bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppressing role of transforming growth factor-beta 1 on cathepsin activity in cultured kidney tubule cells
Authors: Ling H. , Vamvakas S. , Busch G. , Dammrich J. , Schramm L. , Lang F. , Heidland A.
Published: December 2017

Elevated expression and activity of transforming growth factor-beta 1 (TGF-beta 1) have been indicated in various renal diseases, implicating the potential involvement of this growth factor in the accumulation of extracellular matrix. To assess its potential role on protein turnover in tubule cells, we investigated in LLC-PK1 cells the effects of TGF-beta 1 on the activities of lysosomal cysteine proteinases cathepsins B, H, and L + B, which play a major role in the degradation of both cellular protein and extracellular matrix. The results show that 1-10 ng/ml TGF-beta 1 exerted inhibitory effects on cathepsin B and L + B activities, when applied to either the basolateral or apical membrane of these cells (basolateral side: B -23.2%, L + B -19.9%; apical side: B -28.2%, L + B -22.6%). Application of TGF-beta 1 to both sides enhanced suppression of the enzyme activities (B -37.8%, L + B -37.4%). This suppression of cathepsin activities was accompanied by a reduction of cellular protein degradation rate by 20.0% after 24 h and 51.7% after 48 h. Furthermore, TGF-beta 1 stimulated cellular protein synthesis by 50.0% after 48 h. The combined effects on protein turnover resulted in cellular hypertrophy: increases of both protein content and cell size after 48 h. Concerning the underlying mechanism, TGF-beta 1 did not induce a rise in intracellular Ca2+ concentration nor did Ca2+ channel blocker verapamil (10(-6) M) ameliorate the TGF-beta 1-induced inhibition of cathepsin activities. However, TGF-beta 1 raised the pH in lysosomes, which obviously impaired the acidic cysteine proteinases. In conclusion, the TGF-beta 1-induced cellular hypertrophy is caused by both enhanced protein synthesis and reduced protein breakdown. Suppression of cathepsin B and L + B activities mediated probably by an alkalinization in lysosomes is involved in the decreased protein degradation.
Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule
Authors: Roczniak A. , Burns K. D.
Published: December 2017

The proximal tubule contains the target for nitric oxide (NO), soluble guanylate cyclase, and has the capacity for NO production. Inhibition of renal NO synthesis reduces fractional excretion of lithium, suggesting an inhibitory effect of NO on proximal tubule Na+ transport. The present studies determined direct effects of donors of NO in rabbit proximal tubule. In both freshly isolated proximal tubule segments and in primary cultures of proximal tubule cells, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) caused dose-dependent increases in guanosine 3',5'-cyclic monophosphate (cGMP). SNAP was more potent than SNP in stimulating cGMP; this was associated with an enhanced production of nitrite, the stable end-product of NO. In rabbit proximal tubule cells, SNP or SNAP (10(-3) M) significantly inhibited the activity of the apical Na+/H+ exchanger, determined by assay of amiloride-sensitive 22Na+ uptake (% inhibition: SNP, 34.90 +/- 5.52%, P < 0.001; SNAP, 30.77 +/- 8.20%, P < 0.002). To determine the role of cGMP in mediating these effects, proximal tubule cells were incubated with the membrane-permeable analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). Na+/H+ exchange was significantly inhibited by 8-BrcGMP (10(3)M) (% inhibition: 32.40 +/- 9.06%: P < 0.05). The inhibitor of soluble guanylate cyclase, LY-83583, caused partial inhibition of SNP-stimulated cGMP generation and partly blocked the inhibitory effect of SNP on Na+/H+ exchange. Protein kinase A (PKA) activity was not stimulated by SNP, indicating that potential cross-activation of PKA by cGMP did not mediate the effects of NO donors. These data indicate that NO stimulates soluble guanylate cyclase in rabbit proximal tubule and causes inhibition of Na-/H+ exchange. This is at least partly mediated by generation of cGMP. We conclude that NO is an important autocrine or paracrine factor directly regulating Na+ transport in the proximal tubule.
Dual regulation of IL-1 beta-mediated matrix metalloproteinase-9 expression in mesangial cells by NF-kappa B and AP-1
Authors: Yokoo T. , Kitamura M.
Published: December 2017

Glomerular mesangial cells express matrix metalloproteinase-9 (MMP-9) in response to the proinflammatory cytokine interleukin-1 beta (IL-1 beta). To elucidate the signal transduction systems involved, we focused on the role of nuclear factor-kappa B (NF-kappa B) and activator protein-1 (AP-1), since the 5'-flanking region of MMP-9 gene contains binding sequences for these transacting molecules. In rat mesangial cells treated with an inhibitor of NF-kappa B, pyrrolidine dithiocarbamate, induction of MMP-9 by IL-1 beta was suppressed at both mRNA and protein levels. Mesangial cells stably transfected with a transdominant negative mutant of NF-kappa B also showed blunted induction of MMP-9. Transient transfection study with a kappa B reporter plasmid revealed that IL-1 beta indeed activated the kappa B site and that pyrrolidine dithiocarbamate abolished this activation. These results suggest that IL-1 beta induced MMP-9 via the stimulation of NF-kappa B pathway. to examine whether tyrosine kinase is involved in this pathway, mesangial cells were stimulated by IL-1 beta in the presence of a tyrosine kinase inhibitor genistein. This inhibitor dose dependently suppressed the expression of MMP-9, as well as the activation of the kappa B site by IL-1 beta, indicating the involvement of tyrosine kinase in the stimulation of NF-kappa B. Because mesangial cells stimulated by IL-1 beta transiently expressed c-fos and c-jun nRNAs prior to the expression of MMP-9, the role of these genes in mediating the IL-1 beta response was further examined. Transfection of mesangial cells with a c-jun antisense cDNA and treatment with a pharmacological inhibitor of c-Jun/ AP-1, curcumin, revealed that the induction of c-Jun/AP-1 is essential for the expression of MMP-9 by IL-1 beta. Although protein kinase C (PKC) is regarded as a potential inducer of AP-1, stimulation of mesangial cells with phorbol 12-myristate 13-acetate failed to induce MMP-9. Similarly, depletion of intracellular PKC did not obviously affect the induction of MMP-9 by IL-1 beta. These findings demonstrate that dual operation of tyrosine kinase-mediated NF-kappa B stimulation and c-Jun/AP-1 activation is essential to the induction of MMP-9 by IL-1 beta in cultured mesangial cells.
Compartmentation of amino acids in the rat kidney
Authors: Silbernagl S. , Volker K. , Dantzler W. H.
Published: December 2017

