bluuebunny/crossref_metadata_2025 huggingface dataset:
Bradykinin stimulates the ERK→Elk-1→Fos/AP-1 pathway in mesangial cells
Authors: El-Dahr Samir S. , Dipp Susana , Baricos William H.
Published: December 2017

Among its diverse biological actions, the vasoactive peptide bradykinin (BK) induces the transcription factor AP-1 and proliferation of mesangial cells (S. S. El-Dahr, S. Dipp, I. V. Yosipiv, and W. H. Baricos. Kidney Int. 50: 1850–1855, 1996). In the present study, we examined the role of protein tyrosine phosphorylation and the mitogen-activated protein kinases, ERK1/2,in mediating BK-induced AP-1 and DNA replication in cultured rat mesangial cells. BK (10−9to 10−7M) stimulated a rapid increase in tyrosine phosphorylation of multiple proteins with an estimated molecular mass of 120–130, 90–95, and 44–42 kDa. Immunoblots using antibodies specific for ERK or tyrosine-phosphorylated ERK revealed a shifting of p42 ERK2 to a higher molecular weight that correlated temporally with an increase in tyrosine-phosphorylated ERK2. Genistein, a specific tyrosine kinase inhibitor, prevented the phosphorylation of ERK2 by BK. In-gel kinase assays indicated that BK-induced tyrosine phosphorylation of ERK2 is accompanied by fourfold activation of its phosphotransferase activity toward the substrate PHAS-I ( P < 0.05). Furthermore, BK stimulated a 2.5-fold increase ( P < 0.05) in phosphorylation of Elk-1, a transcription factor required for growth factor-induced c-fos transcription. In accord with the stimulation of Elk-1 phosphorylation, BK induced c-fos gene expression and the production of Fos/AP-1 complexes. In addition, thymidine incorporation into DNA increased twofold ( P < 0.05) following BK stimulation. Each of these effects was blocked by tyrosine kinase inhibition with genistein or herbimycin A. Similarly, antisense oligodeoxynucleotide targeting of ERK1/2 mRNA inhibited BK-stimulated DNA synthesis. In contrast, protein kinase C inhibition or depletion had no effect on BK-induced c-fos mRNA, AP-1-DNA binding activity, or DNA synthesis. Collectively, these data demonstrate that BK activates the ERK→Elk-1→AP-1 pathway and that BK mitogenic signaling is critically dependent on protein tyrosine phosphorylation.
Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease
Authors: Sweeney William E. , Avner Ellis D.
Published: December 2017

Evidence from a number of laboratories suggests a potential role for the epidermal growth factor (EGF)-transforming growth factor-α-epidermal growth factor receptor (EGF-R) axis in promoting epithelial hyperplasia and cyst formation in autosomal recessive polycystic kidney disease (ARPKD). As previously reported, in the C57BL-6Jcpk/cpk (CPK), BALB/c-bpk/bpk (BPK), and C3H-orpk/orpk (ORPK) murine models of ARPKD, as well as in human ARPKD and human ADPKD, the EGF-R is mislocated to the apical surface of cystic collecting tubule (CT) epithelial cells. The present studies demonstrate that cells from cystic and control CTs can be isolated and that these cells maintain their in vivo EGF-R phenotype in vitro. Domain-specific high-affinity ligand binding was assessed by standard Scatchard analysis, and selective ligand stimulation of apical vs. basolateral EGF-R in these cells was followed by measurement of receptor autophosphorylation and determination of cell proliferation. These studies demonstrate that in vitro apically expressed EGF-Rs exhibit high-affinity binding for EGF, autophosphorylate in response to EGF, and transmit a mitogenic signal when stimulated by the appropriate ligand.
Relevance of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis
Authors: Wallner Elisabeth I. , Yang Qiwei , Peterson Darryl R. , Wada Jun , Kanwar Yashpal S.
Published: December 2017

Mammalian nephrogenesis begins by the reciprocal interaction of the ureteric bud with the undifferentiated mesenchyme. The mesenchyme differentiates into an epithelial phenotype with the development of the glomerulus and proximal and distal tubules. At the same time, the mesenchyme stimulates the branching morphogenesis of the ureteric bud that differentiates into the collecting ducts. These inductive interactions and differentiation events are modulated by a number of macromolecules, including the extracellular matrix (ECM), integrin receptors, and cell adhesion molecules. Many of these macromolecules exhibit spatiotemporal developmental regulation in the metanephros. Some are expressed in the mesenchyme, whereas others appear in the ureteric bud epithelia. The molecules expressed in the mesenchyme or at the epithelial:mesenchymal interface may serve as ligands while those in the epithelia serve as the receptors. In such a scenario the ligand and the receptor would be ideally suited for epithelial:mesenchymal paracrine/juxtacrine interactions that are also influenced by RGD sequences and Ca2+binding domains of the ECM proteins and their receptors. This review addresses the role of such interactions in metanephric development.
Activation of H+-ATPase by hypotonicity: a novel regulatory mechanism for H+secretion in IMCD cells
Authors: Amlal Hassane , Goel Akhil , Soleimani Manoocher
Published: December 2017

