bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C
Authors: Lu Run , Chan Brenda S. , Schuster Victor L.
Published: December 2017

Conserved from fish to mammals, renal proximal tubule organic anion secretion plays an important role in drug and xenobiotic elimination. Studies with the model substrate p-aminohippurate (PAH) have suggested that a basolateral PAH/α-ketoglutarate exchanger imports diverse organic substrates into the proximal tubule prior to apical secretion. cDNAs encoding PAH transporters have been cloned recently from rat and flounder. Here we report the cloning of a highly similar human PAH transporter (hPAHT) from human kidney. By Northern blot analysis and EST database searching, hPAHT mRNA was detected in kidney and brain. PCR-based monochromosomal somatic cell hybrid mapping placed the hPAHT gene on chromosome 11. When expressed transiently in vitro, hPAHT catalyzed time-dependent and saturable [3H]PAH uptake ( Kmof ∼5 μM). Preincubation with unlabeled α-ketoglutaric or with glutaric acid stimulated tracer PAH uptake, and preincubation with unlabeled PAH stimulated tracer α-ketoglutarate uptake, results that are consistent with PAH/α-ketoglutarate exchange. Several structurally diverse organic anions cis-inhibited PAH uptake. Like rat OAT1 organic anion transporter, hPAHT was inhibited by furosemide, indomethacin, probenecid, and α-ketoglutarate. Unlike OAT1, hPAHT was not inhibited by prostaglandins or methotrexate (MTX). Moreover, tracer PGE2and MTX were not transported, indicating that the substrate specificity for transport by hPAHT is not broad. PAH uptake was inhibited by phorbol 12-myristate 13-acetate (PMA) in a dose- and time-dependent fashion, but not by the inactive 4α-phorbol-12,13 didecanoate. PMA-induced inhibition was blocked by staurosporine. Thus the protein kinase C-mediated inhibition of basolateral organic anion entry previously reported in intact tubules is likely due, at least in part, to direct modulation of the PAH/α-ketoglutarate exchanger.
Developmental expression of sodium entry pathways in rat nephron
Authors: Schmitt Roland , Ellison David H. , Farman Nicolette , Rossier Bernard C. , Reilly Robert F. , Reeves W. Brian , Oberbäumer Ilse , Tapp Rosemarie , Bachmann Sebastian
Published: December 2017

During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Picotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11β-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.
Contribution of endothelin to renal vascular tone and autoregulation in the conscious dog
Authors: Berthold Heike , Münter Klaus , Just Armin , Kirchheim Hartmut R. , Ehmke Heimo
Published: December 2017

Exogenous endothelin-1 (ET-1) is a strong vasoconstrictor in the canine kidney and causes a decrease in renal blood flow (RBF) by stimulating the ETA receptor subtype. The aim of the present study was to investigate the role of endogenously generated ET-1 in renal hemodynamics under physiological conditions. In six conscious foxhounds, the time course of the effects of the selective ETA receptor antagonist LU-135252 (10 mg/kg iv) on mean arterial blood pressure (MAP), heart rate (HR), RBF, and glomerular filtration rate (GFR), as well as its effects on renal autoregulation, were examined. LU-135252 increased RBF by 20% (from 270 ± 21 to 323 ± 41 ml/min, P < 0.05) and HR from 76 ± 5 to 97 ± 8 beats/min ( P< 0.05), but did not alter MAP, GFR, or autoregulation of RBF and GFR. Since a number of interactions between ET-1 and the renin-angiotensin system have been reported previously, experiments were repeated during angiotensin converting enzyme (ACE) inhibition by trandolaprilat (2 mg/kg iv). When ETA receptor blockade was combined with ACE inhibition, which by itself had no effects on renal hemodynamics, marked changes were observed: MAP decreased from 91 ± 4 to 80 ± 5 mmHg ( P < 0.05), HR increased from 85 ± 5 to 102 ± 11 beats/min ( P < 0.05), and RBF increased from 278 ± 23 to 412 ± 45 ml/min ( P< 0.05). Despite a pronounced decrease in renal vascular resistance over the entire pressure range investigated (40–100 mmHg), the capacity of the kidneys to autoregulate RBF was not impaired. The GFR remained completely unaffected at all pressure levels. These results demonstrate that endogenously generated ET-1 contributes significantly to renal vascular tone but does not interfere with the mechanisms of renal autoregulation. If ETAreceptors are blocked, then the vasoconstrictor effects of ET-1 in the kidney are compensated for to a large extent by an augmented influence of ANG II. Thus ET-1 and ANG II appear to constitute a major interrelated vasoconstrictor system in the control of RBF.
Renal and hemodynamic effects of losartan in conscious dogs during controlled mechanical ventilation
Authors: Krebs Martin O. , Kröhn Thorsten , Boemke Willehad , Mohnhaupt Rainer , Kaczmarczyk Gabriele
Published: December 2017

