d([Mg2+]i)/dt, into Mg(2+)-depleted cells determined with a buffer solution of pH 7.4 was 178 +/- 21 nM/s. Mg2+ uptake at pH 8.0 was markedly increased 278 +/- 35 nM/s, whereas transport at pH 6.0 was significantly reduced to 121 +/- 15 nM/s. Mg2+ uptake at pH 7.4 was not stimulated with 20 or 40 mM bicarbonate, nor were the differences in Mg2+ uptake with pH associated with changes in membrane voltage. Mg2+ uptake was stimulated with membrane hyperpolarization at pH 6.0 but not at pH 8.0. Chlorothiazide (10(-4) M), which stimulates Mg2+ uptake by hyperpolarizing the membrane voltage, increased uptake at pH 6.0, 59 +/- 14%, but decreased it at alkaline pH of 8.0, -55 +/- 3%. Accordingly, MDCT cells become refractory to the stimulating effects of hyperpolarization at alkaline pH values. These studies show that protons may directly affect Mg2+ transport in MDCT cells.
Antioxidant expression in experimental hydronephrosis: role of mechanical stretch and growth factors
Authors: Ricardo S. D. , Ding G. , Eufemio M. , Diamond J. R.
Published: December 2017

We assessed whether levels of renal reactive oxygen species (ROS) and antioxidant enzymes are perturbed in rats following unilateral ureteral obstruction (UUO). The mechanism of catalase perturbation was investigated using proximal tubule suspensions following stimulation with transforming growth factor (TGF)-beta and interleukin (IL)-1 and in a proximal tubular cell line (OKC) subjected to cyclic mechanical stretch, which mimics the early hydrodynamic derangement after UUO. Levels of catalase and copperzinc superoxide dismutase (Cu,Zn-SOD) mRNA from 96-h UUO rats showed a 5.5-fold (P < 0.001) and 5.0-fold (P < 0.001) decrease, respectively, compared with the contralateral unobstructed kidney (CUK). Levels of superoxide anion and hydrogen peroxide showed a significant 1.8-fold (P < 0.0001) and 14.0-fold (P < 0.0001) increase, respectively, in 96-h UUO kidney slice cultures. In situ hybridization and immunohistochemistry showed Cu,Zn-SOD and catalase mRNA and protein transcription expressed in proximal tubules of UUO and CUK specimens. Catalase mRNA levels were markedly downregulated following a 1-h exposure of isolated proximal tubules to TGF-beta (0.1–10 ng) and IL-1 (1–5 ng), in comparison to control proximal tubular suspensions. OKC subjected to cyclic mechanical stretch for 1–24 h had marked decrements in catalase mRNA levels, compared with unstretched cells at the same time point. These results indicate that a primary downregulation of proximal tubular Cu,Zn-SOD and catalase expression develops in the proximal tubules of UUO with consequent increments in cortical oxidant levels. These findings suggest that either an early mechanical disturbance produced by UUO or local tubular generation of cytokines can reduce tubular catalase expression. The downregulation of catalase mRNA expression, together with increased oxidant stress in the rat renal cortex post-UUO, may amplify the proinflammatory state of experimental hydronephrosis culminating in tubulointerstitial injury and fibrosis.
Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells
Authors: Katsura T. , Gustafson C. E. , Ausiello D. A. , Brown D.
Published: December 2017

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.
Phosphate transport in renal cell cultures of gy mice: evidence of a single defect in X-linked hypophosphatemia
Authors: Nesbitt T. , Drezner M. K.
Published: December 2017