The effect of hypotonicity on H+-ATPase activity was examined in cultured inner medullary collecting duct (mIMCD-3) cells. mIMCD-3 cells were grown to confluence, loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and assayed for H+-ATPase activity measured as the Na+- and K+-independent intracellular pH (pHi) recovery following an acid load. Exposure of mIMCD-3 cells to a hypotonic solution (150 mosmol/kgH2O) increased pHirecovery by ∼350% ( P < 0.0001). This effect was inhibited by diethylstilbestrol (an inhibitor of H+-ATPase) and was not dependent on external K+, indicating lack of involvement of H+-K+-ATPase. H+-ATPase activation was acute, independent of cell calcium, and was not secondary to Cl−channel activation. The magnitude of H+-ATPase upregulation was dependent on the osmolarity of the media, with maximum stimulation at 150 mosmol/kgH2O. H+-ATPase upregulation in hypotonicity was significantly blocked in the presence of staurosporine or calphostin C or in cells pretreated with phorbol 12-myristate 13-acetate (PMA), indicating involvement of protein kinase C. Hypotonicity inhibited the Na+/H+exchanger activity in mIMCD-3 cells, indicating that its stimulatory effect is specific to H+-ATPase. In conclusion, a novel regulatory mechanism of H+-ATPase by hypotonicity is described. The increased H+-ATPase activity in hypotonicity may be responsible for increased[Formula: see text] reabsorption and maintained acid-base homeostasis in hyposmolar states.
Bradykinin may be involved in neuropeptide Y-induced diuresis, natriuresis, and calciuresis
Authors: Bischoff Angela , Rascher Wolfgang , Michel Martin C.
Published: December 2017

Neuropeptide Y (NPY) can cause diuresis, natriuresis, and calciuresis in rats independently of the pressure-natriuresis mechanism (A. Bischoff and M. C. Michel. Pflügers Arch. 435: 443–453, 1998). Because this is seen in systemic but not intrarenal NPY infusion, we have investigated the possible mediator of tubular NPY effects in anesthetized rats. In the present study, infusion of NPY (2 μg ⋅ kg−1 ⋅ min−1) enhanced renovascular resistance by ≈8 mmHg ⋅ ml−1 ⋅ min and enhanced urine and sodium excretion by ≈450 μl/15 min and ≈60–85 μmol/15 min, respectively. Acute renal denervation did not alter renovascular or tubular NPY effects, indicating that a neuronally released mediator is not involved. Treatment with the angiotensin II-receptor antagonist losartan prevented the decline of the renovascular response with time but did not modify tubular NPY effects. The bradykinin B2-receptor antagonist icatibant accelerated the decline of the renovascular NPY effects with time; concomitantly, it attenuated NPY-induced diuresis and natriuresis and abolished NPY-induced calciuresis. The converting-enzyme inhibitor ramiprilat prevented the decline of the renovascular response with time; concomitantly, it magnified the NPY-induced diuresis, natriuresis, and calciuresis. We conclude that bradykinin may be involved in NPY-induced diuresis, natriuresis, and, in particular, calciuresis.
Immunochemical characterization of Na+/H+exchanger isoform NHE4
Authors: Pizzonia John H. , Biemesderfer Daniel , Abu-Alfa Ali K. , Wu Ming-Shiou , Exner Markus , Isenring Paul , Igarashi Peter , Aronson Peter S.
Published: December 2017

Mammalian Na+/H+exchangers (NHEs) are a family of transport proteins (NHE1–NHE5). To date, the cellular and subcellular localization of NHE4 has not been characterized using immunochemical techniques. We purified a fusion protein containing a portion of rat NHE4 (amino acids 565–675) to use as immunogen. A monoclonal antibody (11H11) was selected by ELISA. It reacted specifically with both the fusion protein and to a 60- to 65-kDa polypeptide expressed in NHE4-transfected LAP1 cells. By Western blot analysis, NHE4 was identified as a 65- to 70-kDa protein that was expressed most abundantly in stomach and in multiple additional epithelial and nonepithelial rat tissues including skeletal muscle, heart, kidney, uterus, and liver. Subcellular localization of NHE4 in the kidney was evaluated by Western blot analysis of membrane fractions isolated by Percoll gradient centrifugation. NHE4 was found to cofractionate with the basolateral markers NHE1 and Na+-K+-ATPase rather than the luminal marker γ-glutamyl transferase. In stomach, NHE4 was detected by immunoperoxidase labeling on the basolateral membrane of cells at the base of the gastric gland. We conclude that NHE4 is a 65- to 70-kDa protein with a broad tissue distribution. In two types of epithelial cells, kidney and stomach, NHE4 is localized to the basolateral membrane.
Differential expression, abundance, and regulation of Na+-phosphate cotransporter genes in murine kidney
Authors: Tenenhouse Harriet S. , Roy Stéphane , Martel Josée , Gauthier Claude
Published: December 2017

Three classes of high-affinity Na+-Picotransporters are expressed in mammalian kidney. These include Npt1 (type I), Npt2 (type II), and the cellular receptors for gibbon ape leukemia virus (Glvr-1) and amphotropic murine retrovirus (Ram-1) (type III). We defined the tissue distribution as well as the relative renal abundance of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs and examined the effects of low-Pidiet, the Hyp mutation, and growth hormone (GH) on their renal expression by ribonuclease protection assay. In normal mouse kidney, Npt1, Npt2, Glvr-1, and Ram-1 accounted for 15 ± 1.0, 84 ± 1.0, 0.5 ± 0.2, and 0.5 ± 0.3% of total Na+-Picotransporter mRNAs, respectively. Evidence was obtained for low-abundance Npt1 mRNA expression in liver and Npt2 mRNA expression in intestine, whereas Glvr-1 and Ram-1 mRNAs were also detected in bone, intestine, heart, and liver. Npt2 mRNA was localized to proximal tubules in the renal outer cortex, whereas Glvr-1 transcripts were detected throughout the kidney by in situ hybridization. The Hyp mutation elicited a significant reduction in renal Npt1 and Npt2 mRNAs (78 ± 8 and 57 ± 3% of normal, respectively), whereas neither low-Pidiet nor GH influenced the renal abundance of Npt1 and Npt2 transcripts. Renal Glvr-1 mRNA expression was significantly increased in Hyp mice and GH-treated mice (145 ± 6 and 165 ± 5% of control, respectively), whereas the renal abundance of Ram-1 transcript was unaffected by either the Hyp mutation, low-Pidiet, or GH treatment. In summary, we demonstrate that Npt2 is the predominant Na+-Picotransporter in mouse kidney, that Npt2 and Glvr-1 have distinct patterns of renal expression, and that the Hyp mutation modulates the renal expression of Npt1, Npt2, and Glvr-1 mRNAs. Our results suggest that increased renal Glvr-1 mRNA may contribute to GH stimulation of renal Na+-Picotransport.
Characterization of angiotensin IV-degrading enzymes and receptors on rat mesangial cells
Authors: Chansel Dominique , Czekalski Stanislas , Vandermeersch Sophie , Ruffet Emmanuel , Fournié-Zaluski Marie-Claude , Ardaillou Raymond
Published: December 2017