Amino acid concentrations ([AA]) were determined in cortical, outer and inner medullary (OM and IM), and papillary tissue of rat kidney (Cti, mmol/kg wet wt), in plasma (Cpl), and in urine. In all regions, Cti values were highest for Tau, Gly, and Glu-, making up 54-65% of the total [AA]:27, 21, and 11 mmol/kg wet wt in cortex, OM, and IM and papilla, respectively. Cortical cell water [AA] values (CcH2O, mmol/kgH2O) were between 12.4 (Tau) and 0.09 (Orn+), representing cell water-to-plasma water ratios (CcH2O/CpH2O) between 134 (Asp-) and 0.9 (Thr and Cit). Short-term water diuresis did not change the total tissue [AA] throughout the kidney. Treatment of the tissue with Triton X-100 instead of sulfosalicylic acid (SSA) resulted in much higher [AA], except for Glu-, Glu-NH2, Tau, and exogenous L-homoarginine+ (hoArg+). When hoArg+ was infused (leading to a Cpl = 5.9 mmol/l), Cti of hoArg+ was similar throughout the kidney (13-22 mmol/kg wet wt). In the presence of hoArg+, CcH2O/CpH2O of Arg+ rose 13-fold. We conclude that 1) AA contribute 20% to cytosolic osmolality in renal cortex, 2) total [AA] decreases from cortex to papilla, 3) cellular uptake of Tau and anionic AA must be rheogenic, whereas cationic AA (except for Arg+ in cortex) are passively distributed, and 4) AA do not seem to contribute quantitatively to short-term medullary osmotic adaptation during diuresis.
Endothelial Ca2+ in afferent arterioles during myogenic activity
Authors: Wagner A. J. , Holstein-Rathlou N. H. , Marsh D. J.
Published: December 2017

We measured endothelial Ca2+ concentration ([Ca2+]) in juxtamedullary afferent arterioles in response to step changes in perfusion pressure. Measurements were made with fura 2 using a fluorescence-imaging system designed to measure Ca2+ in whole tissues and minimize potentially harmful effects of ultraviolet illumination. The system yielded the typical sigmoidal relationship between fluorescence-emission ratio and [Ca2+] in vitro and was sensitive to endothelial Ca2+ transients elicited by bradykinin. Our goal was to determine whether changes in endothelial Ca2+ trigger events that cause myogenic vasoconstriction. Bradykinin, acting via endothelial cells, and sodium nitroprusside (SNP), acting independently, triggered vasodilation; bradykinin but not SNP increased endothelial Ca2+. Increased perfusion pressure caused vasoconstriction and a modest rise in endothelial Ca2+. Because the rise in Ca2+ with bradykinin initiates the vasodilation, the small rise in Ca2+ with pressure cannot cause vasoconstriction. Our results suggest that myogenic constriction is triggered from within vascular smooth muscle cells and that some other phenomenon, most likely increased shear stress, increases endothelial Ca2+ and modulates the myogenic reaction.
CFTR expression in cortical collecting duct cells
Authors: Todd-Turla K. M. , Rusvai E. , Naray-Fejes-Toth A. , Fejes-Toth G.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) is a adenosine 3',5'-cyclic monophosphate-activated chloride channel located in the apical membrane of many epithelial cells, and it may play a significant role in the kidney. Recent functional evidence from our laboratory suggests that CFTR may be expressed by the cortical collecting duct (CCD). Therefore, in the present study, the reverse transcription-polymerase chain reaction (RT-PCR) technique was utilized to detect CFTR mRNA in the M-1 mouse CCD cell line and in immunoselected rabbit CCD cells. Primers were constructed to amplify the cDNA sequence encoding the first nucleotide binding domain of CFTR. CFTR PCR products were obtained from both M-1 and rabbit CCD cDNA preparations. The identify of the product amplified from M-1 cell cDNA was confirmed by restriction digestion analysis. The rabbit CCD PCR product was sequenced, and its deduced amino acid sequence was found to be 97% homologous to the corresponding regions of human CFTR. The level of CFTR cDNA detected after 30 cycles of amplification of CCD cDNA was only 49 +/- 8 (n = 9) times lower than the level of beta-actin PCR product obtained from the same sample, suggesting that the levels of CFTR mRNA present in the CCD are physiologically relevant. Northern analysis, using a cRNA probe corresponding to the amplified region on the mRNA from CCD cells, revealed a single hybridizing species with a size of approximately 6.5 kb. Finally, CFTR PCR was performed with cDNA preparations originating from principal cells (PC), beta-intercalated cells (beta-ICC), and alpha-ICC obtained by fluorescence-activated cell sorting of rabbit CCD. CFTR PCR products were obtained from all three cell types, with the most abundant levels found in beta-ICC. beta-ICC expressed 25-fold (n = 4, P < 0.001) and 4.5-fold (n = 7, P < 0.001) higher levels than PC and alpha-ICC, respectively. This distribution pattern suggests that, within the CCD, CFTR plays a role primarily in beta-ICC function.
Contribution of actin cytoskeletal alterations to ATP depletion and calcium-induced proximal tubule cell injury
Authors: Nurko S. , Sogabe K. , Davis J. A. , Roeser N. F. , Defrain M. , Chien A. , Hinshaw D. , Athey B. , Meixner W. , Venkatachalam M. A. , Weinberg J. M.
Published: December 2017