Although current theory holds that the murine homologs of X-linked hypophosphatemia represent mutations of two closely linked genes with distinct pathophysiological consequences, insufficient data are available to support this hypothesis. We investigated whether an intrinsic defect in renal sodium (Na+)-dependent Pi cotransport truly distinguishes gy from hyp mice. We compared Pi transport in immortalized cells from S1 and S2 segments of the renal proximal convoluted tubule (PCT) of normal and gy mice. Cells from both murine models exhibit characteristics of differentiated PCT cells including gluconeogenesis, alkaline phosphatase activity, and parathyroid hormone (PTH)- and thyrocalcitonin (TCT)-dependent adenosine 3',5'-cyclic monophosphate production. More importantly, kinetic studies reveal that cells from the PCT of gy mice have intrinsically normal Pi transport and support the hypothesis that, as in hyp mice, a humoral abnormality is likely responsible for the renal Pi wasting in this mouse model. These observations are consistent with the conclusion that gy and hyp mice do not represent mutations of two closely linked genes but rather two separate mutations of the same gene.
Osmo-mechanically sensitive phosphatidylinositol signaling regulates a Ca2+ influx channel in renal epithelial cells
Authors: O'Neil R. G. , Leng L.
Published: December 2017

Regulation of dihydropyridine (nifedipine)-sensitive calcium influx was studied in rabbit culture proximal tubule cells using the fura 2 fluorescence ratio technique. "Osmo-mechanically induced" swelling of cells by exposure to hypotonic medium (220 mosmol/kgH2O) caused a rapid rise in intracellular calcium that was predominantly due to influx of calcium via both dihydropyridine-sensitive (nifedipine-sensitive) and -insensitive calcium influx pathways. The dihydropyridine-sensitive pathway was regulated, in part, by the phosphatidylinositol signaling pathway. Inhibition of phospholipase C by treatment with 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC), inhibition of protein kinase C (PKC) by staurosporine, or long-term (24 h) treatment with phorbol 12-myristate 13-acetate (PMA) to downregulate PKC abolished most of the osmo-induced, dihydropyridine-sensitive calcium influx signal. Short-term (seconds) PMA treatment to activate PKC produced a marked stimulation of both dihydropyridine-sensitive and -insensitive calcium influx in isotonic (2- to 3-fold stimulation) and hypotonic (5-fold stimulation) conditions. In contrast, elevation of adenosine 3',5'-cyclic monophosphate (cAMP) by treatment with forskolin or inhibition of protein kinase A (PKA) by treatment with the cAMP analog, Rp-8-CPT-cAMPS (the Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothionate), had little or no influence on calcium influx, including dihydropyridine-sensitive calcium influx. It is concluded that osmo-mechanical stress activates a dihyropyridine-sensitive calcium influx pathway that is predominantly regulated via the phosphatidylinositol signaling pathway and PKC and not through the cAMP/PKA signaling pathway.
Podocyte alpha-actinin induction precedes foot process effacement in experimental nephrotic syndrome
Authors: Smoyer W. E. , Mundel P. , Gupta A. , Welsh M. J.
Published: December 2017

Attachment of podocytes to the glomerular basement membrane is thought to be mediated primarily by alpha 3/beta 1-integrins and by cytoskeletal proteins including actin, talin, vinculin, and alpha-actinin. We analyzed the expression of those molecules in rat glomeruli at several time points during induction of podocyte foot process effacement and nephrotic syndrome with puromycin aminonucleoside (PAN). PAN injection resulted in marked induction of glomerular alpha-actinin (40% increase vs. paired controls, P < 0.01), which clearly preceded development of podocyte foot process effacement and proteinuria and localized almost exclusively to podocytes. Delayed induction of glomerular alpha 3-integrin (44% increase vs. paired controls, P < 0.01) following foot process effacement was also observed but was not restricted to podocytes. No significant changes in glomerular vinculin, talin, beta 1-integrin, or total actin expression occurred at any time point during disease development. We conclude that foot process effacement is preceded by induction of alpha-actinin in podocytes in experimental nephrotic syndrome. Altered expression of this actin cross-linking protein in podocytes may have a pathogenic role in foot process effacement in nephrotic syndrome.
Modeling of progressive glomerular injury in humans with lupus nephritis
Authors: Buckheit J. B. , Olshen R. A. , Blouch K. , Myers B. D.
Published: December 2017