Because mesangial cells (MC) are a target and a degradation site for angiotensin II (ANG II), we characterized the degrading enzymes and receptors of ANG IV, a metabolite of ANG II, on these cells. ANG IV was metabolized into its NH2-terminal deleted peptides, ANG II-(4–8), ANG II-(5–8), and ANG II-(6–8) by rat MC. Total protection of ANG IV was obtained only when PC-18, a specific aminopeptidase N (APN) inhibitor, and JFH-27A, a mixed inhibitor of dipeptidylaminopeptidase (DAP) and neutral endopeptidase (NEP), were simultaneously added. In contrast, thiorphan, an NEP inhibitor, was inactive. These results demonstrate the exclusive role of APN and DAP in ANG IV degradation.125I-labeled ANG IV binding was studied in the presence of PC-18 and JFH-27A to suppress ligand degradation. Under these conditions, ANG IV-specific receptors could be demonstrated with a KDof 1.8 nM and a density of 55 fmol/mg. In contrast with MC, no evidence for ANG IV receptors could be obtained in freshly isolated glomeruli. ANG IV stimulated cytosolic calcium concentration in MC, whereas its NH2-terminal deleted metabolites were inactive. Therefore, ANG IV must be protected from degradation by APN and DAP in studies on the nonimmediate biological effects of this peptide.
Cyclosporin A increases hypoxia and free radical production in rat kidneys: prevention by dietary glycine
Authors: Zhong Zhi , Arteel Gavin E. , Connor Henry D. , Yin Ming , Frankenberg Moritz V. , Stachlewitz Robert F. , Raleigh James A. , Mason Ronald P. , Thurman Ronald G.
Published: December 2017

The major side effect of cyclosporin A is severe nephrotoxicity. It is likely that cyclosporin A causes vasoconstriction leading to hypoxia-reperfusion injury; therefore, these experiments were designed to attempt to obtain physical evidence for hypoxia and free radical production in kidney following cyclosporin A. Rats were treated daily with cyclosporin A (25 mg/kg ig) for 5 days, and pimonidazole, a hypoxia marker, was injected 2 h after the last dose of cyclosporin A. A dose of α-(4-pyridyl-1-oxide)- N- tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free radicals. Cyclosporin A doubled serum creatinine and decreased glomerular filtration rates by 65% as expected. Pimonidazole adduct binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A increased 4-POBN/radical adducts nearly sixfold in the urine but did not alter levels in the serum. Glycine, which causes vasodilatation and prevents cyclosporin A toxicity, minimized hypoxia and blocked free radical production; however, it did not alter cyclosporin A blood levels. These results demonstrate for the first time that cyclosporin A causes hypoxia and increases production of a new free radical species exclusively in the kidney. Therefore, it is concluded that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation, effects which can be prevented effectively by dietary glycine.
Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function
Authors: Blanco Gustavo , Mercer Robert W.
Published: December 2017

The Na-K-ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both its α- and β-subunits. At present, as many as four different α-polypeptides (α1, α2, α3, and α4) and three distinct β-isoforms (β1, β2, and β3) have been identified in mammalian cells. The stringent constraints on the structure of the Na pump isozymes during evolution and their tissue-specific and developmental pattern of expression suggests that the different Na-K-ATPases have evolved distinct properties to respond to cellular requirements. This review focuses on the functional properties, regulation, and possible physiological relevance of the Na pump isozymes. The coexistence of multiple α- and β-isoforms in most cells has hindered the understanding of the roles of the individual polypeptides. The use of heterologous expression systems has helped circumvent this problem. The kinetic characteristics of different Na-K-ATPase isozymes to the activating cations (Na+and K+), the substrate ATP, and the inhibitors Ca2+and ouabain demonstrate that each isoform has distinct properties. In addition, intracellular messengers differentially regulate the activity of the individual Na-K-ATPase isozymes. Thus the regulation of specific Na pump isozymes gives cells the ability to precisely coordinate Na-K-ATPase activity to their physiological requirements.
Inhibitory effect of calyculin A, a Ser/Thr protein phosphatase type I inhibitor, on renin secretion
Authors: Park Chun Sik , Kim Mi Hyun , Leem Chae Hun , Jang Yeon Jin , Kim Hae Won , Kim Hyoun Sik , Hong Yoo Sun
Published: December 2017

