bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization and regulation of endothelial NO synthase mRNA expression in rat kidney
Authors: Ujiie K. , Yuen J. , Hogarth L. , Danziger R. , Star R. A.
Published: December 2017

Nitric oxide (NO) has effects on renal blood flow, glomerular filtration rate, renin secretion, and renal sodium excretion. Four isoforms of nitric oxide synthase (NOS) have been cloned to date. However, the molecular identity of NOS present in the renal vasculature is unknown. Endothelial NOS (NOS-III) is regulated both acutely by cell calcium and chronically by shear stress. To determine if renal blood vessels and the glomerulus express NOS-III mRNA, we used degenerate polymerase chain reaction (PCR) to clone a portion of rat NOS-III. We then assayed NOS-III mRNA in microdissected renal structures by reverse transcriptase-PCR. NOS-III mRNA was expressed at high levels in glomeruli, arcuate vessels, and interlobular artery/afferent arterioles. NOS-III mRNA was detected inconsistently in proximal tubules, thick ascending limbs, and cortical and inner medullary collecting ducts. Previous studies have shown that chronic oral treatment with the NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) decreases NO synthesis and causes hypertension. To determine if the systemic blockade occurs only by competitive inhibition, we determined the effect of L-NAME on glomerular NOS-III mRNA. L-NAME administration (5 days) decreased NOS-III mRNA in the glomerulus to 25 +/- 12% of control levels. We conclude that endothelial NOS-III mRNA is preferentially expressed in the glomerulus and renal vasculature, where it can modulate renal blood flow and glomerular filtration rate. Furthermore, glomerular NOS-III may be modulated at the level of mRNA abundance in vivo by systemic L-NAME.(ABSTRACT TRUNCATED AT 250 WORDS)
Role of arginine in health and in renal disease
Authors: Reyes A. A. , Karl I. E. , Klahr S.
Published: December 2017

In addition to participating in protein synthesis in cells and tissues, L-arginine is essential for the synthesis of urea, creatine, creatinine, nitric oxide, and agmatine and influences hormonal release and the synthesis of pyrimidine bases. This places L-arginine, its precursors and its metabolites at the center of the interaction of different metabolic pathways and interorgan communication. Thus L-arginine participates in changing the internal environment in different but simultaneous ways, ranging from disposal of protein metabolic waste, muscle metabolism, vascular regulation, immune system function, and neurotransmission, to RNA synthesis and hormone-mediated regulation of the internal milieu. In normal rats, inhibition of the nitric oxide pathway results in systemic hypertension and decreased glomerular filtration rate and effective renal plasma flow. If the inhibition of this pathway is sustained, then glomerulosclerosis and death from uremia follow. Dietary intervention with L-arginine has resulted in amelioration of a number of experimental kidney diseases, such as those caused by subtotal nephrectomy, diabetic, nephropathy, cyclosporin A administration, salt-sensitive hypertension, ureteral obstruction, puromycin amino-nucleoside nephrosis, kidney hypertrophy due to high-protein feeding, and glomerular thrombosis due to administration of lipopolysaccharide. The present review addresses the current evidence for the beneficial effects of dietary intervention with L-arginine in a number of experimental renal diseases and describes the basis for the concept of L-arginine deficiency (absolute or relative) in certain settings in which supplementation of the diet with this amino acid may be beneficial.
Apical membrane and intracellular distribution of endogenous alpha 2A-adrenergic receptors in MDCK cells
Authors: Okusa M. D. , Lynch K. R. , Rosin D. L. , Huang L. , Wei Y. Y.
Published: December 2017