We studied glomerular function longitudinally for 36-120 mo in 21 patients undergoing treatment for diffuse, proliferative lupus nephritis. We determined glomerular filtration rate (GFR) and glomerular oncotic pressure (IIGC) and computed the two-kidney ultrafiltration coefficient (Kf) at 6- to 12-mo intervals. The relationships and cross talk among the three variables over time were then analyzed by eigenfunction regression and canonical correlations. We also performed a morphometric analysis of serial biopsies and computed single-nephron Kf in patent glomeruli at baseline and after 36-94 mo of follow-up. Patients were divisible into progressors (n = 12) or nonprogressors (n = 9) according to the presence or absence, respectively, of an irrevocable decline in GFR over time. Examination of longitudinal variables revealed GFR to be strongly related to Kf in all patients and inversely related to IIGC in progressors. By serial morphometric analysis we observed a threefold increase in the prevalence of global sclerosis in progressors but unchanged prevalence in nonprogressors. Whereas single-nephron Kf of remnant glomeruli increased to supernormal levels in nonprogressors, the absence of this compensatory phenomenon in progressors permitted GFR and Kf to decline in parallel with the declining number of functional glomeruli.
Alterations in dopamine DA1 receptor and G proteins in renal proximal tubules of old rats
Authors: Kansra V. , Hussain T. , Lokhandwala M. F.
Published: December 2017

The present study examines the effect of dopamine DA1-receptor agonists on the renal proximal tubular Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity and quantitates DA1 receptors and the coupled G proteins in Fischer 344 model of adult (6 mo old) and old (23 mo old) rats. Dopamine and the preferential DA1-receptor agonist, SKF-38393, produced a concentration-dependent inhibition of Na(+)-K(+)-ATPase activity in proximal tubules from adult rats, whereas the enzyme activity was unaffected by these agonists in the old rats. The binding of DA1-receptor antagonist [3H]Sch-23390 in the proximal tubular basolateral membranes showed a marked decrease (approximately 47%) in the receptor numbers in old compared with adult rats, whereas dissociation constant (Kd) values in old compared with adult rats were not significantly different. Dopamine and SKF-38393 stimulated 35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding in adult rats, but there was no significant effect on the binding in the old rats. Quantification of G2 alpha and Gq/11 alpha using Western analysis revealed a significant increase in quantities of both the G proteins in old rats. The data suggest that a reduction in DA1 receptor number and subsequently reduced G protein activation may be the causative factors for the impairment in DA1 receptor-mediated inhibition of Na(+)-K(+)-ATPase activity in the proximal tubules of old rats.
Plasma membrane calcium ATPase isoform expression in cultured rat mesangial cells
Authors: Rouse D. , Abramowitz J. , Zhou X. , Kamijo T. , Gonzalez J. , Wang J. , Vaziri N. D. , Suki W. N.
Published: December 2017

The maintenance of intracellular ionized calcium (iCa2+) in the submicromolar range is important for mesangial cell (MC) function, and, as in most mammalian cells, plasma membrane Ca(2+)-ATPases (PMCA) play an important role in the homeostatic process. Molecular studies have demonstrated four PMCA isoforms, each with multiple splice variants. The present study examines the expression of PMCA isoforms and calmodulin-binding region splice variants in cultured MC from Sprague-Dawley rats and from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats before and after the onset of hypertension in SHR. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analyses, we have demonstrated PMCA1, -3, and -4, but not PMCA2, to be present in MC from these rat strains. Splice variant analysis revealed PMCA1a and -1b, PMCA3a, -3b, and -3c, and PMCA4a and -4b to be expressed in MC from all three strains. The relative quantities of PMCA1 and PMCA4 mRNA were not different in age-matched SHR vs. WKY rats, correlating with similar iCa2+ measurements. The expression of all three isoforms declined with age in SHR and WKY.
Calcitonin activates an Na(+)-independent HCO3(-)-dependent pathway in the rabbit distal convoluted tubule
Authors: Dagher G. , Thomas S. R. , Griffiths N. , Siaume-Perez S. , Sauterey C.
Published: December 2017