We have recently shown that several putative selective inhibitors of Ca2+-calmodulin-dependent myosin light chain kinase (MLCK), such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], reversibly stimulate renin secretion [C. S. Park, S.-H. Chang, H. S. Lee, S.-H. Kim, J. W. Chang, and C. D. Hong. Am. J. Physiol. 271 ( Cell Physiol. 40): C242–C247, 1996]. We hypothesized that Ca2+inhibits renin secretion, via phosphorylation of 20-kDa myosin light chain (MLC20), by activating MLCK. In the present studies, we have investigated the types of protein phosphatase (PP) involved in the control of renin secretion through inhibition of MLC dephosphorylation using inhibitors of various types of serine/threonine-specific protein phosphatases. Cyclosporin A, a putative inhibitor of PP type 2 (calcineurin), was without effect. Calyculin A and okadaic acid, putative selective inhibitors of both PP type 1 (PP1) and type 2A (PP2A), significantly inhibited renin secretion under control conditions. Calyculin A had inhibitory effects at least 10-fold more potent than okadaic acid, suggesting that PP1, rather than PP2A, is involved in the control of renin secretion. Furthermore, calyculin A blocked the reversal of renin secretion preinhibited by raised intracellular Ca2+concentrations in a concentration-dependent manner. Calyculin A (10−6M) significantly inhibited renin secretion stimulated by lowering intracellular Ca2+concentrations and blocked the stimulatory effect of ML-9 on renin secretion. Taking all of these results into consideration, we hypothesize that dephosphorylation of MLC20by Ca2+-independent PP1 stimulates renin secretion, whereas phosphorylation of MLC20by Ca2+-calmodulin-dependent MLCK inhibits it. This hypothesized regulatory model of renin secretion predicts that the rate of renin secretion at a given time is determined by the ratio of phosphorylated to dephosphorylated MLC20, which is, in turn, determined by the dynamic balance between activity of MLCK and MLC phosphatase.
Cycles and separations in a model of the renal medulla
Authors: Thomas S. Randall
Published: December 2017

This study gives the first quantitative analysis of net steady-state transmural fluxes of water, urea, and NaCl in a numerical model of the rat renal medulla in antidiuresis, revealing the model’s predictions of water, urea, and NaCl cycling patterns. These predictions are compared both to in vivo micropuncture data from the literature and to earlier qualitative proposals (e.g., K. V. Lemley and W. Kriz. Kidney Int. 31: 538–548, 1987) of cycling and exchange patterns based on medullary anatomy and available permeability and transport parameter measurements. The analysis is based on our most recent three-dimensional model [X. Wang, S. R. Thomas, and A. S. Wexler. Am. J. Physiol. 274 ( Renal Physiol. 43): F413–F424, 1998]. In general agreement with earlier proposed patterns, this analysis predicts the following: 1) important water short-circuiting from descending structures to ascending vasa recta in most medullary regions, 2) massive urea recycling in the upper inner medulla, 3) a progressive increase of the ratio of urea to total osmoles along the corticopapillary axis, 4) urea dumped from the collecting ducts (CD) into the deep papilla is returned to the cortex essentially via outer medullary short vasa recta, bearing witness to a shift from the long descending limbs and vasa recta of the inner medulla (IM) to short structures in the outer medulla (OM). The analysis also shows that the known radial heterogeneity of the inner stripe (IS) implies unequal osmolalities in long descending limbs, vasa recta, and CDs entering the IM across the OM/IM border and explains the model’s unorthodox osmolality profile along the CD. In conflict with micropuncture evidence of a doubling of urea flow in superficial Henle’s loops (SHL) between the end proximal and early distal tubule (T. Armsen and H. W. Reinhardt. Pflügers Arch. 326: 270–280, 1971), the model predicts net urea loss from SHL due to the model’s inclusion of nonneglible measured urea permeability of medullary thick ascending limbs [M. A. Knepper, Am. J. Physiol. 245 ( Renal Fluid Electrolyte Physiol. 14): F634–F639, 1983]. We present a suite of adjusted model permeabilities that improves agreement with the micropuncture data on this point. In conclusion, this modeling analysis of solute and water recycling serves as a quantitative check on qualitative propositions in the literature and allows closer critical comparison of model behavior with published experimental results than was heretofore possible.
Protein kinase C regulation of p-glycoprotein-mediated xenobiotic secretion in renal proximal tubule
Authors: Miller David S. , Sussman Caroline R. , Renfro J. Larry
Published: December 2017

Fluorescence microscopy, fluorescent substrates [daunomycin and a fluorescent cyclosporin A (CSA) derivative] and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of p-glycoprotein in killifish renal proximal tubules. PKC activators, phorbol ester (phorbol 12-myristate 13-acetate, PMA) and dioctylglycerol, reduced luminal drug accumulation, and protein kinase inhibitors, staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), increased luminal accumulation; a PMA analog that does not activate PKC was without effect. PMA effects were blocked by staurosporine. The increase in luminal fluorescence caused by staurosporine was blocked by the p-glycoprotein substrate, CSA, indicating that this component of transport was indeed mediated by p-glycoprotein. Neither PMA, dioctylglycerol, nor protein kinase inhibitors altered cellular drug accumulation. Finally, in primary cultures of flounder proximal tubule cells, PMA decreased transepithelial [3H]daunomycin secretion. This pharmacological approach demonstrates that in teleost renal proximal tubule, p-glycoprotein-mediated xenobiotic secretion is negatively correlated with changes in PKC activity, a finding that conflicts with results from studies using mammalian tumor cells that express p-glycoprotein.
Increased decorin mRNA in diabetic mouse kidney and in mesangial and tubular cells cultured in high glucose
Authors: Mogyorosi Andras , Ziyadeh Fuad N.
Published: December 2017