The purpose of the current studies was to characterize the endogenous alpha 2-adrenergic receptor (AR) subtypes present in Madin-Darby canine kidney (MDCK) cells and to determine their level of expression and pattern of distribution. By saturation binding analysis with [3H]MK-912, MDCK cells expressed high levels of alpha 2-ARs with a maximum receptor density (Bmax) of 798 +/- 55 fmol/mg protein and an equilibrium dissociation constant (Kd) of 0.98 +/- 0.32 nM. Competitive binding studies using prazosin, oxymetazoline, phentolamine, and epinephrine to displace [3H]MK-912 demonstrated inhibition constant (Ki) values of 1,270 +/- 250, 5.0 +/- 0.4, 5.5 +/- 0.3, and 392 +/- 150 nM (n = 3), respectively. In Northern blot analysis we found that MDCK cells expressed transcripts encoding alpha 2A-AR and not alpha 2B-AR or alpha 2C-AR. Surface binding experiments suggested that approximately 60% of alpha 2A-ARs are distributed at the cell surface domain. Specific binding of [3H]MK-912 to soluble apical and basolateral surface proteins isolated by surface biotinylation indicated the expression of surface alpha 2A-ARs was limited to the apical domain of MDCK cells. No alpha 2A-ARs were detected on the basolateral surface. We conclude that endogenous alpha 2A-ARs are targeted to the apical domain of MDCK cells and that the intracellular compartment may contain ARs as a reservoir for de novo cell surface expression or, alternatively, may represent internalized receptors.
Hormonal regulation of MAP kinase in cultured rat inner medullary collecting tubule cells
Authors: Heasley L. E. , Senkfor S. I. , Winitz S. , Strasheim A. , Teitelbaum I. , Berl T.
Published: December 2017

Mitogen-activated protein (MAP) kinase is a widely expressed protein serine/threonine kinase that serves as a convergence point for many signaling pathways including receptor tyrosine kinases, G protein-coupled receptors, and protein kinase C (PKC). The hormonal regulation of MAP kinase was studied in cultured established rat inner medullary collecting tubule (RIMCT) cells. Neither vasopressin nor beta-adrenergic agonists stimulated MAP kinase, despite clear stimulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. In contrast, carbachol, ATP, and epidermal growth factor (EGF), which are known to antagonize vasopressin action in the RIMCT, stimulated the MAP kinase pathway. This stimulation was mimicked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, which directly activates PKC. The potency with which EGF and carbachol activated MAP kinase was similar to the potency with which they inhibited vasopressin-stimulated cAMP accumulation. To assess the role of Gi proteins in these stimulatory events, RIMCT cells were pretreated with pertussis toxin to inhibit Gi-mediated signaling. Pertussis toxin did not influence ATP- or EGF-stimulated MAP kinase, but completely inhibited carbachol stimulation, suggesting that Gi proteins mediate muscarinic stimulation. Prolonged exposure of RIMCT cells to high phorbol ester concentrations to downregulate PKC ablated carbachol- and ATP-stimulated MAP kinase, but not EGF-stimulated MAP kinase, suggesting that PKC is a component of the network involved in MAP kinase activation by purinergic and muscarinic agonists. Investigation of the sidedness of the hormonal stimulations indicated that EGF-stimulated MAP kinase was highly polarized, occurring exclusively from the basolateral surface, whereas carbachol stimulated MAP kinase similarly from either cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in mRNAs for enzymes of glutamine metabolism in kidney and liver during ammonium chloride acidosis
Authors: Schoolwerth A. C. , deBoer P. A. , Moorman A. F. , Lamers W. H.
Published: December 2017

Changes in protein and mRNAs for enzymes of glutamine metabolism were determined in rat kidney cortex at different times after induction of NH4Cl acidosis. After NH4Cl, phosphoenolpyruvate carboxykinase (PEPCK) mRNA increased 16-fold by 10 h (P < 0.05) and then returned to control levels by 30 h. In situ hybridization (ISH) showed that PEPCK mRNA was confined to medullary rays; after NH4Cl, expression of PEPCK expanded throughout the cortex, reaching a maximal intensity at 10 h. Phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GDH) mRNAs increased 8- and 2.6-fold, respectively (both P < 0.05), by 10 h before decreasing; the increased expression was confirmed by ISH. Immunohistochemistry showed that increased PEPCK, PDG, and GDH protein occurred at variable times after the rise in mRNAs. The increase was confined to proximal tubules and was sustained, a finding noted also by Western blot analysis. In contrast, glutamine synthase protein and mRNA, confined to deep cortex and outer medullar, did not change after NH4Cl. These studies reveal striking changes in PEPCK and PDG mRNAs in rat renal cortex during acidosis. The ISH pattern suggested that increased amounts of PEPCK were synthesized in recruited cells which contained little enzyme under physiological conditions. mRNA levels for PEPCK, PDG, and GDH peaked at 10 h before returning to control levels. Despite the decrease in mRNAs, a sustained increase in proteins was noted.
IL-1 beta stabilizes COX II mRNA in renal mesangial cells: role of 3'-untranslated region
Authors: Srivastava S. K. , Tetsuka T. , Daphna-Iken D. , Morrison A. R.
Published: December 2017