The actin cytoskeleton of rabbit proximal tubules was assessed by deoxyribonuclease (DNase) binding, sedimentability of detergent-insoluble actin, laser-scanning confocal microscopy, and ultrastructure during exposure to hypoxia, antimycin, or antimycin plus ionomycin. One-third of total actin was DNase reactive in control cells prior to deliberate depolymerization, and a similar proportion was unsedimentable from detergent lysates during 2.5 h at 100,000 g. Tubules injured by hypoxia or antimycin alone, without glycine, showed Ca(2+)-dependent pathology of the cytoskeleton, consisting of increases in DNase-reactive actin, redistribution of pelletable actin, and loss of microvilli concurrent with lethal membrane damage. In contrast, tubules similarly depleted of ATP and incubated with glycine showed no significant changes of DNase-reactive actin or actin sedimentability for up to 60 min, but, nevertheless, developed substantial loss of basal membrane-associated actin within 15 min and disruption of actin cores and clubbing of microvilli at durations > 30 min. These structural changes that occurred in the presence of glycine were not prevented by limiting Ca2+ availability or pH 6.9. Very rapid and extensive cytoskeletal disruption followed antimycin-plus-ionomycin treatment. In this setting, glycine and pH 6.9 decreased lethal membrane damage but did not ameliorate pathology in the cytoskeleton or microvilli; limiting Ca2+ availability partially protected the cytoskeleton but did not prevent lethal membrane damage. The data suggest that both ATP depletion-dependent but Ca(2+)-independent, as well as Ca(2+)-mediated, processes can disrupt the actin cytoskeleton during acute proximal tubule cell injury; that both types of change occur, despite protection afforded by glycine and reduced pH against lethal membrane damage; and that Ca(2+)-independent processes primarily account for prelethal actin cytoskeletal alterations during simple ATP depletion of proximal tubule cells.
Upregulated renal adenosine A1 receptors augment PKC and glucose transport but inhibit proliferation
Authors: Coulson R. , Proch P. S. , Olsson R. A. , Chalfant C. E. , Cooper D. R.
Published: December 2017

Adenosine A1 receptor densities were increased in cultured LLC-PK1 and OK cells by chronic treatment with the adenosine receptor antagonists 1,3,7-trimethylxanthine (caffeine, 1 mM) and 1,3-dimethyl-8-cyclopentylxanthine [cyclopentyltheophylline (CPT), < or = 0.4 mM], respectively. The A1 receptor number per cell was increased twofold by 10-day treatments with 1 mM caffeine or 0.1 mM CPT, and the sodium-coupled glucose uptake was augmented twofold by 1 mM caffeine and sevenfold by 0.1 microM CPT (higher doses of CPT were progressively less stimulatory). Glucose uptake was blocked by acute (2-h) treatment with CPT, adenosine deaminase, or calphostin C. Caffeine (1 mM) or CPT (> or = 0.1 mM) inhibited cell proliferation for the first 10 days, then cell growth assumed a normal proliferative rate despite continued presence of antagonist. Cytosolic protein kinase C (PKC) beta-isoform immunoactivity and PKC-beta II mRNA were elevated at least twofold during 10 days of 0.1 mM CPT or 1 mM caffeine treatment. The sustained elevation in sodium-glucose symport and PKC activity observed with adenosine receptor antagonists was similar to acute (2-h) effects of the adenosine A1 agonist R(-)-N6-phenylisopropyladenosine (R-PIA, 0.1-1 microM). Moreover, cell proliferation was increased by adenosine (0.1 microM R-PIA), whereas Na-K-adenosinetriphosphatase activity was unaltered with chronic antagonist or acute adenosine treatments. Caffeine treatment may have some non-adenosine A1 receptor-mediated actions, because it slightly (30%) augmented protein kinase A activity. It is concluded that chronic exposure of proximal tubule cells to caffeine or CPT augments PKC and sodium-glucose transport but retards cell proliferation mainly via adenosine A1 receptor-mediated mechanisms.
Extracellular cGMP inhibits transepithelial sodium transport by LLC-PK1 renal tubular cells
Authors: Chevalier R. L. , Fang G. D. , Garmey M.
Published: December 2017

Both atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP) inhibit tubular sodium reabsorption by generation of guanosine 3',5'-cyclic monophosphate (cGMP). To determine the role of extracellular cGMP in this response, monolayers of porcine renal tubular LLC-PK1 cells were incubated for 5 min with ANP, SNP, cGMP, or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) (10 nM to 0.1 mM). Transepithelial sodium transport was measured as amiloride-inhibitable short-circuit current (Isc). Incubation of cell monolayers with 1 microM of ANP, cGMP, or 8-BrcGMP inhibited Isc by > 70%, as did SNP at 100 microM (P < 0.01). Adenosine 3',5'-cyclic monophosphate (0.1 mM) had no significant effect. Incubation of monolayers with 1 microM LY-83583 (an inhibitor of guanylyl cyclase), 10 microM probenecid (an organic anion transport inhibitor), or preincubation with 1 microgram/ml nocodazole (a microtubule disrupter) reduced extracellular accumulation of cGMP (P < 0.05) and abolished the SNP-mediated reduction of Isc. However, addition of these inhibitors did not affect reduction of Isc by exogenous cGMP. We conclude that SNP inhibits sodium transport by LLC-PK1 monolayers through generation of cGMP but that extrusion of cGMP out the cell is necessary for its effect.
Thrombin stimulates association of src homology domain containing adaptor protein Nck with pp125FAK
Authors: Choudhury G. G. , Marra F. , Abboud H. E.
Published: December 2017