The core protein of the proteoglycan decorin binds and neutralizes transforming growth factor-β (TGF-β). Activation of TGF-β is crucial to tissue injury in diabetic nephropathy, but it is not currently known whether decorin plays a role in this disease. Mouse kidney cortex demonstrates more than a twofold increase in decorin mRNA after 1, 2, 3, and 6 wk of streptozotocin diabetes. Various mouse and rat renal cell types are studied in culture under normal or high-glucose conditions. Mouse glomerular mesangial and proximal tubular epithelial cells constitutively express decorin, and high glucose (450 mg/dl) increases decorin mRNA fourfold compared with 100 mg/dl glucose. Unlike rat mesangial cells, rat glomerular epithelial and endothelial cells do not constitutively express decorin, and no induction is observed in high glucose. When mouse mesangial and proximal tubular cells are exposed to TGF-β1 (1 ng/ml), decorin mRNA is significantly decreased. Our findings suggest that the increased decorin expression in the diabetic kidney may counteract the hypertrophic and prosclerotic effects of increased TGF-β levels and that a negative feedback loop may exist between decorin and TGF-β.
PGE2stimulates Mg2+uptake in mouse distal convoluted tubule cells
Authors: Dai Long-Jun , Bapty Brian , Ritchie Gordon , Quamme Gary A.
Published: December 2017

Prostaglandins have diverse effects on renal electrolyte reabsorption, inhibiting NaCl absorption in the thick ascending limb and modulating sodium and calcium transport in cortical collecting cells. It is unclear what effect, if any, prostaglandins have on tubular magnesium handling. The effects of prostaglandin E2(PGE2) were studied on immortalized mouse distal convoluted tubule (MDCT) cells by measuring cellular cAMP formation with radioimmunoassays and Mg2+uptake with fluorescence techniques. Intracellular free Mg2+concentration ([Mg2+]i) was measured on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+uptake, MDCT cells were first Mg2+depleted to 0.22 ± 0.01 mM by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the changes in [Mg2+]iwere determined. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 173 ± 8 nM/s. Indomethacin, 5 μM, diminished basal Mg2+uptake, suggesting that endogenous prostaglandins may stimulate Mg2+entry in control cells. PGE2stimulated Mg2+entry in a concentration-dependent manner with maximal response of 311 ± 12 nM/s, at a concentration of 10−7M, which represented an 80 ± 3% increase in uptake rate above control values. This was associated with a sixfold increase in intracellular cAMP generation. PGE2-stimulated Mg2+uptake was completely inhibited with the Rp diastereoisomer of adenosine 3′,5′-cyclic monophosphothionate (Rp-cAMPS), a protein kinase A inhibitor, and U-73122, a phospholipase C inhibitor, and partially by chelerythrine, a protein kinase C inhibitor. Accordingly, PGE2-mediated Mg2+entry rates involve multiple intracellular signaling pathways. These studies demonstrate that PGE2stimulates Mg2+uptake in a cell line of MDCT.
Upregulation of H+-ATPase in the distal nephron during potassium depletion: structural and functional evidence
Authors: Bailey Matthew A. , Fletcher Robert M. , Woodrow David F. , Unwin Robert J. , Walter Stephen J.
Published: December 2017

In the present study, we have investigated the effects of dietary potassium depletion on the activity and distribution of the H+-ATPase in the distal nephron of the Sprague-Dawley rat. H+-ATPase activity was assessed from the change in transepithelial potential difference ( Vte) in response to bafilomycin A1during perfusion of the late distal tubule in vivo, with solutions containing inhibitors of known ion channels. Bafilomycin A1caused a negative deflection in Vtein control animals, an effect that was significantly enhanced during potassium depletion ( P < 0.01). The distribution of H+-ATPase within the population of intercalated cells was assessed using a specific monoclonal antibody (E11). Hypokalemia was associated with a highly significant redistribution of the staining pattern ( P < 0.001), with an increase in the percentage of cells displaying immunoreactivity in the apical membrane. These results indicate that dietary potassium depletion increases electrogenic H+-ATPase activity in the rat distal tubule; this may be associated with increased insertion of pumps into the apical membrane.
Prostanoid signaling, localization, and expression of IP receptors in rat thick ascending limb cells
Authors: Hébert Richard L. , O’Connor Tim , Neville Chris , Burns Kevin D. , Laneuville Odette , Peterson Linda N.
Published: December 2017

It is widely held that only one prostacyclin (IP) receptor exists that can couple to guanine stimulatory nucleotide binding proteins (Gs) leading to activation of adenyl cyclase. Although IP receptor mRNA is expressed in vascular arterial smooth muscle cells and platelets, with lower level expression in mature thymocytes, splenic lymphocytes, and megakaryocytes, there is no molecular evidence for IP receptor expression in renal epithelial cells. The purpose of the present study was to obtain molecular evidence for the expression and localization of the IP receptor and to study the signaling pathways of IP receptor in rat medullary thick ascending limb (MTAL). Biochemical studies showed that IP prostanoids do not increase cAMP in rat MTAL. However, in the presence of vasopressin, inhibition of cAMP formation by prostacyclin (PGI2) analogs is pertussis toxin sensitive and does not activate protein kinase C. In situ hybridization studies localized IP receptor mRNA expression to MTAL in the rat kidney outer medulla. The results of RT-PCR of freshly isolated RNA from MTAL, with primers specific for the mouse IP receptor cDNA, produced an amplification product of the correct predicted size that contained an expected Nco I endonuclease restriction site. We conclude that rat renal epithelial cells express the IP receptor, coupled to inhibition of cAMP production.
Effects of physiological hyperinsulinemia on systemic, renal, and hepatic substrate metabolism
Authors: Meyer Christian , Dostou Jean , Nadkarni Veena , Gerich John
Published: December 2017