We stimulated rat mesangial cells for different time intervals with interleukin-1 beta (IL-1 beta) and phorbol 12-myristate 13-acetate, prepared cytoplasmic extracts, and examined these extracts for the presence of RNA binding proteins by gel mobility shift assays. Here we report that the 3'-untranslated region (3'-UNTR) of the prostaglandin endoperoxide synthase II (COX II) gene is responsible for posttranscriptional regulation of the response to IL-1 beta. Two cytosolic transacting factors of 65 and 45 kDa, respectively, have been detected that bind to the 3'-UNTR. Competition with excess RNA and acid phosphatase treatment of the cytoplasmic extract suggest the binding is specific and that phosphorylation is required for these rapid binding events. These experiments suggest that IL-1 beta induces the phosphorylation of cytosolic factors, which bind to the 3'-UNTR of COX II mRNA, and stabilizes the message.
Renal transepithelial phosphate secretion: luminal membrane voltage and Ca2+ dependence
Authors: Lu M. , Barber L. E. , Renfro J. L.
Published: December 2017

The role of apical membrane electrical potential, the possibility of K+ channel involvement, and the role of extracellular Ca2+ in transepithelial P(i) secretion were examined in primary monolayer cultures of flounder renal proximal tubule cells in Ussing chambers. Exposure to 200 nM thapsigargin (TG) significantly increased net P(i) secretion. In TG-stimulated tissues, substitution of 100 mM KCl for 100 mM NaCl in the luminal medium depolarized the apical membrane potential from -64 +/- 2.8 to -26 +/- 3.9 mV and strongly inhibited net P(i) secretion. In 32P(i)-preloaded tissues, cell-to-lumen exit of 32P(i) was significantly decreased to approximately 50% of control by high luminal K+ while cell-to-peritubular bath movement was unchanged. Addition of BaCl2 (2 mM) or charybdotoxin (20 nM) to the luminal surface significantly reduced TG-stimulated net P(i) secretion. The elevation of bath Ca2+ from 2 to 5 mM significantly increased secretory flux and decreased reabsorptive flux. The effect of TG on net P(i) secretion was reduced by the Ca2+ channel blocker verapamil (VE, 100 microM) to 65% of control and by calmodulin antagonist W-7 (20 microM) to 35% of control but it was not blocked by the protein kinase inhibitor H-7 (100 microM). VE also significantly inhibited the P(i) secretion induced by acidification of the peritubular bathing medium. The data indicate that transepithelial P(i) secretion induced by TG is significantly influenced by apical membrane electrical polarity, which may be regulated in part by Ca(2+)-activated K+ channels.
Arginine metabolism in experimental glomerulonephritis: interaction between nitric oxide synthase and arginase
Authors: Cook H. T. , Jansen A. , Lewis S. , Largen P. , O'Donnell M. , Reaveley D. , Cattell V.
Published: December 2017

L-Arginine is metabolized by two pathways: 1) by nitric oxide synthase (NOS) to nitric oxide (NO) and 2) by arginase forming urea and L-ornithine. Inflammatory responses may involve a balance between the pathways, as NO is cytotoxic and vasodilatory and L-ornithine is a promoter of cell proliferation and matrix synthesis. In experimental glomerulonephritis we have previously shown that NOS is activated in nephritic glomeruli. We have now examined both pathways of L-arginine metabolism to study competition for L-arginine, temporal variation, and the sources of NOS and arginase. Acute in situ glomerulonephritis was induced in rats, and glomeruli were studied at 1, 4, and 7 days. Both NOS and arginase activities were present. There was temporal variation: NOS activity was highest on day 1 and arginase activity on day 4; both declined by day 7. Competition between the pathways was demonstrated by increased urea synthesis in the presence of NG-monomethyl-L-arginine, an inhibitor of NOS. Measurement of NOS and arginase activities in macrophages isolated from nephritic glomeruli showed that these cells were a major source of glomerular NOS but not arginase activity. In contrast, high arginase activity but low NO production was identified in cultured rat glomerular mesangial cells. These studies show differential temporal variation in expression of NOS and arginase pathways of arginine metabolism in experimental glomerulonephritis. We have found two factors that may contribute to this: 1) competition for substrate L-arginine between the two pathways and 2) different cellular sources. We hypothesize that the balance between these pathways is a mechanism regulating injury, hemodynamics, and mesangial cell proliferation.
Diverse modulations of chloride channels in renal proximal tubules
Authors: Darvish N. , Winaver J. , Dagan D.
Published: December 2017