Thrombin stimulates mitogenesis and tyrosine phosphorylation of several proteins in glomerular mesangial cells [T. Force, J. M. Kyriakis, J. Avruch, and J. V. Bonventre, J. Biol. Chem. 266: 6650-6656, 1991; and G. Grandaliano, G. Ghosh Choudhury, P. Biswas, and H. E. Abboud, Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36: F528-F536, 1994]. However, none of the tyrosine phosphorylated proteins have been identified. Here we show that thrombin stimulates phosphorylation of four major proteins of molecular masses 170, 125, 97, and 47 kDa in antiphosphotyrosine immunoprecipitates in vitro. Immunoblot analysis of antiphosphotyrosine immunoprecipitates from lysates of thrombin-treated cells with anti-Nck antibody revealed the presence of this src homology domain-containing adaptor molecule in the tyrosine-phosphorylated protein fraction. In addition, in thrombin-treated cells, direct immunoblotting of Nck immunoprecipitates with antiphosphotyrosine antibody showed no tyrosine phosphorylation of Nck. In these immunoprecipitates, we detected a 125-kDa tyrosine-phosphorylated protein. We identified this protein as pp125FAK (FAK, focal adhesion kinase) after analyzing Nck immunoprecipitates by anti-FAK immunoblotting. Treatment of mesangial cells with thrombin resulted in stimulation of the tyrosine kinase activity of pp125FAK in vitro. We conclude that activation of the cytoplasmic protein tyrosine kinase pp125FAK by thrombin stimulates its association with the src homology domain-containing adaptor protein Nck. This indicates that Nck is a direct target for FAK in the thrombin-induced signal transduction pathway.
Regulation of renal oatp mRNA expression by testosterone
Authors: Lu R. , Kanai N. , Bao Y. , Wolkoff A. W. , Schuster V. L.
Published: December 2017

A recently cloned cDNA encodes the so-called "organic anion-transporting polypeptide" (i.e., oatp), which is expressed in rat liver and in the kidney S3 proximal tubule. functional characterization of the cloned transporter indicates that estradiol 17 beta-D-glucuronide is a major substrate. Because the urinary excretion of glucuronidated steroids differs between males and females, we hypothesized that renal oatp expression may be under sex hormone control. Total RNA was isolated from male or female kidneys and probed with a digoxigenin-labeled oatp antisense riboprobe. Expression of oatp mRNA expression was quantitated by densitometry from Northern blots. Male kidneys expressed at least six distinct oatp transcripts (approximately 4.0, 3.2, 2.9, 2.6, 1.7, and 1.2 kb). Of these, the 3.2-kb band was consistently the strongest. In female rats, renal oatp mRNA expression was markedly less, such that only the 3.2-kb band was consistently detectable. Administering testosterone to female rats increased, and administering estradiol (E2) to male rats decreased, the steady-state levels of renal oatp mRNA. Gonadectomized male and female rats, as well as adrenalectomized male rats, were given pharmacological hormone replacement (testosterone, E2, or dexamethasone, respectively) by subcutaneous osmotic minipump. Castration of male rats produced a dramatic drop in the steady-state level of all six renal oatp transcripts. These were returned to normal by testosterone replacement. In contrast, there was no regulation of hepatic oatp mRNA expression by testosterone. Renal oatp mRNA expression in female rats was mildly increased by oophorectomy. Administration of E2 to oophorectomized females moderately suppressed renal oatp mRNA expression. Adrenalectomy produced a small decrease in oatp expression, but dexamethasone replacement failed to return expression to normal. We conclude that renal oatp mRNA expression is under strong (stimulatory) testosterone control and perhaps weaker (inhibitory) estrogen control. We speculate that this regulation of renal oatp expression is important in modulating the renal tubular secretion of conjugated E2.
Acid/base transport in a model of the proximal tubule brush border: impact of carbonic anhydrase
Authors: Krahn T. A. , Weinstein A. M.
Published: December 2017

A mathematical model of the brush border of the proximal tubule (T. A. Krahn, P. S. Aronson, and A. M. Weinstein. Bull. Math. Biol, 56: 459-490, 1994) has been extended by the inclusion of CO2 and H2CO3 as diffusible species and by the inclusion of finite rate constants for the hydration of CO2. This permits the simulation of carbonic anhydrase (CA) activity and its inhibition. We confirm the result of our previous study, which is that, in the presence of CA, the unstirred layer has only a modest effect on the observed formic acid permeability. CA inhibition results in disequilibrium pH gradients, and the effect of these gradients on formic acid permeability depends on the presence of other membrane transport proteins. We also examined the impact of CA activity on the flux of total CO2 through the brush border. Under physiological conditions, CA inhibition depressed NaHCO3 reabsorption through the brush border by interfering with the HCO3(-)-facilitated diffusion of CO2. However, the determination of brush-border CO2 permeability, using an imposed CO2 gradient, was relatively uninfluenced by CA activity. Finally, we inserted a kinetic representation of the Na+/H+ exchanger into the brush-border model. Even when luminal and cytosolic diffusion coefficients were increased 1,000-fold, there was no effect on brush-border Na+ flux. This suggests that variations in the unstirred layer cannot be responsible for the flow dependence of Na+ reabsorption.
Apical Na+ conductance in maturing rabbit principal cell
Authors: Satlin L. M. , Palmer L. G.
Published: December 2017