To determine the effect of physiological hyperinsulinemia on renal and hepatic substrate metabolism, we assessed systemic and renal glucose release and uptake, systemic and renal gluconeogenesis from glutamine, and certain aspects of systemic and renal glutamine and free fatty acid (FFA) metabolism. These were assessed under basal postabsorptive conditions and during 4-h hyperinsulinemic euglycemic clamp experiments in nine normal volunteers using a combination of isotopic techniques and renal balance measurements. Hepatic glucose release (HGR) and glutamine gluconeogenesis were calculated as the difference between systemic and renal measurements. Infusion of insulin suppressed systemic glucose release and glutamine gluconeogenesis by ∼50% during the last hour of the insulin infusion ( P < 0.001). Renal glucose release and glutamine gluconeogenesis decreased from 2.3 ± 0.4 to 0.9 ± 0.2 ( P < 0.002) and from 0.52 ± 0.07 to 0.14 ± 0.03 μmol ⋅ kg−1 ⋅ min−1( P < 0.001), respectively. HGR and glutamine gluconeogenesis decreased from 8.7 ± 0.4 to 4.5 ± 0.5 ( P < 0.001) and from 0.35 ± 0.02 to 0.27 ± 0.03 μmol ⋅ kg−1 ⋅ min−1( P < 0.002), respectively. Renal glucose uptake (RGU) increased from 1.61 ± 0.19 to 2.18 ± 0.25 μmol ⋅ kg−1 ⋅ min−1( P = 0.029) but accounted for only ∼5% of systemic glucose disposal (40.6 ± 4.3 μmol ⋅ kg−1 ⋅ min−1). Both systemic and renal FFA clearance increased approximately fourfold ( P < 0.001 for both). Nevertheless, renal FFA uptake decreased ( P = 0.024) and was inversely correlated with RGU ( r = −0.582, P = 0.011). Finally, insulin increased systemic glutamine release ( P = 0.007), uptake ( P < 0.005), and clearance ( P < 0.001) but left renal glutamine uptake and release unaffected ( P > 0.4 for both).
Concerted actions of IL-1β inhibit Na+absorption and stimulate anion secretion by IMCD cells
Authors: Husted Russell F. , Zhang Chong , Stokes John B.
Published: December 2017

Increasing evidence indicates that factors other than adrenocorticoid hormones can influence long-term regulation of Na+transport by inner medullary collecting duct (IMCD) cells. We now report that, of 14 interleukins tested, only interleukin-1α (IL-1α) and IL-1β inhibited Na+transport by primary cultures of rat IMCD. IL-1β reduced both basal and mineralocorticoid (MC)-stimulated Na+transport by 50–70%; its effect on glucocorticoid (GC)-stimulated Na+transport was significantly less. IL-1β continued to blunt MC stimulation of Na+transport even after it had been removed from the medium for 24 h. The onset of action to inhibit Na+transport was within 20 min. The acute effect from the basolateral surface was greater than that from the apical surface, but the effect from each surface was additive. In addition to its inhibitory effect on Na+transport, chronic IL-1β exposure increased both basal and cAMP-stimulated anion secretion rates. IL-1β had no acute effect on anion secretion. Monolayers chronically treated with IL-1β had an increased capacity to secrete fluid, as predicted from its effects on ion transport. Inhibitors of cyclooxygenase did not blunt the actions of IL-1β. Furthermore, IL-1β did not produce a rise in intracellular Ca2+. These results suggest novel signaling pathways induced by IL-1β regulating Na+and Cl−transport by the IMCD.
Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone
Authors: Sariban-Sohraby Sarah , Svoboda Michal , Mies Frédérique
Published: December 2017

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPγS revealed a class of binding sites with an apparent Kdvalue of 100 nM and a Bmaxof 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmaxby 50%. The mRNA levels for Gαi-3, Gα0, Gαs, and Gαqwere not increased after aldosterone. The patterns of small MrG proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [α-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Giproteins and stimulates the expression of a specific GTP-binding protein of Mr59.5 kDa.
Regulation of cAMP-dependent chloride channels in DC1 immortalized rabbit distal tubule cells in culture
Authors: Rubera Isabelle , Tauc Michel , Verheecke-Mauze Catherine , Bidet Michel , Poujeol Chantal , Touret Nicolas , Cuiller Béatrice , Poujeol Philippe
Published: December 2017

Cl− conductances were studied in an immortalized cell line (DC1) derived from rabbit distal bright convoluted tubule (DCTb). The DC1 clone was obtained after transfection of primary cultures of DCTb with pSV3 neo. RT-PCR experiments showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in the DC1 cell line. Using the whole cell patch-clamp technique, we recorded a linear Cl− conductance activated by forskolin (FK). This conductance was insensitive to DIDS and corresponded to a CFTR-like channel conductance. Fluorescence experiments with 6-methoxy-1-(3-sulfonatopropyl)quinolinium (SPQ) showed that FK induced an increase in Cl− efflux and influx in DC1 cells similar to that observed in cultured DCTb cells.125I−efflux experiments performed on DC1 cells grown on collagen-coated filters showed that exposure of the monolayer to FK led to an increased125I−loss through the apical membrane only. The addition of 10 μM adenosine activated a linear conductance identical to that recorded with FK and corresponding to the CFTR-like conductance. This conductance was also activated by 5′-( N-ethylcarboxamido)adenosine and CGS-21680 and inhibited in the presence of 8-cyclopentyl-1,3-diproxylxanthine (DPCPX). This Cl− conductance could also be activated by guanosine 5′- O-(3-thiotriphosphate) (GTPγS). The addition of protein kinase A (PKA) inhibitor to the pipette solution inhibited the development of the current activated by CGS-21680. Finally,125I−efflux showed that adenosine induced an apical efflux mediated through basolateral A2 receptors. Overall, the data show that the DC1 cell line expressed an apical CFTR Cl− conductance that could be activated by adenosine via A2A receptors located in the basolateral membrane and involving G protein and PKA pathways.
Effect of experimentally induced hypothyroidism on sulfate renal transport in rats
Authors: Sagawa Kazuko , Murer Heini , Morris Marilyn E.
Published: December 2017