Cl- selective channels were detected and characterized in apical membranes of cultured rat renal proximal convoluted tubule cells (PCT) using patch-clamping methods. Subpopulations of Cl- channels modulated by cyclic nucleotides, Ca2+, or voltage were identified. Two different 30-pS, voltage-independent, Cl- channels modulated by adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ were seen most frequently. The cAMP-dependent channel was activated by membrane-permeable analogues of cAMP, dibutyryl-cAMP or 8-bromo-cAMP. Catalytic subunit of protein kinase A (PKA) applied to detached inside-out patches, activated the channel as well, suggesting activation via phosphorylation. Channel activity was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, by 4,4-dinitrostilbene-2,2-disulfonic acid, and by SCN-. Permeability sequence for different halides was Cl- > I > F with a Cl(-)-to-cation permeability ratio (PCl/Pcation) of 7:1. The Ca(2+)-sensitive channel was not activated by cAMP nor by PKA. A third anionic selective channel encountered infrequently is voltage dependent and has a unitary conductance of 145 pS, with a PCl/Pcation value of 9:1. This diversity of Cl- channels may underlie the rich repertoire of physiological functions attributed to Cl- channels.
Influence of sodium diet on L-NAME effects on renin release and renal vasoconstriction
Authors: Gardes J. , Gonzalez M. F. , Alhenc-Gelas F. , Menard J.
Published: December 2017

The intervention of the L-arginine-NO pathway in renal vasodilation and renin secretion was studied in an isolated perfused rat kidney model. NG-nitro-L-arginine methyl ester (L-NAME, 1-25 microM), an inhibitor of nitric oxide (NO) synthesis, caused a dose-dependent increase in perfusion pressure (PP) and a dose-dependent decrease in renal perfusate flow. Renin was inhibited independently of the rise in PP, since the effect of L-NAME on renin release was the same when PP was maintained constant. Exposure of rats to low [salt depleted (SD)] or high [salt repleted (SR)] salt intake for 1 mo influenced the renal vascular response to L-NAME (3 microM). Isolated SR rat kidney vasculature vasoconstricted to a greater extent after inhibition of NO synthase than did that of SD kidneys. A similar fall in renin release was observed after L-NAME in both groups, despite a higher renin secretion rate in SD than in SR rats. These results suggest that NO-dependent vasodilation counteracts the renal vasoconstrictor effect of sodium loading.
Acute acidosis inhibits liver amino acid transport: no primary role for the urea cycle in acid-base balance
Authors: Boon L. , Blommaart P. J. , Meijer A. J. , Lamers W. H. , Schoolwerth A. C.
Published: December 2017

To examine further the role of the liver in acid-base homeostasis, we studied hepatic amino acid uptake and urea synthesis in rats in vivo during acute acidosis and alkalosis, induced by infusion of 1.8 mmol of HCl or NaHCO3 over 3 h. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences of amino acids and urinary urea and NH4+ excretion were measured. In acidosis, urinary urea excretion was reduced 36% (P < 0.01), whereas urinary NH4+ excretion increased ninefold (P < 0.01), but the sum of urea and NH4+ excretion was unchanged. Total hepatic amino acid uptake, as determined from arteriovenous differences, was decreased by 63% (P < 0.01) in acidosis, with the major effect being noted with alanine and glycine. Only glutamine was released in both acidosis and alkalosis but was not significantly different in the two conditions. Since intracellular concentrations of readily transportable amino acids were not different at low pH despite accelerated protein degradation, these results indicate that hepatic amino acid transport was inhibited markedly and sufficiently to explain the observed decrease in urea synthesis. Total hepatic vein amino acid content was greater in acidosis than alkalosis (P < 0.01). Directly or indirectly, by conversion to glutamine elsewhere, these increased amino acids were degraded in kidney and accounted for the ninefold increase in urinary NH4+ excretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of interactions between myogenic and TGF mechanisms using nonlinear analysis
Authors: Chon K. H. , Chen Y. -M. , Marmarelis V. Z. , Marsh D. J. , Holstein-Rathlou N. -H.
Published: December 2017