Net Na+ absorption in microperfused rabbit cortical collecting ducts (CCDs) is low during the 1st wk of postnatal life, increasing substantially thereafter [L. M. Satlin. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F57-F65, 1994]. To establish whether the low rate of Na+ absorption observed immediately after birth is due to a low apical Na+ permeability of the neonatal principal cell, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to estimate conductance, number (N), and open probability (Po) of apical Na+ channels in principal cells. With LiCl in the pipette and a NaCl or potassium gluconate solution, warmed to 37 degrees C, in the bath, inward currents with a conductance of approximately 11 pS (n = 23) were observed in 17% of cell-attached patches at 1 wk, 41% of patches at 2 wk, and 43% of patches at 5 wk. The mean N per patch in the 1st wk (0.22 +/- 0.09; n = 36) was significantly less than that observed in the 2nd (1.38 +/- 0.39; n = 34) and 5th (1.24 +/- 0.37; n = 21) wk of life. Po, studied at positive pipette voltages, was significantly lower in the 1st wk (0.085 +/- 0.035; n = 5) than in the 2nd wk (0.345 +/- 0.063; n = 9) and 5th wk (0.291 +/- 0.058; n = 4). To confirm that the 11-pS channel represented the amiloride-sensitive apical Na+ channel, cell-attached patches in CCDs isolated from 2-wk-old rabbits were studied with 0.5 microM amiloride added to the LiCl pipette solution. Amiloride led to > 90% reductions in mean open and closed times of the 11-pS conductance, consistent with blockade of the channel. These data indicate that N and Po of apical amiloride-sensitive Na+ channels in principal cells increase significantly after birth.
Calcium entry and mobilization signaling pathways in ANG II-induced renal vasoconstriction in vivo
Authors: Ruan X. , Arendshorst W. J.
Published: December 2017

The purpose of this study was to determine the relative importance of calcium signaling pathways in angiotensin II (ANG II)-induced renal vasoconstriction in vivo. Nifedipine was used to antagonize dihydropyridine-sensitive voltage-dependent calcium channels; BAY K 8644 was employed to activate these calcium channels. Intracellular calcium mobilization was evaluated using TMB-8 or heparin to inhibit calcium release from sarcoplasmic reticulum. Renal blood flow was measured by electromagnetic flowmetry in anesthetized euvolemic Wistar-Kyoto rats. The animals were pretreated with indomethacin to avoid interactions with prostaglandins. ANG II (2 ng) or BAY K 8644 (1 microgram) was injected into the renal artery to produce a transient 30–50% decrease in renal blood flow without affecting arterial pressure. Coadministration of nifedipine with BAY K 8644 produced dose-dependent inhibition of the maximum renal vasoconstriction elicited by BAY K 8644. The calcium-channel antagonist had similar effects on ANG II-induced renal vasoconstriction. Nifedipine exerted maximum inhibition by blocking 50% of the peak ANG II response. To evaluate intracellular calcium mobilization, TMB-8 or heparin was coadministered with ANG II. Each agent produced dose-dependent inhibition of up to 50% of the maximum renal vasoconstriction produced by ANG II. The inhibitory effects of nifedipine and TMB-8 were additive; neither agent had an effect when ANG II AT1 receptors were antagonized with losartan. These observations indicate that one-half of the ANG II-induced constriction of renal resistance vessels is mediated by voltage-dependent L-type calcium channels responsive to the dihydropyridine nifedipine. The remaining 50% of the renal vasoconstriction elicited by ANG II is mediated by inositol 1,4,5-trisphosphate-mediated calcium mobilization from intracellular sources. The additive nature of the inhibitory effects indicates distinct mechanisms involving calcium mobilization and calcium entry signaling pathways that are of equal importance in ANG II activation of AT1 receptors to trigger constriction of renal resistance vessels under basal conditions
Hypertonic activation and recovery of system A amino acid transport in renal MDCK cells
Authors: Chen J. G. , Coe M. , McAteer J. A. , Kempson S. A.
Published: December 2017

Amino compounds are abundant within the renal inner medulla, but their possible role during hypertonic stress is not clear. Renal epithelial Madin-Darby canine kidney cells were used to examine the osmoregulation of system A transport, a major Na(+)-dependent process for neutral amino acid transport. System A activity was markedly increased after 6 h of hypertonic challenge, and intracellular alanine content increased more than twofold. The activation of system A was reversed after 24 h of hypertonic challenge. This downregulation was accompanied by the activation of betaine transport, as measured by gamma-aminobutyric acid uptake. Extracellular betaine prevented the early activation of system A. The hypertonic activation of system A was blocked by actinomycin D and cycloheximide. When cells were returned to isotonic medium after hypertonic activation, the recovery of system A transport also was partially inhibited by actinomycin D and puromycin. The results are consistent with the possibility that hypertonicity, by disrupting a repressor protein, leads to increased synthesis of a system A-related protein. The isotonic recovery may require synthesis of new repressor proteins.
Salt intake modulates the developmental expression of renal kallikrein and bradykinin B2 receptors
Authors: el-Dahr S. , Yosipiv I. V. , Muchant D. G. , Chevalier R. L.
Published: December 2017