Decreased serum sulfate concentrations are observed in hypothyroid patients. However, the mechanism involved in thyroid hormone-induced alterations of renal sulfate homeostasis is unknown. The objectives of this investigation were to determine the effect of 6-propyl-2-thiouracil (PTU)-induced hypothyroidism in rats on 1) the in vivo serum concentrations, renal clearance, and renal reabsorption of sulfate, 2) the in vitro renal transport in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, and 3) the cellular mechanism of the hypothyroid-induced alteration in sulfate renal transport. Serum sulfate concentrations, renal fractional reabsorption of sulfate, and creatinine clearance were decreased significantly in the hypothyroid group. The Vmaxvalues for sodium-sulfate cotransport in BBM were significantly decreased in the kidney cortex from the hypothyroid animals (0.90 ± 0.31 vs. 0.49 ± 0.08 nmol ⋅ mg−1⋅ 10 s−1, n = 5–6, P < 0.05) without changes in Km. There were no significant differences in Vmaxand Kmfor sulfate/anion exchange transport in BLM. Sodium-dependent sulfate transporter (NaSi-1) mRNA and protein levels were significantly lower in the kidney cortex from hypothyroid rats. Hypothyroidism did not alter the membrane motional order (fluidity) in BBM and BLM, which indicates that the changes in the membrane fluidity do not represent the mechanism for the altered renal transport. These results demonstrate that PTU-induced hypothyroidism decreases sodium-sulfate cotransport by downregulation of the NaSi-1 gene.
Adaptation of NHE-3 in the rat thick ascending limb: effects of high sodium intake and metabolic alkalosis
Authors: Laghmani Kamel , Chambrey Régine , Froissart Marc , Bichara Maurice , Paillard Michel , Borensztein Pascale
Published: December 2017

The present studies examined the effects of chronic NaCl administration and metabolic alkalosis on NHE-3, an apical Na+/H+exchanger of the rat medullary thick ascending limb of Henle (MTAL). NaCl administration had no effect on NHE-3 mRNA abundance as assessed by competitive RT-PCR, as well as on NHE-3 transport activity estimated from the Na+-dependent cell pH recovery of Na+-depleted acidified MTAL cells, in the presence of 50 μM Hoe-694, which specifically blocks NHE-1 and NHE-2. Two models of metabolic alkalosis were studied, one associated with high sodium intake, i.e., NaHCO3administration, and one not associated with high sodium intake, i.e., chloride depletion alkalosis (CDA). In both cases, the treatment induced a significant metabolic alkalosis that was associated with a decrease in NHE-3 transport activity (−27% and −25%, respectively). Negative linear relationships were observed between NHE-3 activity and plasma pH or bicarbonate concentration. NHE-3 mRNA abundance and NHE-3 protein abundance, assessed by Western blot analysis, also decreased by 35 and 25%, respectively, during NaHCO3-induced alkalosis, and by 47 and 33%, respectively, during CDA. These studies demonstrate that high sodium intake has per se no effect on MTAL NHE-3. In contrast, chronic metabolic alkalosis, regardless of whether it is associated with high sodium intake or not, leads to an appropriate adaptation of NHE-3 activity, which involves a decrease in NHE-3 protein and mRNA abundance.
Urea transport processes are induced in rat IMCD subsegments when urine concentrating ability is reduced
Authors: Kato Akihiko , Sands Jeff M.
Published: December 2017

Infusing urea into low-protein-fed mammals increases urine concentration within 5–10 min. To determine which urea transporter may be responsible, we measured urea transport in perfused IMCD3segments [inner medullary collecting duct (IMCD) segments from the deepest third of the IMCD] from low-protein-fed rats. Basal facilitated urea permeability increased 78%, whereas active urea secretion was completely inhibited. This suggests that upregulation of facilitated urea transport may mediate the rapid increase in urine concentration. Next, expression of active urea transporter(s) in perfused IMCDs was determined in rats with other causes of reduced urine concentrating ability. In untreated and water diuretic rats, IMCD1segments showed no active urea transport, nor did IMCD2segments from untreated or hypercalcemic rats. In IMCD1segments from hypercalcemic rats, active urea reabsorption was induced. The induced active urea reabsorption was completely inhibited by replacing perfusate Na+with N-methyl-d-glucamine (NMDG+). Active urea secretion was completely inhibited in IMCD3segments from hypercalcemic rats. In contrast, water diuresis stimulated active urea secretion in IMCD2segments. The induced active urea secretion was inhibited by phloretin, ouabain, triamterene, or replacing perfusate Na+with NMDG+. In conclusion, the response of active urea transporters to reductions in urine concentrating ability follows two paradigms: one occurs with hypercalcemia or a low-protein diet, and the second occurs only in water diuresis.
Cloning and characterization of a naturally occurring soluble form of TGF-β type I receptor
Authors: Choi Mary E.
Published: December 2017