Pages F160–F173: K. H. Chon, Y.-M. Chen, V. Z. Marmarelis, D. J. Marsh, and N.-H. Holstein-Rathlou. “Detection of interactions between myogenic and TGF mechanisms using nonlinear analysis.” Pages F168–F169: The original Figs. 8–11 do not give sufficient detail of the contour plots. New, higher resolution images are reproduced here with the original legends.
Potentiated 1,25(OH)2D3-induced 24-hydroxylase gene expression in uremic rat intestine
Authors: Koyama H. , Inaba M. , Nishizawa Y. , Ishimura E. , Imanishi Y. , Hini M. , Furuyama T. , Takagi H. , Morii H.
Published: December 2017

24-Hydroxylase has been considered a major enzyme regulating metabolism of circulating 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3]. To understand the metabolism of 1,25(OH)2D3 in chronic renal failure, we examined 1,25(OH)2D3-induced 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) gene expression in the intestine of uremic rats. Northern blot and dot blot analyses showed that the induction of duodenal 24-hydroxylase gene expression was 2.0- to 3.8-fold greater in uremic rats than in sham-operated rats (P < 0.05, Student's t-test) at 6 h after 1,25(OH)2D3 administration. Gene induction of calbindin D9k by 1,25(OH)2D3 was not augmented in uremic group. In situ hybridization analysis revealed that the induction of 24-hydroxylase mRNA by 1,25(OH)2D3 was observed exclusively in the columnar epithelium of the crypt and the lower part of the villi, suggesting that the stage of epithelial cell differentiation is a major determinant of 1,25(OH)2D3-induced 24-hydroxylase gene expression. In uremia, 1,25(OH)2D3-induced 24-hydroxylase gene expression was accelerated selectively, possibly because of poorly differentiated epithelial cells.
Transforming growth factor-beta expression in macrophages during hypercholesterolemic states
Authors: Ding G. , van Goor H. , Frye J. , Diamond J. R.
Published: December 2017

Macrophage infiltration into the glomerular mesangium is a prominent feature of various glomerulopathies. Recent evidence suggests that infiltrating macrophages may play a role in propagating initial glomerular injury to the development of glomerulosclerosis via transforming growth factor-beta (TGF-beta)-stimulating matrix accumulation. Rats with the acute puromycin aminonucleoside (PA) nephrosis exhibit an elevated gene expression of glomerular TGF-beta 1; however, the cellular origin of this upregulation is unknown. Using polymerase chain reaction (PCR), we detected that the TGF-beta 1 isoform is expressed in glomerular macrophages isolated from experimental rats made hypercholesterolemic by either diet or by induction of PA nephrosis. Peritoneal macrophages from nephrotic or dietary-hypercholesterolemic animals also exhibited a significant increment in the expression of TGF-beta 1 mRNA on Northern analysis, in contrast to similar cells obtained from normal control rats. PCR analysis of glomerular RNA also detected the expression of the TGF-beta 2 mRNA isoform. TGF-beta 2 mRNA expression was not observed in isolated glomerular macrophages from either glomeruli of PA-nephrotic rats or from glomeruli of animals with dietary hypercholesterolemia. Expression of the TGF-beta 3 mRNA isoform was only observed by PCR in J774 A.1 cells. Thus the as a cellular source for the enhanced expression of TGF-beta 1 during the acute nephrotic phase of our toxic, progressive glomerulopathy model and within several days of inducing only hypercholesterolemia by dietary means.
Acid extrusion in S3 segment of rabbit proximal tubule. II. Effect of basolateral CO2/HCO3-
Authors: Chen L. K. , Boron W. F.
Published: December 2017