In 12 conscious dogs, we investigated whether the angiotensin II-receptor antagonist losartan increases renal sodium excretion and urine volume during controlled mechanical ventilation (CMV) with positive end-expiratory pressure. In four experimental protocols, the dogs were extracellular volume (ECV) expanded (electrolyte solution, 0.5 ml ⋅ kg−1 ⋅ min−1iv) or not and received losartan (100 μg ⋅ kg−1 ⋅ min−1iv) or not. They breathed spontaneously during the 1st and 4th hour and received CMV with positive end-expiratory pressure (mean airway pressure 20 cmH2O) during the 2nd and 3rd hours. In the expansion group, dogs with losartan excreted ∼18% more sodium (69 ± 7 vs. 38 ± 5 μmol ⋅ min−1 ⋅ kg−1) and 15% more urine during the 2 h of CMV because of a higher glomerular filtration rate (5.3 ± 0.3 vs. 4.5 ± 0.2 ml ⋅ min−1 ⋅ kg−1) and the tubular effects of losartan. In the group without expansion, sodium excretion (2.0 ± 0.6 vs. 2.6 ± 1.0 μmol ⋅ min−1 ⋅ kg−1) and glomerular filtration rate (3.8 ± 0.3 vs. 3.8 ± 0.4 ml ⋅ min−1 ⋅ kg−1) did not change, and urine volume decreased similarly in both groups during CMV. Plasma vasopressin and aldosterone increased in both groups, and plasma renin activity increased from 4.9 ± 0.7 to 7.8 ± 1.3 ng ANG I ⋅ ml−1 ⋅ h−1during CMV in nonexpanded dogs without losartan. Mean arterial pressure decreased by 10 mmHg in nonexpanded dogs with losartan. In conclusion, losartan increases sodium excretion and urine volume during CMV if the ECV is expanded. If the ECV is not expanded, a decrease in mean arterial blood pressure and/or an increase in aldosterone and vasopressin during CMV attenuates the renal effects of losartan.
AVP inhibits LPS- and IL-1β-stimulated NO and cGMP via V1receptor in cultured rat mesangial cells
Authors: Umino Tetsuo , Kusano Eiji , Muto Shigeaki , Akimoto Tetsu , Yanagiba Satoru , Ono Shuichi , Amemiya Morimasa , Ando Yasuhiro , Homma Sumiko , Ikeda Uichi , Shimada Kazuyuki , Asano Yasushi
Published: December 2017

The present study examined how arginine vasopressin (AVP) affects nitric oxide (NO) metabolism in cultured rat glomerular mesangial cells (GMC). GMC were incubated with test agents and nitrite, and intracellular cGMP content, inducible nitric oxide synthase (iNOS) mRNA, and iNOS protein were analyzed by the Griess method, enzyme immunoassay, and Northern and Western blotting, respectively. AVP inhibited lipopolysaccharide (LPS)- and interleukin-1β (IL-1β)-induced nitrite production in a dose- and time-dependent manner, with concomitant changes in cGMP content, iNOS mRNA, and iNOS protein. This inhibition by AVP was reversed by V1- but not by oxytocin-receptor antagonist. Inhibition by AVP was also reproduced on LPS and interferon-γ (IFN-γ). Protein kinase C (PKC) inhibitors reversed AVP inhibition, whereas PKC activator inhibited nitrite production. Although dexamethasone and pyrrolidinedithiocarbamate (PDTC), inhibitors of nuclear factor-κB, inhibited nitrite production, further inhibition by AVP was not observed. AVP did not show further inhibition of nitrite production with actinomycin D, an inhibitor of transcription, or cycloheximide, an inhibitor of protein synthesis. In conclusion, AVP inhibits LPS- and IL-1β-induced NO production through a V1receptor. The inhibitory action of AVP involves both the activation of PKC and the transcription of iNOS mRNA in cultured rat GMC.
Tonic and phasic influences of nitric oxide on renal blood flow autoregulation in conscious dogs
Authors: Just Armin , Ehmke Heimo , Wittmann Uwe , Kirchheim Hartmut R.
Published: December 2017

The aim of this study was to investigate the influence of the mean level and phasic modulation of NO on the dynamic autoregulation of renal blood flow (RBF). Transfer functions were calculated from spontaneous fluctuations of RBF and arterial pressure (AP) in conscious resting dogs for 2 h under control conditions, after NO synthase (NOS) inhibition [ NG-nitro-l-arginine methyl ester hydrochloride (l-NAME)] and afterl-NAME followed by a continuous infusion of an NO donor [ S-nitroso- N-acetyl-dl-penicillamine (SNAP)]. After l-NAME ( n = 7) AP was elevated, heart rate (HR) and RBF were reduced. The gain of the transfer function above 0.08 Hz was increased, compatible with enhanced resonance of the myogenic response. A peak of high gain around 0.03 Hz, reflecting oscillations of the tubuloglomerular feedback (TGF), was not affected. The gain below 0.01 Hz, was elevated, but still less than 0 dB, indicating diminished but not abolished autoregulation. Afterl-NAME and SNAP ( n = 5), mean AP and RBF were not changed, but HR was slightly elevated. The gain above 0.08 Hz and the peak of high gain at 0.03 Hz were not affected. The gain below 0.01 Hz was elevated, but smaller than 0 dB. It is concluded that NO may help to prevent resonance of the myogenic response depending on the mean level of NO. The feedback oscillations of the TGF are not affected by NO. NO contributes to the autoregulation below 0.01 Hz due to phasic modulation independent of its mean level.
Localization of rat CLC-K2 chloride channel mRNA in the kidney
Authors: Yoshikawa Momono , Uchida Shinichi , Yamauchi Atsushi , Miyai Akiko , Tanaka Yujiro , Sasaki Sei , Marumo Fumiaki
Published: December 2017

To gain insight into the physiological role of a kidney-specific chloride channel, CLC-K2, the exact intrarenal localization was determined by in situ hybridization. In contrast to the inner medullary localization of CLC-K1, the signal of CLC-K2 in our in situ hybridization study was highly evident in the superficial cortex, moderate in the outer medulla, and absent in the inner medulla. To identify the nephron segments where CLC-K2 mRNA was expressed, we performed in situ hybridization of CLC-K2 and immunohistochemistry of marker proteins (Na+/Ca2+exchanger, Na+-Cl−cotransporter, aquaporin-2 water channel, and Tamm-Horsfall glycoprotein) in sequential sections of a rat kidney. Among the tubules of the superficial cortex, CLC-K2 mRNA was highly expressed in the distal convoluted tubules, connecting tubules, and cortical collecting ducts. The expression of CLC-K2 in the outer and inner medullary collecting ducts was almost absent. In contrast, a moderate signal of CLC-K2 mRNA was observed in the medullary thick ascending limb of Henle’s loop, but the signal in the cortical thick ascending limb of Henle’s loop was low. These results clearly demonstrated that CLC-K2 was not colocalized with CLC-K1 and that its localization along the nephron segments was relatively broad compared with that of CLC-K1.
Renal arteriolar Na+/Ca2+exchange in salt-sensitive hypertension
Authors: Nelson Lawrence D. , Unlap M. Tino , Lewis James L. , Bell P. Darwin
Published: December 2017