Transforming growth factor-β1 (TGF-β1) has been implicated to play an important role both in the process of normal development and in the pathogenesis of a wide variety of disease processes, including those of the kidney. TGF-β1 regulates diverse cellular functions via a heteromeric signaling complex of two transmembrane serine/threonine kinase receptors (types I and II). Several distinct type I receptors have been described and are thought to determine specificity of the TGF-β response and confer multifunctionality. This report reveals the cloning of a novel, naturally occurring soluble form of TGF-β type I receptor, designated sTβR-I, from a rat kidney cDNA library. In vivo expression of a mRNA transcript encoding the sTβR-I, which lacks the transmembrane and cytoplasmic domains, is confirmed by RT-PCR followed by Southern blot analysis and by RNase protection assay. The sTβR-I mRNA abundance is greater in the neonatal rat kidney compared with the adult rat kidney. Furthermore, sTβR-I is a functional protein capable of binding TGF-β1 ligands in the presence of a TGF-β type II receptor on the cell surface, as determined by affinity cross-linking with125I-labeled TGF-β1. Studies using p3TP-Lux reporter construct reveal that this novel protein may function as a potentiator of TGF-β signaling. The discovery of a sTβR-I provides an additional level of complexity to the TGF-β receptor system.
Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle’s loop
Authors: Kim Gheun-Ho , Ecelbarger Carolyn A. , Mitchell Carter , Packer Randall K. , Wade James B. , Knepper Mark A.
Published: December 2017

To investigate whether the enhancement of thick ascending limb (TAL) NaCl transport in response to long-term increases in circulating vasopressin concentration is associated with increased expression levels of the apical Na-K-2Cl cotransporter in the rat TAL, we have carried out immunoblotting and immunofluorescence studies using affinity-purified, peptide-directed antibodies. Semiquantitative immunoblotting studies demonstrated a marked increase (193% of controls) in Na-K-2Cl cotransporter band density in response to restriction of water intake to 15 ml/day for 7 days. In contrast, the expression levels of two other apical proteins of the TAL (the type 3 Na/H exchanger and Tamm-Horsfall protein) were unchanged in the outer medulla. A 7-day subcutaneous infusion of the V2receptor-selective vasopressin analog, 1-desamino-[8-d-arginine]vasopressin (DDAVP), to Brattleboro rats also markedly increased Na-K-2Cl cotransporter expression in the outer medulla (183% of controls). Immunofluorescence localization in outer medullary tissue sections confirmed the increase in Na-K-2Cl cotransporter expression in response to DDAVP. We conclude that vasopressin strongly upregulates the expression of the Na-K-2Cl cotransporter of the TAL and that it is likely to play an important role in the long-term regulation of the countercurrent multiplication system.
Exaggerated Ca2+signaling in preglomerular arteriolar smooth muscle cells of genetically hypertensive rats
Authors: Iversen Bjarne M. , Arendshorst William J.
Published: December 2017

Experiments were conducted to gain insight into mechanisms responsible for exaggerated renal vascular reactivity to ANG II and vasopressin (AVP) in spontaneously hypertensive rats (SHR) during the development of hypertension. Cytosolic calcium concentration ([Ca2+]i) was measured by ratiometric fura 2 fluorescence and a microscope-based photometer. Vascular smooth muscle cells (SMC) from preglomerular arterioles were isolated and dispersed using an iron oxide-sieving method plus collagenase treatment. ANG II and AVP produced rapid and sustained increases in [Ca2+]i. ANG II elicited similar dose-dependent increases in [Ca2+]iin SMC from SHR and Wistar-Kyoto rats (WKY). In contrast, AVP caused almost twofold larger responses in afferent arteriolar SMC from SHR. ANG II effects were inhibited by the AT1receptor antagonist losartan. AVP action was blocked by the V1receptor antagonist [d(CH2)5,Tyr(NH2)9]AVP. In SMC pretreated with nifedipine, neither ANG II nor AVP elicited [Ca2+]iresponses. Poststimulation nifedipine reversed elevated [Ca2+]ito basal levels. Short-term reductions in external [Ca2+]i(EGTA) mimicked the nifedipine effects. Our study shows that AT1and V1receptors stimulate [Ca2+]iby a common mechanism characterized by preferential action on voltage-gated L-type channels sensitive to dihydropyridines. Calcium signaling elicited by AT1receptors does not differ between SHR and WKY; thus the in vivo exaggerated reactivity may be dependent on interactions with other cell types, e.g., endothelium. In contrast, AVP produced larger changes in [Ca2+]iin arteriolar SMC from SHR, and such direct effects can account for the exaggerated renal blood flow responses.
Renal sulfate secretion is carbonic anhydrase dependent in a marine teleost, Pleuronectes americanus
Authors: Renfro J. Larry , Maren Thomas H. , Zeien Cristina , Swenson Erik R.
Published: December 2017

Though chemical assays indicate that carbonic anhydrase (CA) activity is present in marine teleost nephrons, CA inhibitors have no effect on urine pH or bicarbonate excretion, parameters typically CA dependent in almost all vertebrate groups. Because marine teleost renal sulfate secretion is associated with bicarbonate anion exchange, we investigated the effect of CA inhibition on transepithelial sulfate transport by flounder renal tubule primary monolayer cultures (PTC) and on renal sulfate secretion (Q˙so 4) by intact flounder. Both methazolamide and ethoxzolamide (10 μM) inhibited PTC secretory flux by ∼50%; reabsorptive sulfate flux, Na-dependent glucose transport, and transepithelial electrical resistance were unaffected. A CA inhibitor restricted to the extracellular space (10 μM polyoxyethylene-aminobenzolamide, 3.7 kDa) had no effect on PTC sulfate transport. Intravenous administration of methazolamide reducedQ˙so 4almost 40% and had no effect on glomerular filtration rate (GFR), urine flow rate, or Pi excretion rate. Serum pH was significantly reduced 0.2 units, whereas urine pH was unchanged. Together, the in vitro and in vivo results indicate that CA facilitates renal sulfate secretion in the seawater teleost.