Monitoring intracellular absorbance spectra of the pH-sensitive dye dimethylcarboxyfluorescein we studied intracellular pH (pHi) regulation in the isolated perfused S3 segment of the rabbit proximal tubule. In the preceding study [Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F179-F192, 1995.], we demonstrated that simultaneously adding CO2/HCO3- to both lumen and bath stimulated two acid-extruding mechanisms, one dependent on luminal Na+ (a Na+/H+ exchanger) and one independent of Na+ (presumably a H+ pump). Here, we examine the effects of adding CO2/HCO3- to the lumen only or to the bath only. Over a broad pHi range, the total rate of pHi recovery from an acid load in the presence of Na+ was not increased by luminal CO2/HCO3-. On the other hand, basolateral CO2/HCO3- increased the pHi recovery rate to an even greater extent than had bilateral CO2/HCO3-. Regarding the Na(+)-independent pHi recovery mechanism, we found that luminal CO2/HCO3- failed to increase the pHi recovery rate compared with controls also studied in the absence of Na+. Neither did luminal CO2/HCO3- significantly affect the lag time between the maximal acid load and the initiation of the Na(+)-independent pHi recovery (approximately 147 vs. approximately 212 s in controls). On the other hand, adding CO2/HCO3- to only the bath substantially increased the rate of Na(+)-independent pHi recovery, which generally was greater than that observed with bilateral CO2/HCO3-. CO2/HCO3- only in the bath also reduced the lag time to approximately 51 s, which is not significantly different from the value of approximately 36 s observed with bilateral CO2/HCO3-. Our data are consistent with the hypothesis that basolateral (but not luminal) CO2/HCO3- stimulates both a luminal Na+/H+ exchanger and a luminal H+ pump.
Short-term response of nonurea organic osmolytes in human kidney to a water load and water deprivation
Authors: Sizeland P. C. , Chambers S. T. , Lever M. , Bason L. M. , Robson R. A.
Published: December 2017

The cells of the inner medulla of the mammalian kidney accumulate high concentrations of nonurea organic osmolytes. The organic osmolytes found in the kidney include glycine betaine and sorbitol. This study was designed to measure changes in the urinary excretion of glycine betaine and sorbitol and the plasma concentration of glycine betaine in response to an acute water load (20 ml/kg) or acute water deprivation in young healthy males. In response to a water load the urinary excretion of glycine betaine and sorbitol increased parallel with or shortly after urinary urea excretion. The increase in urinary urea and sorbitol excretions preceded maximum minute volume, whereas peak glycine betaine excretion was closely related to maximum urine minute volume. Subsequently, urea, sorbitol, and glycine betaine excretion rates returned to baseline. In contrast, during water deprivation no change in glycine betaine, sorbitol, and urea urinary excretions occurred during the study period. Plasma glycine betaine concentration was stable during both diuresis and antidiuresis. We conclude that the organic osmolytes glycine betaine and sorbitol are components of a physiological and dynamic system in response to an acute water diuresis.
In vitro production of angiotensin II by isolated glomeruli
Authors: Atiyeh B. A. , Arant B. S. , Henrich W. L. , Seikaly M. G.
Published: December 2017

The glomerulus has several components of the renin-angiotensin system (RAS). The purpose of this study was to evaluate the ability of glomeruli isolated from adult Wistar-Kyoto rats to produce angiotensin II (ANG II). When isolated glomeruli were incubated in Krebs buffer, the peak concentration of immunoreactive angiotensin (ANG) in the incubation medium, representing simultaneous production and degradation, occurred after 15 min of incubation (3.98 +/- 0.34 pg.mg protein-1.15 min-1, of which 18% was ANG II. When 125I-labeled ANG II was incubated with isolated glomeruli, the half-life of ANG II was 6.06 min. Hence, we estimated ANG II production at 3.77 +/- 0.21 pg.mg protein-1.15 min-1. When angiotensinogen-rich serum was added to the incubation medium, ANG concentration at 15 min increased by 500-fold (1,978 +/- 44 pg.mg protein-1.15 min-1, P < 0.001). ANG concentration in the glomerular incubate responded to perturbations known to alter systemic RAS. Enalaprilat, chymostatin, propranolol, and renin antiserum decreased ANG concentration in glomerular incubate, whereas salt depletion increased this (P < 0.05). We conclude that the rat glomerulus can generate ANG II independent of neural, hormonal, or vascular control.
Biotinylation and assessment of membrane polarity: caveats and methodological concerns
Authors: Gottardi C. J. , Dunbar L. A. , Caplan M. J.
Published: December 2017

Studies of epithelial membrane polarity have been greatly facilitated through the use of the N-hydroxysuccinimide-biotin surface labeling technique (M. Sargiacomo, M. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. J. Membr. Biol. 107: 277-286, 1989). We have used this technique in studies on the sorting and targeting of ion-transporting adenosinetriphosphatase molecules in polarized epithelial cells. Through efforts to optimize this technique in our experimental system, we have encountered several experimental conditions and circumstances where biotinylation is extremely inefficient and the assessment of membrane polarity which it provides is misleading. We demonstrate that the pH and ionic strength of the biotinylation buffer can dramatically affect biotin incorporation and that protocol-dependent variations in the recovery of biotinylated proteins can result in misrepresentation of the actual apical/basolateral distribution of a protein. Conditions and protocols that may improve the sensitivity and accuracy of this technique are discussed.
Water channel expression in human ADPKD kidneys
Authors: Bachinsky D. R. , Sabolic I. , Emmanouel D. S. , Jefferson D. M. , Carone F. A. , Brown D. , Perrone R. D.
Published: December 2017