Calcitonin is known to stimulate Ca2+ reabsorption and natriuresis and to increase adenosine 3',5'-cyclic monophosphate levels in early distal tubule, but its effects on acid-base transport mechanisms are not well characterized in this segment. We found that recovery of cell pH (pH1) from an induced acid load (using NH4+) in single isolated segments of the initial portion ("bright") of the rabbit distal convoluted tubule (DCTb) was due to an ethylisopropylamiloride-sensitive Na+/H+ exchanger both in the absence and presence of HCO3-, but we found no evidence for participation of other mechanisms such as an H+ pump or an HCO3(-)-dependent mechanism. Introduction of calcitonin stimulated an Na(+)-independent, HCO3(-)-dependent mechanism (0.17 +/- 0.04 pH units/min, n = 14) that reestablishes normal pHi after an induced acid load. This mechanism was observed only in the presence of CO2/HCO3- and was not inhibited by N-ethylmaleimide (1 mM), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM), or Sch-28080 nor stimulated by glutamine (2 mM) or ketoglutarate (0.5 mM), but it was dependent on chloride. We conclude that, in the DCTb, salmon calcitonin activates a latent Na(+)-independent, HCO3(-)-dependent mechanism.
Flow versus pressure in the control of renin release in conscious dogs
Authors: Nafz B. , Berthold H. , Ehmke H. , Hackenthal E. , Kirchheim H. R. , Persson P. B.
Published: December 2017

In Goldblatt hypertension, renal artery stenosis reduces renal arterial pressure (RAP) and renal blood flow (RBF) and thereby increases plasma renin activity (PRA) levels. Although it is clear that reduction in RAP stimulates renin, the decrease in RBF may contribute to higher PRA as well. However, it has hitherto never been possible to dissociate a decrease in RBF from a concomitant decrease in RAP. To overcome this restriction, we used two protocols. 1) RAP was reduced in a single step to 70 +/- 0.2 mmHg (N = 8). RBF followed the sudden fall in RAP within 15 s but subsequently took on initial levels. In contrast, renal venous PRA increased from 0.95 +/- 0.22 to 5.6 +/- 1.4 ng angiotensin I.ml-1.h-1 (P < 0.05) and remained at higher values even after RBF had regained control conditions. 2) Resonance between RAP and RBF was induced by superimposing slow sinusoidal RAP waves with a period length of 450 s (N = 9), leading to a phase shift of roughly 180 degrees (time delay, 241 +/- 12 s), i.e., RBF was maximal at minimal RAP. Under these conditions, renin release was only dependent on decrements in RAP (delay of only 27 +/- 8 s). In conclusion, RBF played no major role in renin release.
Unexpected protection of glomerular mesangial cells from oxidant-triggered apoptosis by bioflavonoid quercetin
Authors: Yokoo T. , Kitamura M.
Published: December 2017

Bioflavonoid quercetin is known as an anti-cancer agent that induces apoptosis of tumor cells. Currently, however, little is understood about the effect of this drug on the function of normal cells. In this report, we address an unexpected, novel action of quercetin against apoptosis. Pretreatment with quercetin protected mesangial cells from hydrogen peroxide (H2O2)-induced apoptosis. A similar effect was observed in other cell types including LLC-PK1 epithelial cells and NRK49F fibroblasts. To explore the molecular mechanisms involved, we tested the effect of quercetin on c-Jun/activator protein-1 AP-1), the crucial mediator for H2O2-initiated apoptosis. Northern blot analysis revealed that quercetin suppressed the c-jun expression by H2O2. This was correlated with blunted activation of 12-O-tetradecanoylphorbol 13-acetate response element (TRE) in response to H2O2. These results suggested that quercetin inhibited apoptosis via intervention in the c-Jun/AP-1 pathway. To further investigate the action of quercetin, its effect on tyrosine kinases was studied. Immunoblot analysis revealed that H2O2 induced tyrosine phosphorylation. Quercetin inhibited this process in a dose-dependent manner. Inactivation of tyrosine kinases was an event upstream of c-Jun/AP-1, because tyrosine kinase inhibitors suppressed both activation of c-Jun/AP-1 and induction of apoptosis by H2O2. These findings elucidated the novel action of quercetin as an apoptosis inhibitor. This cytoprotective effect was found to be via suppression of the tyrosine kinase-c-Jun/AP-1 pathway triggered by oxidant stress.
Role of Rho and myosin phosphorylation in actin stress fiber assembly in mesangial cells
Authors: Kreisberg J. I. , Ghosh-Choudhury N. , Radnik R. A. , Schwartz M. A.
Published: December 2017