The mechanisms involved in the postnatal induction of renal kallikrein gene transcription and enzymatic activity are unknown. The present study was designed to test the hypothesis that salt (NaCl) intake regulates the ontogeny of renal kallikrein gene expression and enzymatic activity and urinary kallikrein excretion. Newborn rats were artificially fed via a gastric tube with a milk formula containing either normal (25 meq/l, same as in maternal milk) or high (145 meq/l) NaCl content from day 7 to 14 of postnatal life. High-salt feeding decreased renal kallikrein mRNA levels (P < 0.05) and kallikrein-like activity (P < 0.05) compared with rat pups on normal salt intake. However, urinary kallikrein excretion (Ukal) was not different on chronic high vs. normal salt intake. Furthermore, acute volume expansion (0.9% saline, 1% body wt iv) did not alter Ukal in either group of developing rats. In adult rats, 1% NaCl in the drinking water for 10 days decreased renal active kallikrein contents (P < 0.05) but did not alter kallikrein mRNA levels compared with pair-fed rats on normal salt diet. Acute volume expansion in adult rats decreased active Ukal in the high-salt group only (P < 0.05). High-salt feeding upregulated bradykinin B2 receptor mRNA in the developing rats (P < 0.05). We conclude that chronic salt loading suppresses the postnatal rise in renal kallikrein gene expression and enzymatic activity, indicating that sodium intake is an important factor in the maturation of renal kallikrein synthesis. The data also suggest that bradykinin B2 receptor gene expression in the developing kidney may be subject to reciprocal feedback regulation by endogenous kallikrein-kinin activity.
Glycosphingolipid synthesis and proliferation in a renal cell line grown in high glucose
Authors: el-Khatib M. , Radin N. S. , Shayman J. A.
Published: December 2017

We evaluated the role of sphingolipids as potential mediators of the renal epithelial growth response to growth in high-glucose media. The mouse cortical tubule (MCT) cell line was studied under high-glucose (450 mg/dl) and normal glucose (100 mg/dl) conditions. In cells plated at low-density, high-glucose media stimulated cell proliferation as measured by DNA, protein, and cell number and [3H]thymidine incorporation with a corresponding increase in glucosylceramide (GlcCer). The GlcCer synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocked the proliferative response to high glucose in association with a decrease in endogenous GlcCer and an increase in ceramide concentrations. Addition of N-acetylsphingosine, a short-chain homologue of natural ceramides, increased the levels of endogenous ceramides and inhibited proliferation. In contrast, the beta-glucosidase inhibitor conduritol B epoxide resulted in increased cell GlcCer but did not increase proliferation. We conclude that MCT cells proliferate when grown in the presence of high glucose. GlcCer levels increase and ceramide levels decrease in concert with the proliferative response. Pharmacologically increasing endogenous levels of ceramide inhibits the proliferative response to high glucose, but increasing endogenous levels of GlcCer does not stimulate proliferation.
Cloning and expression of the rabbit prostaglandin EP4 receptor
Authors: Breyer R. M. , Davis L. S. , Nian C. , Redha R. , Stillman B. , Jacobson H. R. , Breyer M. D.
Published: December 2017

Prostaglandin E2 (PGE2) is an important regulator of systemic hemodynamics and epithelial ion transport. To further investigate the mechanism of PGE2 action, a clone encoding a PGE2 receptor was isolated from a rabbit kidney cortex cDNA library. Expression of the full-length cDNA in COS-1 cells yielded a ligand-binding profile typical for a butaprost-insensitive Gs-coupled E-prostanoid (EP) receptor. Misoprostol-free acid, a receptor-selective PGE analogue, produced concentration-dependent increases in adenosine 3',5'-cyclic monophosphate production. The data are consistent with the receptor being an EP4 subtype. Ribonuclease protection assays demonstrated that this receptor gene is highly expressed in intestine, uterus, and thymus, with lower but significant expression in kidney, whole adrenal, lung, spleen, and stomach. In situ hybridization in kidney revealed intense hybridization to glomeruli and urothelium of the renal pelvis. This prostanoid receptor was also highly expressed in the duodenal epithelium and adrenal cortex. The tissue distribution suggests a functional role for this receptor in mediating glomerular effects of PGE2 and effects on aldosterone secretion, intestinal transport, and immune function.
Upregulation of endothelial constitutive NOS: a major role in the increased NO production in cirrhotic rats
Authors: Martin P. Y. , Xu D. L. , Niederberger M. , Weigert A. , Tsai P. , St John J. , Gines P. , Schrier R. W.
Published: December 2017

Nitric oxide (NO) is postulated to mediate the peripheral arterial vasodilation in cirrhosis. However, it is not known which isoform of the nitric oxide synthase (NOS) is involved in the increased production of NO. This study was therefore undertaken to examine the expression of the NOS isoforms in arteries of cirrhotic rats compared with controls. Cirrhosis was induced by CCl4, and vessels were harvested for immunoblots using antibodies against inducible NOS (iNOS) and endothelial constitutive NOS (ecNOS). Endothelial cells were used as controls for ecNOS, and vascular smooth muscle cells treated with lipopolysaccharide or septic rats were used for iNOS controls. The results demonstrated an upregulation of ecNOS in both the aortas and mesenteric arteries of cirrhotic compared with control rats. Chronic inhibition of NOS decreased ecNOS in cirrhotic vessels. Although iNOS mRNA was found by reverse transcription-polymerase chain reaction in arteries of cirrhotic rats, iNOS protein was not detectable by immunoblotting compared with septic rats, suggesting a low vascular level of this isoform. In conclusion, the ecNOS seems to play a major role in the increased NO production in cirrhotic rats, whereas the role of iNOS remains elusive.
Natriuresis caused by increased carotid Na+ concentration after renal denervation
Authors: Emmeluth C. , Goetz K. L. , Drummer C. , Gerzer R. , Forssmann W. G. , Bie P.
Published: December 2017