The present studies were performed to assess Na+/Ca2+exchange activity in afferent and efferent arterioles from Dahl/Rapp salt-resistant (R) and salt-sensitive (S) rats. Renal arterioles were obtained by microdissection from S and R rats on either a low-salt (0.3% NaCl) or high-salt (8.0% NaCl) diet. On the high-salt diet, S rats become markedly hypertensive. Cytosolic calcium concentration ([Ca2+]i) was measured in fura 2-loaded arterioles bathed in a Ringer solution in which extracellular Na (Nae) was varied from 150 to 2 mM (Na was replaced with N-methyl-d-glucamine). Baseline [Ca2+]iwas similar in afferent arterioles of R and S rats fed low- and high-salt diet. The change in [Ca2+]i(Δ[Ca2+]i) during reduction in Naefrom 150 to 2 mM was 80 ± 10 and 61 ± 3 nM (not significant) in afferent arterioles from R rats fed the low- and high-salt diet, respectively. In afferent arterioles from S rats on a high-salt diet, Δ[Ca2+]iduring reductions in Naefrom 150 to 2 mM was attenuated (39 ± 4 nM) relative to the Δ[Ca2+]iof 79 ± 13 nM ( P < 0.05) obtained in afferent arterioles from S rats on a low-salt diet. In efferent arterioles, baseline [Ca2+]iwas similar in R and S rats fed low- and high-salt diets, and Δ[Ca2+]iin response to reduction in Naewas also not different in efferent arterioles from R and S rats fed low- or high-salt diets. Differences in regulation of the exchanger in afferent arterioles of S and R rats were assessed by determining the effects of protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA, 100 nM) on Δ[Ca2+]iin response to reductions in Naefrom 150 to 2 mM. PMA increased Δ[Ca2+]iin afferent arterioles from R rats but not from S rats. These results suggest that Na+/Ca2+exchange activity is suppressed in afferent arterioles of S rats that are on a high-salt diet. In addition, there appears to be a defect in the PKC-Na+/Ca2+exchange pathway that might contribute to altered [Ca2+]iregulation in this important renal vascular segment in salt-sensitive hypertension.
Expression of c-ret promotes morphogenesis and cell survival in mIMCD-3 cells
Authors: O’Rourke Dawn A. , Sakurai Hiroyuki , Spokes Katherine , Kjelsberg Crystal , Takahashi Masahide , Nigam Sanjay , Cantley Lloyd
Published: December 2017

c-Ret, a protein tyrosine kinase receptor, and its ligand glial-derived neurotropic factor (GDNF) are critical for early regulation of ureteric bud development and nephrogenesis. To address whether c-ret directly initiates epithelial cell morphogenesis, the c-ret receptor was expressed in murine inner medullary collecting duct cells (mIMCD-3, a cell line of ureteric bud origin, which has no detectable endogenous c-ret expression). Stable expression of wild-type c-ret was found to yield a constitutively tyrosine-phosphorylated receptor, with no change after the addition of GDNF. Examination of mRNA from these cells demonstrated the message for endogenous GDNF, suggesting that c-ret was potentially being constitutively activated by an autocrine mechanism. When mIMCD-3 cells stably expressing the phosphorylated c-ret receptor were cultured in a type I collagen matrix, they exhibited little GDNF-independent or -dependent branching process formation at early time points compared with the known morphogen hepatocyte growth factor (HGF) (48 h; control, 0.33 ± 0.33; GDNF, 1.0 ± 0.58, P = nonsignificant; and HGF, 6.33 ± 0.33 processes/20 cell clusters, P < 0.001), whereas extended culture (7 days) under serum-free conditions revealed a marked increase in cell survival and the spontaneous development of rudimentary branching process formation. Extended culture (7 days) of c-ret-expressing clones in type I collagen with the epithelial morphogens HGF and/or epidermal growth factor (EGF) resulted in the development of complex three-dimensional spiny cysts, whereas parental mIMCD-3 cells died under these conditions. We conclude that activated c-ret appears to mediate epithelial morphogenesis by prolonging cell survival and, in conjunction with activation of the morphogenic receptors c-met and the EGF receptor, initiates the events required for very early branching morphogenesis.
Bradykinin inhibits ceramide production and activates phospholipase D in rabbit cortical collecting duct cells
Authors: Liu Gele , Kleine Leonard , Nasrallah Rania , Hébert Richard L.
Published: December 2017