Treatment of renal glomerular mesangial cells with adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents induces actin stress fiber disassembly, myosin light chain (MLC) dephosphorylation, loss of adhesion to the substratum and cell shape change [J. I. Kreisberg and M. A. Venkatachalam. Am. J. Physiol. 251 (Cell Physiol. 20): C505-C511, 1986]. Thrombin and vasopressin block the effects of cAMP. Because these agents are known to promote stress fiber formation via the small GTP-binding protein Rho, we investigated the effect of an activated variant of Rho on the response to cAMP elevation. Microinjecting V14-Rho completely blocked the effect of cAMP elevation on cell shape and the actin cytoskeleton, whereas inactivating Rho with botulinum C3 exoenzyme induced stress fiber disruption and cell retraction that was indistinguishable from that caused by elevations in intracellular levels of cAMP. Disruption of actin stress fibers by cAMP has previously been ascribed to MLC dephosphorylation; however, both C3 and cytochalasin D also caused dephosphorylation of MLC, whereas blocking MLC dephosphorylation failed to block the cAMP-induced loss of actin stress fibers. We conclude that Rho can modulate the effects of cAMP elevation and suggest that MLC dephosphorylation may be a consequence of actin stress fiber disassembly.
Acid-induced stimulation of Na-Pi cotransport in OK cells: molecular characterization and effect of dexamethasone
Authors: Jehle A. W. , Forgo J. , Biber J. , Lederer E. , Krapf R. , Murer H.
Published: December 2017

Alterations in systemic acid/base balance affect renal Pi excretion. In the present study, the effects of an acidic pH on apical Na-dependent Pi (Na-Pi) cotransport were analyzed using OK cells (opossum kidney cell line). Cells were maintained at either pH 7.4 or 7.1 (altered HCO3- concentration at constant PCO2). Incubation in acidic medium led to an increase in Na-Pi cotransport activity, which was characterized by a transient, initial response (2-4 h, 25% increase) followed by a sustained response (24 h, 75% increase). Increased Na-Pi cotransport activity (24 h) was sensitive to inhibition by parathyroid hormone. Actinomycin D did not abolish the acid-induced increases (initial and sustained responses). Cycloheximide abolished the increase in Na-Pi cotransport observed after 24 h. The increase in Na-Pi cotransport (24 h) was prevented by dexamethasone (2 x 10(-6) M). Western blots showed a twofold (3 h) and two- to threefold (24 h) increase in NaPi-4 protein after acid exposure. Cycloheximide prevented the late increase in NaPi-4 protein abundance. Also dexamethasone reduced the increase in specific protein content. In conclusion, the exposure of OK cells to an acidic medium causes a stimulation of the NaPi-4 cotransporter that is prevented by dexamethasone.
Is the secretory K channel in the rat CCT ROMK?
Authors: Palmer L. G. , Choe H. , Frindt G.
Published: December 2017

The biophysical properties of low-conductance secretory K (SK) channels in the apical membrane of the rat cortical collecting tubule were examined to compare these properties with those of the cloned renal K channels of the ROMK family expressed in oocytes. At room temperature, with the tubule superfused with 140 mM K and 110 mM cation in the pipette, the inward single-channel conductance of the SK channels was 36 +/- 1 pS for K, 41 +/- 2 pS for NH4, and 22 +/- 3 pS for Tl. The reversal potential was nearly the same for K and Tl in the pipette but was shifted by -60 mV for NH4. The kinetics of the channel when K was the permeant ion could be described by a single open state (mean open time, 24 ms) and two closed states (mean closed times, 1.6 and 65 ms). The kinetics of SK changed when Tl was the permeant ion (mean open times of 6.6 ms and no long closed state) and when NH4 was the permeant ion (mean open time of 3.0 ms and a more prevalent long closed state). Thus the gating kinetics of the channel depend strongly on the nature of the conducted ion. The properties of SK channels were quite similar to those of ROMK2 expressed in Xenopus oocytes and measured under similar conditions, suggesting that these channels are identical.