The renal effects of a selective estimated 3 mM increase in the concentration of Na+ in blood perfusing the brain was investigated in conscious dogs with surgically denervated kidneys. In split-infusion experiments the concentration of Na+ in carotid plasma was increased by a bilateral carotid infusion of hypertonic NaCl combined with an infusion of distilled water into the caval vein. In control experiments the same load of NaCl and water was administered as an isotonic solution into the carotid and jugular vessels. Peak rate of Na+ excretion was significantly higher during split infusion (156 +/- 19 mumol/min) compared with control (89 +/- 14 mumol/min). Renal excretion of urodilatin increased in both series. Renal excretion of endothelin immunoreactivity increased significantly more during split infusion (20 +/- 6 pg/min) than during control (9 +/- 3 pg/min). It is concluded that the natriuretic response to minute increases in Na+ concentration of carotid plasma is intact after renal denervation. Furthermore, endothelin may be involved in the excess excretion observed.
Gentamicin inhibits carrier-mediated dipeptide transport in kidney
Authors: Skopicki H. A. , Zikos D. , Sukowski E. J. , Fisher K. A. , Peterson D. R.
Published: December 2017

The effect of gentamicin on transport of pyroglutamylhistidine (pGlu-His) was examined in rabbit renal brush-border membrane vesicles (BBMV). Gentamicin, an aminoglycoside antibiotic, is limited in its usage because of nephrotoxicity characterized in part by transport defects in the proximal tubule. Since there is no information regarding the effects of gentamicin on renal peptide carriers, uptake of [3H]pGlu-His was measured in BBMV following either in vivo or in vitro exposure to the antibiotic. One hour after in vivo administration, the maximal rate (Vmax) for pGlu-His transport was significantly reduced in isolated membrane vesicles washed free of the drug, but the apparent Michaelis constant (Km) was unaltered. Coincubation of membranes with gentamicin during measurements of pGlu-His uptake had a similar effect, causing a significant decrease in the Vmax but not the Km of transport. The addition of 5 mM magnesium to the uptake medium prevented the in vitro but not the in vivo effect. The data indicate that high doses of gentamicin inhibit the capacity but not the affinity of dipeptide transport in the kidney, prior to morphological changes which typify acute tubular necrosis. The in vitro effect is rapid and involves a direct action of gentamicin on the brush-border membrane. The in vivo experiments show that toxicity may be prolonged and remains following removal of the drug from the renal brush border.
Spatial and temporal variations in pacemaking and conduction in the isolated renal pelvis
Authors: Lammers W. J. , Ahmad H. R. , Arafat K.
Published: December 2017

In renal pelvis preparations isolated from the sheep, the location of the pacemaker and the pathway of conduction of the electrical impulse in the pelvis were analyzed in detail. An electrophysiological acquisition system was used allowing simultaneous recordings from 240 extracellular electrodes. Reconstruction of the spread of activity showed that the site of the pelvis pacemaker was, in virtually all cases, located at the pelvicalyceal border and never in the body of the pelvis or in the area of the pelviureteric junction. One single pacemaker was responsible for a particular spread of activation, and fusion of activity originating from two or more pacemakers did not place. Furthermore, spontaneous shifts of the pacemaker could occur from one site to another along the pelvicalyceal border. Conduction from the site of the current pacemaker to the pelviureteric junction and the ureter was slow, inhomogeneous, and contorted. Multiple instances of partial or total conduction block were seen at all levels in the pelvis and were not restricted to the pelviureteric junction. The occurrence of the conduction block did not seem to be related to the length of the preceding interval, implying that the refractory period did not play a major role in the genesis of intrapelvic conduction block. In conclusion, high-resolution mapping of the renal pelvis is possible and reveals location and behavior of the pacemaker and documents inhomogeneities in conduction and conduction block.
Evidence of calcium phosphate supersaturation in the loop of Henle
Authors: Asplin J. R. , Mandel N. S. , Coe F. L.
Published: December 2017

We have used published rat micropuncture data to construct a matrix of ion concentrations along the rat nephron. With an iterative computer model of known ion interactions, we calculated relative supersaturation ratios in all nephron segments. The collecting ducts and urine showed expected supersaturation with stone-forming salts. Fluid in the thin segment of the loop of Henle may be supersaturated with calcium carbonate and calcium phosphate under certain conditions. Because calculations cannot predict the actual course of crystallization, we made solutions to mimic, in vitro, presumed conditions in the loop of Henle. The solid phases that formed were analyzed by X-ray powder diffraction, electron microprobe, and infrared spectroscopy. All samples were identified as poorly crystallized or immature apatite. The descending limb of Henle's loop creates a unique condition as it extracts water but not sodium, bicarbonate, calcium, or phosphate, giving a calcium concentration at the bend of 3 mM, pH 7.4, and a phosphate concentration that varies from 0.8 to 48 mM, depending on parathyroid hormone and dietary phosphate. We conclude that conditions in the thin segment potentially could create a solid calcium phosphate phase, which may initiate nucleation of calcium oxalate salts in the collecting ducts, potentiating nephrolithiasis and nephrocalcinosis.
Evidence for dual signaling pathways for V2 vasopressin receptor in rat inner medullary collecting duct
Authors: Ecelbarger C. A. , Chou C. L. , Lolait S. J. , Knepper M. A. , DiGiovanni S. R.
Published: December 2017