Recent reports suggest that inflammatory cytokines, growth factors, and vasoconstrictor peptides induce sphingomyelinase (SMase) activity. This results in the hydrolysis of sphingomyelin (SM) into ceramide, which is implicated in various cellular functions. Although ceramide regulates phospholipase D (PLD) activity, there is controversy about this relationship. Thus we investigated whether the effect of bradykinin (BK), a proinflammatory factor and vasodilator, was mediated by ceramide signal transduction and by PLD. In rabbit cortical collecting duct (RCCD) cells, BK increased SM levels and decreased ceramide levels in a time-dependent manner. Thus SMase activity was inhibited by BK. Also, the production of ceramide was regulated in a concentration-dependent manner. The BK-B1antagonist [Lys-des-Arg9,Leu8]BK did not affect ceramide signal transduction but the BK-B2antagonist (Hoe-140) blocked the effect of BK on SMase, suggesting that the BK-B2receptor mediates BK-induced inhibition of ceramide generation. Our results show that exogenous SMase significantly hydrolyzed endogenous SM to form ceramide and weakly activated PLD. In contrast, BK induced a significant activation of PLD. However, additive effects of BK and ceramide on PLD activity were not observed. We concluded that in RCCD cells, the BK-induced second messengers ceramide and phosphatidic acid were generated by distinct signal transduction mechanisms, namely the SMase and PLD pathways.
CCAAT/enhancer binding protein-βtrans-activates murine nitric oxide synthase 2 gene in an MTAL cell line
Authors: Gupta Ashish K. , Kone Bruce C.
Published: December 2017

Nitric oxide production by nitric oxide synthase 2 (NOS2) has been implicated in epithelial cell injury from oxidative and immunologic stress. The NOS2 gene is transcriptionally activated by lipopolysaccharide (LPS) and cytokines in medullary thick ascending limb of Henle’s loop (MTAL) cells and other cell types. The 5′-flanking region of the NOS2gene contains a consensus element for CCAAT/enhancer binding proteins (C/EBP) at −150 to −142 that we hypothesized contributes to NOS2 trans-activation in the mouse MTAL cell line ST-1. Gel shift assays demonstrated LPS + interferon-γ (IFN-γ) induction of C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the complexes were comprised of C/EBPβ, but not C/EBPα, C/EBPδ, or C/EBPε. NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box exhibited lower activities in response to LPS + IFN-γ compared with wild-type NOS2 promoter constructs. Overexpression of a C/EBP-specific dominant-negative mutant limited LPS + IFN-γ activation of the NOS2 promoter. In trans-activation assays, overexpression of C/EBPβ stimulated basal NOS2 promoter activity. Thus C/EBPβ appears to be an important trans-activator of the NOS2 gene in the MTAL.
Type II cAMP-dependent protein kinase regulates electrogenic ion transport in rabbit collecting duct
Authors: Qi Zhonghua , Hao Chuan-Ming , Salter Kelli , Redha Reyadh , Breyer Matthew D.
Published: December 2017

cAMP mediates many of the effects of vasopressin, prostaglandin E2, and β-adrenergic agents upon salt and water transport in the renal collecting duct. The present studies examined the role of cAMP-dependent protein kinase (PKA) in mediating these effects. PKA is a heterotetramer comprised of two regulatory (R) subunits and two catalytic (C) subunits. The four PKA isoforms may be distinguished by their R subunits that have been designated RIα, RIβ, RIIα, and RIIβ. Three regulatory subunits, RIα, RIIα, and RIIβ, were detected by immunoblot and ribonuclease protection in both primary cultures and fresh isolates of rabbit cortical collecting ducts (CCDs). Monolayers of cultured CCDs grown on semipermeable supports were mounted in an Ussing chamber, and combinations of cAMP analogs that selectively activate PKA type I vs. PKA type II were tested for their effect on electrogenic ion transport. Short-circuit current ( Isc) was significantly increased by the PKA type II-selective analog pairs N6-monobutyryl-cAMP plus 8-(4-chlorophenylthio)-cAMP or N6-monobutyryl-cAMP plus 8-chloro-cAMP. In contrast the PKA type I-selective cAMP analog pair [ N6-monobutyryl-cAMP plus 8-(6-aminohexyl)-amino-cAMP] had no effect on Isc. These results suggest PKA type II is the major isozyme regulating electrogenic ion transport in the rabbit collecting duct.
Endothelin mediates renal vascular memory of a transient rise in perfusion pressure due to NOS inhibition
Authors: Zhang Xin-Zhou , Baylis Chris
Published: December 2017

We investigated the renal responses to NO synthase (NOS) inhibition with N-monomethyl-l-arginine (l-NMA; 30 mg/kg) in anesthetized rats in which renal perfusion pressure (RPP) to the left kidney was mechanically adjusted. Acutel-NMA increased blood pressure (BP, ∼20%) and renal vascular resistance (RVR) rose (∼50%) in the right kidneys that were always exposed to high RPP. In group 1, the left kidney was exposed to a transient increase (5 min) in RPP which was then normalized, and the rise in RVR was similar to the right kidney. In group 2 the left kidney was never exposed to high RPP, and the rise in RVR was attenuated relative to the right kidney. In group 3, rats were pretreated with the endothelin (ET) receptor antagonist Bosentan, immediately before exposure of the left kidney to a transient increase in RPP, and the rise in RVR was also attenuated relative to the right kidney. NOS inhibition resulted in a natriuresis and diuresis in the right kidneys, and ∼50% of the natriuresis persisted in the left kidney of group 2, in the absence of any rise in RPP. ET antagonism completely prevented the natriuresis and diuresis in response to acutel-NMA in both left and right kidneys. These data suggest that transient exposure to high RPP by NOS inhibition prevents an appropriate vasodilatory response when RPP is lowered, due to the intrarenal action of ET.
Stoichiometry and Na+binding cooperativity of rat and flounder renal type II Na+-Picotransporters
Authors: Forster Ian C. , Loo Donald D. F. , Eskandari Sepehr
Published: December 2017

The stoichiometry of the rat and flounder isoforms of the renal type II sodium-phosphate (Na+-Pi) cotransporter was determined directly by simultaneous measurements of phosphate (Pi)-induced inward current and uptake of radiolabeled Piand Na+in Xenopus laevis oocytes expressing the cotransporters. There was a direct correlation between the Pi-induced inward charge and Piuptake into the oocytes; the slope indicated that one net inward charge was transported per Pi. There was also a direct correlation between the Pi-induced inward charge and Na+influx; the slope indicated that the influx of three Na+ions resulted in one net inward charge. This behavior was similar for both isoforms. We conclude that for both Na+-Picotransporter isoforms the Na+:Pistoichiometry is 3:1 and that divalent Piis the transported substrate. Steady-state activation of the currents showed that the Hill coefficients for Piwere unity for both isoforms, whereas for Na+, they were 1.8 (flounder) and 2.5 (rat). Therefore, despite significant differences in the apparent Na+binding cooperativity, the estimated Na+:Pistoichiometry was the same for both isoforms.
Localization of PEPT1 and PEPT2 proton-coupled oligopeptide transporter mRNA and protein in rat kidney
Authors: Shen Hong , Smith David E. , Yang Tianxin , Huang Yuning G. , Schnermann Jürgen B. , Brosius Frank C.
Published: December 2017

To determine the renal localization of oligopeptide transporters, Northern blot analyses were performed and polyclonal antisera were generated against PEPT1 and PEPT2, the two cloned rat H+/peptide transporters. Under high-stringency conditions, a 3.0-kb mRNA transcript of rat PEPT1 was expressed primarily in superficial cortex, whereas a 3.5-kb mRNA transcript of PEPT2 was expressed primarily in deep cortex/outer stripe of outer medulla. PEPT1 antisera detected a specific band on immunoblots of renal and intestinal brush-border membrane vesicles (BBMV) with an apparent mobility of ∼90 kDa. PEPT2 antisera detected a specific broad band of ∼85 kDa in renal but not in intestinal BBMV. PEPT1 immunolocalization experiments showed detection of a brush border antigen in S1 segments of the proximal tubule and in the brush border of villi from all segments of the small intestine. In contrast, PEPT2 immunolocalization was primarily confined to the brush border of S3 segments of the proximal tubule. All other nephron segments in rat were negative for PEPT1 and PEPT2 staining. Overall, our results conclusively demonstrate that although PEPT1 is expressed in early regions of the proximal tubule (pars convoluta), PEPT2 is specific for the latter regions of proximal tubule (pars recta).
Expression of the polymeric immunoglobulin receptor and excretion of secretory IgA in the postischemic kidney
Authors: Rice James C. , Spence Jeff S. , Megyesi Judit , Goldblum Randall M. , Safirstein Robert L.
Published: December 2017

The humoral mucosal immune response of the kidney involves the transport of secretory IgA (S-IgA) through renal epithelial cells by the polymeric immunoglobulin receptor (pIgR). The pIgR is cleaved and released as free secretory component (FSC) or attached to IgA (S-IgA). We examined the effects of an ischemic model of acute renal failure (ARF) on the expression of pIgR and the secretion of FSC and S-IgA in the urine. Kidney pIgR mRNA levels decreased in ischemic animals by 55% at 4 h and by 85% at 72 h compared with controls. pIgR protein expression in the medullary thick ascending limb (TAL) decreased within 24 h and was nearly undetectable by 72 h. Urinary S-IgA and FSC concentrations decreased by 60% between days 3 and 6. pIgR mRNA and pIgR protein in the kidney returned to ∼90% of control levels and urinary FSC and S-IgA concentrations returned to ∼55% of control levels by day 7. We demonstrate that ischemic ARF decreases renal mucosal S-IgA transport in vivo and may contribute to the increased incidence of urinary tract infections.
Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration
Authors: Salomonsson Max , Arendshorst William J.
Published: December 2017

This study provides new information about the relative importance of Ca2+mobilization and entry in the renal vascular response to adrenoceptor activation. We measured renal blood flow (RBF) in Sprague-Dawley rats in vivo using electromagnetic flowmetry. We measured intracellular free Ca2+concentration ([Ca2+]i) in isolated afferent arterioles utilizing ratiometric photometry of fura-2 fluorescence. Renal arterial injection of NE produced a transient decrease in RBF. The response was attenuated, in a dose-dependent manner, up to ∼50% by nifedipine, an antagonist of L-type Ca2+entry channels. Inhibition of Ca2+mobilization by 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8) inhibited the renal vascular effects of NE in a dose-dependent manner, with maximal blockade of ∼80%. No additional attenuation was observed when nifedipine and TMB-8 were administered together. In microdissected afferent arterioles, norepinephrine (NE; 10−6M) elicited an immediate square-shaped increase in [Ca2+]i, from 110 to 240 nM. This in vitro response was blocked by nifedipine (10−6M) and TMB-8 (10−5M) to a degree similar to that of the in vivo experiments. A nominally calcium-free solution blocked 80–90% of the [Ca2+]iresponse to NE. The increased [Ca2+]ielicited by depolarization with medium containing 50 mM KCl was totally blocked by nifedipine. In contrast, TMB-8 had no effect. Our results indicate that both Ca2+entry and mobilization play important roles in the renal vascular Ca2+and contractile response to adrenoceptor activation. The entry and mobilization mechanisms activated by NE may interact. That a calcium-free solution caused a larger inhibition of the NE effects on afferent arterioles than nifedipine suggests more than one Ca2+entry pathway.
cAMP-dependent and -independent downregulation of type II Na-Picotransporters by PTH
Authors: Pfister Markus F. , Forgo Jutka , Ziegler Urs , Biber Jürg , Murer Heini
Published: December 2017