Previous studies have demonstrated that both the V2-receptor agonist, 1-desamino-8-D-arginine vasopressin (dDAVP), and the V1a-receptor agonist, [Phe2, Orn8]vasotocin (PO-VT), increase intracellular calcium concentration ([Ca2+]i) in the rat inner medullary collecting duct (IMCD). The present studies were done to clarify the receptor subtype(s) responsible for calcium mobilization. Measurements of [Ca2+]i, using fura 2 in microdissected IMCD segments, confirmed that arginine vasopressin (AVP), dDAVP, and PO-VT stimulate an increase in [Ca2+]i and that the response to all three agents could be blocked by the specific V2-receptor antagonist, [d(CH2)5(1),D-Ile2, Ile4, Arg8]vasopressin (II-VP). These results would suggest that all three agents acted through the V2 receptor. Furthermore, we showed that PO-VT increased cAMP production in IMCD suspensions and water permeability in isolated perfused tubules. These responses were also blocked by II-VP, indicating that PO-VT is also a V2 agonist in the IMCD. Finally, we utilized the quantitative reverse transcription-polymerase chain reaction technique of Wiesner (Nucleic Acids Res. 20: 5863-5864, 1992) to evaluate V1a and V2 mRNA levels in rat collecting duct. In terminal IMCD, we estimated > 30 copies/cell for V2 receptor mRNA but less than 1 copy/cell of V1a receptor mRNA, thus there is littler or no V1a mRNA expression in the terminal IMCD. These results suggest that calcium mobilization in response to vasopressin analogues is associated with the V2 receptor and that the V2 receptor is linked to both adenylyl cyclase and calcium mobilization in the rat IMCD.
Interleukin-1 beta activates c-jun NH2-terminal kinase subgroup of mitogen-activated protein kinases in mesangial cells
Authors: Guan Z. , Tetsuka T. , Baier L. D. , Morrison A. R.
Published: December 2017

We investigated whether JNK is activated by interleukin-1 beta (IL-1 beta) in mesangial cells. We performed in-gel kinase assays with His-c-jun-(1-79), which contains the amino-terminal activation domain of c-jun and a mutant His-c-jun in which Ser-63 and Ser-73 of His-c-jun were mutated to Ala as the substrates. JNK1 (p45) and JNK2 (p54) isoforms phosphorylated His-c-jun in mesangial cells. IL-1 beta produced a time- and concentration-dependent increase in JNK activity. IL-1 beta did not phosphorylated the mutant, His-c-jun. The IL-1 beta-activated JNK activity was independent of serum and suppressed by neither tyrosine kinase inhibitors nor protein kinase C inhibitors. JNK was also stimulated by anisomycin and okadaic acid but not by phorbol 12-myristate 13-acetate. The protein synthesis inhibitors and okadaic acid potentiated the IL-1 beta-induced JNK activity. Together, these studies indicate that the novel JNK group of protein kinases may play an important role in the signal transduction pathway initiated by proinflammatory cytokines, such as IL-1 beta in mesangial cells.
Molecular cloning and functional expression of a sodium-dicarboxylate cotransporter from human kidney
Authors: Pajor A. M.
Published: December 2017

The renal Na(+)-dicarboxylate cotransporter reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the glomerular filtrate. The present study describes the cloning and characterization of the human renal Na(+)-dicarboxylate cotransporter, hNaDC-1. The amino acid sequence of hNaDC-1 is 78% identical to the rabbit renal Na(+)-dicarboxylate cotransporter, NaDC-1, and 42% identical to the rat renal Na(+)-sulfate transporter, NaSi-1. The carboxy terminus of hNaDC-1 protein contains two N-glycosylation sites that appear to be utilized. Xenopus oocytes injected with hNaDC-1 cRNA expressed a low-affinity Na(+)-dependent dicarboxylate transporter with Michaelis constant (Km) for succinate around 0.4 mM. The transport of succinate by hNaDC-1 was insensitive to the pH of the medium, whereas the transporter of citrate was stimulated by acidic pH. Northern blot analysis indicates that hNaDC-1 mRNA is found in both kidney and intestine. The gene for hNaDC-1 was localized to chromosome 17. This study provides the first demonstration that the human kidney contains a low-affinity Na(+)-dicarboxylate cotransporter with properties that resemble those of the Na(+)-dicarboxylate cotransporter on the apical membrane of the rabbit renal proximal tubule.
Functional roles of apical membrane Na+/H+ exchange in rat medullary thick ascending limb
Authors: Good D. W. , Watts B. A.
Published: December 2017

The medullary thick ascending limb (MTAL) of the rat actively absorbs both HCO3- and ammonium. The roles of apical membranes Na+/H+ exchange in these processes and in determining steady-state intracellular pH (pHi) were examined in MTAL perfused in vitro with solutions containing 146 mM Na+ and 25 mM HCO3- (pH 7.4). Addition of 1 mM amiloride or 50 microM ethylisopropylamiloride (EIPA) to the lumen decreased HCO3- absorption (JHCO3) from 10.6 +/- 0.5 to 2.3 +/- 0.3 pmol.min-1.mm-1 (P < 0.001) and pHi from 7.10 +/- 0.02 to 6.86 +/- 0.03 (P < 0.001). The combination of lumen Na+ replacement plus amiloride abolished JHCO3. Chronic metabolic acidosis (CMA) caused a 32% increase in JHCO3 that was inhibited by luminal amiloride. Addition of 4 mM NH4Cl to perfusate and bath markedly decreased pHi (from 7.10 to 6.70) but did not stimulate luminal H+ secretion as assessed by HCO3- absorption. With 4 mM NH4Cl in perfusate and bath, luminal addition of amiloride decreased pHi from 6.70 +/- 0.06 to 6.50 +/- 0.05 (P < 0.005) but had no effect on net ammonium absorption. These results demonstrate that 1) apical membrane Na+/H+ exchange mediates virtually all of HCO3- absorption and is an important determinant of steady-state pHi in the MTAL; 2) the adaptive increase in HCO3- absorption in CMA is mediated by an increase in apical membrane Na+/H+ exchange; 3) ammonium markedly acidifies the cells but does not stimulate luminal acidification, suggesting that pHi is not a predominant influence on apical Na+/H+ exchange activity and that H+ generated in the cells as the result of transcellular ammonium absorption is extruded across the basolateral membrane; and 4) apical membrane Na+/H+ exchange is not important for ammonium absorption.