Parathyroid hormone (PTH) leads to the inhibition of Na-Picotransport activity and to the downregulation of the number of type II Na-Picotransporters in proximal tubules, as well as in opossum kidney (OK) cells. PTH is known also to lead to an activation of adenylate cyclase and phospholipase C in proximal tubular preparations, as well as in OK cells. In the present study, we investigated the involvement of these two regulatory pathways in OK cells in the PTH-dependent downregulation of the number of type II Na-Picotransporters. We have addressed this issue by using pharmacological activators of protein kinase A (PKA) and protein kinase C (PKC), i.e., 8-bromo-cAMP (8-BrcAMP) and β-12- O-tetradecanoylphorbol 13-acetate (β-TPA), respectively, as well as by the use of synthetic peptide fragments of PTH that activate adenylate cyclase and/or phospholipase C, i.e., PTH-(1–34) and PTH-(3–34), respectively. Our results show that PTH signal transduction via cAMP-dependent, as well as cAMP-independent, pathways leads to a membrane retrieval and degradation of type II Na-Picotransporters and, thereby, to the inhibition of Na-Picotransport activity. Thereby, the cAMP-independent regulatory pathway leads only to partial effects (∼50%).
Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins
Authors: Tsukamoto Tatsuo , Nigam Sanjay K.
Published: December 2017

After the simulation of anoxia by ATP depletion of MDCK cell monolayers with metabolic inhibitors, the tight junction (TJ) is known to become structurally perturbed, leading to loss of the permeability barrier. Peripheral TJ proteins such as zonula occludens 1 (ZO-1), ZO-2, and cingulin become extremely insoluble and associate into large macromolecular complexes (T. Tsukamoto and S. K. Nigam. J. Biol. Chem. 272: 16133–16139, 1997). For up to 3 h, this process is reversible by ATP repletion. We now show that the reassembly process depends on tyrosine phosphorylation. Recovery of transepithelial electrical resistance in ATP-replete monolayers was markedly inhibited by the tyrosine kinase inhibitor, genistein. Indirect immunofluorescence revealed a decrease in staining of occludin, a membrane component of the TJ, in the region of the TJ after ATP depletion, which reversed after ATP repletion; this reversal process was inhibited by genistein. Examination of the Triton X-100 solubilities of occludin and several nonmembrane TJ proteins revealed a shift of occludin and nonmembrane TJ proteins into an insoluble pool following ATP depletion. These changes reversed after ATP repletion, and the movement of insoluble occludin, ZO-1, and ZO-2 back into the soluble pool was again via a genistein-sensitive mechanism. Rate-zonal centrifugation analyses of detergent-soluble TJ proteins showed a reversible increase in higher density fractions following ATP depletion-repletion, although this change was not affected by genistein. In32P-labeled cells, dephosphorylation of all studied TJ proteins was observed during ATP depletion, followed by rephosphorylation during ATP repletion; rephosphorylation of occludin was inhibited by genistein. Furthermore, during the ATP repletion phase, tyrosine phosphorylation of Triton X-100-insoluble occludin, which is localized at the junction, as well as ZO-2, p130/ZO-3 (though not ZO-1), and other proteins was evident; this tyrosine phosphorylation was completely inhibited by genistein. This indicates that tyrosine kinase activity is necessary for TJ reassembly during ATP repletion and suggests an important role for the tyrosine phosphorylation of occludin, ZO-2, p130/ZO-3, and possibly other proteins in the processes involved in TJ (re)formation.
Localization of an organic anion transporter-GFP fusion construct (rROAT1-GFP) in intact proximal tubules
Authors: Sweet Douglas H. , Miller David S. , Pritchard John B.
Published: December 2017

The organic anion transporter, rROAT1, is a dicarboxylate/organic anion exchanger, a function associated with the basolateral membrane in rat proximal tubule. To directly establish the subcellular localization of rROAT1 in renal epithelia, we made a rROAT1-green fluorescent protein (GFP) fusion construct (rROAT1-GFP). Plasma membrane-associated fluorescence was observed in rROAT1-GFP-expressing Xenopus oocytes examined by confocal microscopy. Uptake of 3H-labeled p-aminohippurate (PAH) increased 2.5-fold in rROAT1-GFP-expressing Xenopus oocytes, and this increase was abolished by 1 mM probenecid. Thus the construct was capable of specific organic anion transport. Cultured renal epithelial cell lines (MDCK and LLC-PK1) transfected with the vector pEGFP-C3 showed a diffuse, evenly distributed cytoplasmic signal. However, when transfected with pEGFP-C3/rROAT1 (vector coding for rROAT1-GFP), both cell lines showed predominantly plasma membrane fluorescence. The expression and distribution of rROAT1-GFP in intact renal proximal tubules was also investigated. Isolated killifish ( Fundulus heteroclitus) renal tubules transfected with pEGFP-C3/rROAT1 showed marked basal and lateral membrane-associated fluorescence, but no detectable signal in the nucleus or the apical pole of tubule cells. Tubules transfected with pEGFP-C3 showed diffuse cytoplasmic fluorescence. Function of the rROAT1-GFP construct was demonstrated in transfected killifish tubules by fluorescein transport assay. These results demonstrate the basolateral subcellular localization of rROAT1 in polarized renal epithelia and validate a new technique for localizing cloned transporters within intact renal tubules.
Characterization of Na+/ HCO 3 − cotransporter isoform NBC-3
Authors: Amlal Hassane , Burnham Charles E. , Soleimani Manoocher
Published: December 2017

Na+-[Formula: see text]cotransporters mediate the transport of[Formula: see text] into or out of the cell. Two Na+-[Formula: see text]cotransporters (NBC) have been identified previously, which are referred to as NBC-1 and NBC-2. A cDNA library from uninduced human NT-2 cells was screened with an NBC-2 cDNA probe. Several clones were identified and isolated. Sequence analysis of these clones identified a partial coding region (2 kb) of a novel NBC (called here NBC-3), which showed 53% and 72% identity with NBC-1 and NBC-2, respectively. Northern blot analysis revealed that NBC-3 encodes a 4.4-kb mRNA with a tissue distribution pattern distinct from NBC-1 and NBC-2. NBC-3 is highly expressed in brain and spinal column, with moderate levels in trachea, thyroid, and kidney. In contrast with NBC-1, NBC-3 shows low levels of expression in pancreas and kidney cortex. In the kidney, NBC-3 expression is predominantly limited to the medulla. Cultured mouse inner medullary collecting duct (mIMCD-3) cells showed high levels of NBC-1 and low levels of NBC-3 mRNA expression. Subjecting the mutagenized mIMCD-3 cells to sublethal acid stress decreased the mRNA expression of NBC-1 by ∼90% but increased the Na+-dependent[Formula: see text] cotransport activity by ∼7-fold (as assayed by DIDS-sensitive, Na+-dependent,[Formula: see text]-mediated intracellular pH recovery). This increase was associated with ∼5.5-fold enhancement of NBC-3 mRNA levels. NBC showed significant affinity for Li+in the mutant but not the parent mIMCD-3 cells. On the basis of the widespread distribution of NBC-3, we propose that this isoform is likely involved in cell pH regulation by transporting [Formula: see text] from blood to the cell. We further propose that enhanced expression of NBC-3 in severe acid stress could play an important role in cell survival by mediating the influx of [Formula: see text] into the cells.
HCO 3 − reabsorption in renal collecting duct of NHE-3-deficient mouse: a compensatory response
Authors: Nakamura Suguru , Amlal Hassane , Schultheis Patrick J. , Galla John H. , Shull Gary E. , Soleimani Manoocher
Published: December 2017

Mice with a targeted disruption of Na+/H+exchanger NHE-3 gene show significant reduction in[Formula: see text] reabsorption in proximal tubule, consistent with the absence of NHE-3. Serum[Formula: see text], however, is only mildly decreased (P. Schulties, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282–285, 1998), indicating possible adaptive upregulation of[Formula: see text]-absorbing transporters in collecting duct of NHE-3-deficient (NHE-3 −/−) mice. Cortical collecting duct (CCD) and outer medullary collecting duct (OMCD) were perfused, and total CO2(net[Formula: see text] flux, JtCO2) was measured in the presence of 10 μM Schering 28080 (SCH, inhibitor of gastric H+-K+-ATPase) or 50 μM diethylestilbestrol (DES, inhibitor of H+-ATPase) in both mutant and wild-type (WT) animals. In CCD, JtCO2increased in NHE-3 mutant mice (3.42 ± 0.28 in WT to 5.71 ± 0.39 pmol ⋅ min−1⋅ mm tubule−1in mutants, P < 0.001). The SCH-sensitive net[Formula: see text] flux remained unchanged, whereas the DES-sensitive [Formula: see text] flux increased in the CCD of NHE-3 mutant animals. In OMCD, JtCO2increased in NHE-3 mutant mice (8.8 ± 0.7 in WT to 14.2 ± 0.6 pmol ⋅ min−1⋅ mm tubule−1in mutants, P < 0.001). Both the SCH-sensitive and the DES-sensitive [Formula: see text] fluxes increased in the OMCD of NHE-3 mutant animals. Northern hybridizations demonstrated enhanced expression of the basolateral Cl−/[Formula: see text]exchanger (AE-1) mRNA in the cortex. The gastric H+-K+-ATPase mRNA showed upregulation in the medulla but not the cortex of NHE-3 mutant mice. Our results indicate that[Formula: see text] reabsorption is enhanced in CCD and OMCD of NHE-3-deficient mice. In CCD, H+-ATPase, and in the OMCD, both H+-ATPase and gastric H+-K+-ATPase contribute to the enhanced compensatory[Formula: see text] reabsorption in NHE-3-deficient animals.
A numerical model of the renal distal tubule
Authors: Chang Hangil , Fujita Toshiro
Published: December 2017

A numerical model of the rat distal tubule was developed to simulate water and solute transport in this nephron segment. This model incorporates the following: 1) Na-Cl cotransporter, K-Cl cotransporter, Na channel, K channel, and Cl channel in the luminal membrane; 2) Na-K-ATPase, K channel, and Cl channel in the basolateral membrane; and 3) conductances for Na, K, and Cl in the paracellular pathway. Transport rates were calculated using kinetic equations. Axial heterogeneity was represented by partitioning the model into two subsegments with different sets of model parameters. Model equations derived from the principles of mass conservation and electrical neutrality were solved numerically. Values of the model parameters were adjusted to minimize a penalty function that was devised to quantify the difference between model predictions and experimental results. The developed model could simulate the water and solute transport of the distal tubule in the normal state, as well as in conditions including thiazide or amiloride application and various levels of sodium load and tubular flow rate.
Neuronal NOS contributes to biphasic autoregulatory response during enhanced TGF activity
Authors: Ichihara Atsuhiro , Navar L. Gabriel
Published: December 2017

To assess the afferent arteriolar autoregulatory response during increased activity of the tubuloglomerular feedback (TGF) mechanism and to delineate the contribution of neuronal nitric oxide synthase (nNOS) to this response, afferent arteriolar diameter responses to changes in renal perfusion pressure (RPP) were monitored in vitro using the blood-perfused rat juxtamedullary nephron preparation. At RPP of 100 mmHg, basal afferent arteriolar diameter averaged 21.1 ± 1.4 μm ( n = 9). The initial and sustained constrictor responses of afferent arterioles to a 60-mmHg increase in RPP averaged 14.8 ± 1.4% and 13.3 ± 1.3%, respectively. Acetazolamide treatment, which enhances TGF responsiveness by increasing distal nephron volume delivery, significantly decreased basal afferent arteriolar diameter by 8.2 ± 0.5% and enhanced the initial response (25.5 ± 2.3%) to a 60-mmHg increase in RPP but did not alter the sustained response (14.3 ± 1.5%). In another series of experiments, nNOS inhibition with 10 μM S-methyl-l-thiocitrulline (l-SMTC) significantly decreased afferent arteriolar diameter from 20.3 ± 1.3 to 18.3 ± 1.1 μm ( n = 7) and enhanced both the initial (34.4 ± 3.5%) and sustained constrictor responses (27.6 ± 2.9%) to a 60-mmHg increase in RPP. Treatment with acetazolamide further enhanced both initial (56.4 ± 3.0%) and sustained responses (54.6 ± 2.7%). Interruption of distal delivery by transection of the loops of Henle prevented the enhanced responses to increases in RPP elicited with either acetazolamide orl-SMTC. These results indicate that nNOS contributes to the counteracting resetting process of biphasic afferent arteriolar constrictor responses to increases in RPP through a TGF-dependent mechanism.
Nitrotyrosine formation with endotoxin-induced kidney injury detected by immunohistochemistry
Authors: Bian Ka , Davis Karen , Kuret Jeff , Binder Lester , Murad Ferid
Published: December 2017

The presence of nitrotyrosine in the kidney has been associated with several pathological conditions. In the present study, we investigated nitrotyrosine formation in rat kidney after animals received endotoxin for 24 h. With lipopolysaccharide (LPS) treatment, immunohistochemical data demonstrated intense nitrotyrosine staining throughout the kidney. In spite of marked nitrotyrosine formation, the architectural appearance of tubules, glomeruli, and capillaries remained intact when examined by reticulin staining. Our data suggested that the marked staining of nitrotyrosine in proximal tubular epithelial cells was in the subapical compartment where the endocytic lysosomal apparatus is located. Thus a large portion of nitrotyrosine may come from the hydrolysis of nitrated proteins that are reabsorbed by the proximal tubule during the LPS treatment. We also found the colocalization of nitric oxide synthase (NOS-1) and nitrotyrosine within the macula densa of LPS-treated rats by using a double fluorescence staining method. In renal arterial vessels, vascular endothelial cells were more strongly stained for nitrotyrosine than vascular smooth muscle cells. Control animals without LPS treatment showed much less renal staining for nitrotyrosine. The general distribution of nitrotyrosine staining in control rat renal cortex is in the proximal and convoluted tubules, whereas the endothelial cells of vasa recta are major areas of nitrotyrosine staining in inner medulla. The renal distribution of nitrotyrosine in control and LPS-treated animals suggests that protein nitration may participate in renal regulation and injury in ways that are yet to be defined.
Renal cellular response to ureteral obstruction: role of maturation and angiotensin II
Authors: Chevalier Robert L. , Thornhill Barbara A. , Wolstenholme Jennifer T.
Published: December 2017

Renal angiotensin II (ANG II) is increased as a result of unilateral ureteral obstruction (UUO), and angiotensin AT2 receptors predominate over AT1 receptors in the early postnatal period. To examine the renal cellular response to 3-day UUO in the neonatal and adult rat, AT1and AT2 receptors were inhibited by losartan and PD-123319, respectively. Additional rats received exogenous ANG II, 0.5 mg ⋅ kg−1 ⋅ day−1. Renal cellular proliferation and apoptosis were quantitated by proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling technique, respectively. In the neonate, UUO reduced proliferation and increased tubular apoptosis. Losartan had no detectable cellular effect, whereas PD-123319 increased cellular proliferation and suppressed apoptosis, and exogenous ANG II stimulated apoptosis. In the adult, UUO increased cellular proliferation as well as apoptosis, whereas losartan, PD-123319, and exogenous ANG II did not alter the cellular response. In conclusion, UUO impairs renal growth in the neonate by reducing proliferation and stimulating apoptosis, at least in part through angiotensin AT2 receptors. UUO stimulates both renal cellular proliferation and apoptosis in the adult, but these effects are independent of ANG II. We speculate that the unique early responses of the developing kidney to urinary tract obstruction are mediated by a highly activated renin-angiotensin system and preponderance of AT2 receptors.
Glucose transporters control gene expression of aldose reductase, PKCα, and GLUT1 in mesangial cells in vitro
Authors: Henry Douglas N. , Busik Julia V. , Brosius Frank C. , Heilig Charles W.
Published: December 2017

The process linking increased glucose utilization and activation of metabolic pathways leading to end-organ damage from diabetes is not known. We have previously described rat mesangial cells that were transduced to constitutively express the facilitative glucose transporter 1 (GLUT1, MCGT1 cells) or bacterial β-galactosidase (MCLacZ, control cells). Glucose transport was rate limiting for extracellular matrix production in the MCGT1 cells. In the present work, we investigated the effect of GLUT1 overexpression in mesangial cells on aldose reductase (AR), protein kinase Cα (PKCα), and native GLUT1 transcript levels, to determine whether changes in GLUT1 alone could regulate their expression in the absence of high extracellular glucose concentrations. MCGT1 cells grown in normal (8 mM) or elevated (20 mM) glucose had elevated abundance of AR, PKCα, and the native GLUT1 transcripts compared with control cells. AR protein levels, AR activity, sorbitol production, and PKCα protein content were also greater in the MCGT1 cells than in control cells grown in the same media. This is the first report of the concomitant activation of AR, PKCα, and GLUT1 genes by enhanced GLUT1 expression. We conclude that increased GLUT1 expression leads to a positive feedback of greater GLUT1 expression, increased AR expression and activity with polyol accumulation, and increased total and active PKCα protein levels, which leads to detrimental stimulation of matrix protein synthesis by diabetic mesangial cells.