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) results in part from the transport of solute and fluid into the lumen of the cyst. In proximal tubules and thin descending limbs of normal kidneys, the high transepithelial water permeability of these segments is due to the presence of the water channel protein, aquaporin-CHIP (AQP-CHIP, i.e., AQP-1). The collecting ducts of normal kidneys express another member of this gene family, the aquaporin collecting duct (AQP-CD, i.e., AQP-2). The expression and distribution of these two members of the aquaporin gene family were examined in ADPKD and normal human kidneys. In both tissues, Western blotting with the anti-AQP-CHIP antibody revealed a major 28-kDa band. By immunofluorescence, AQP-CHIP was present in proximal tubules and thin descending limbs of Henle of both normal and ADPKD kidneys. In the latter, AQP-CHIP was detected in the epithelia lining 71% of cysts. Many cysts were positive for the proximal tubule marker gp330 (44%). Some cysts expressing AQP-CHIP did not stain for gp330, suggesting a descending thin limb origin, and a few cysts were negative for both markers. In normal human kidney, Western blotting with the anti-AQP-CD antibody revealed a band at 28 kDa. AQP-CD was localized to collecting ducts and did not show colocalization with gp330 in normal human kidney. In ADPKD kidney, AQP-CD was expressed by only 8% of cysts. In summary, water channels, primarily AQP-CHIP, are expressed in epithelial cells lining cysts in approximately 80% of cysts in ADPKD kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)
Pressure dependency of canine intrarenal blood flow within the range of autoregulation
Authors: Lerman L. O. , Bentley M. D. , Fiksen-Olsen M. J. , Strick D. M. , Ritman E. L. , Romero J. C.
Published: December 2017

The mechanism of pressure-induced natriuresis remains controversial. To assess whether intracortical or medullary renal blood flows (RBF) change with changes in renal perfusion pressure (RPP), global and regional RBFs were measured using the dynamic spatial reconstructor, a fast computed tomography scanner, in eight anesthetized dogs (group B) within the range of RBF autoregulation (RPP of 153.5 and 114.4 mmHg). Similar measurements were obtained in seven control dogs (group A) in which RPP was not manipulated. In group B, only inner medullary perfusion decreased (from 0.84 to 0.51 ml/min per cm3 of tissue, P = 0.03) with reduction of RPP, whereas global renal, intracortical, and outer medullary perfusions remained unaltered. In group A there was no change in global or regional renal perfusion. The change in inner medullary perfusion in group B (-34.7%) was significantly different (P = 0.021) from that in group A (+27.4%). Global, cortical, and total medullary RBFs (ml/min) and volumes did not change in either group. These results suggest that with changes in RPP, the only detectable change in intrarenal perfusion occurs in the inner medulla.
Role of endothelin receptor subtypes in the in vivo regulation of renal function
Authors: Clavell A. L. , Stingo A. J. , Margulies K. B. , Brandt R. R. , Burnett J. C.
Published: December 2017

Endothelin (ET) is a potent vasoconstrictor peptide of endothelial origin, which at low doses results in renal vasoconstriction and diuresis with variable actions on sodium excretion. The current study conducted in four groups of anesthetized dogs was designed to define the role of the ETA and ETB receptor subtypes in the renal actions of low-dose exogenous ET. Group 1 (n = 4) animals served as time controls. In group 2 (n = 6) a systemic ET-1 (5 ng.kg-1.min-1) infusion mediated renal vasoconstriction, antinatriuresis with increases in proximal fractional reabsorption of sodium, and diuresis with a decrease in urine osmolality. In group 3 (n = 6) intrarenal BQ-123 (4 micrograms.kg-1.min-1), a selective ETA antagonist, abolished the systemic ET-1-mediated changes in renal hemodynamics and unmasked a natriuretic action at the level of the proximal tubule. In contrast, the diuretic response of ET was not altered by BQ-123. In group 4 (n = 6) intrarenal sarafotoxin 6-c, a selective ETB receptor agonist, resulted in a diuretic response without a change in sodium excretion. These studies suggest that the ETA receptor contributes to the renal vasoconstriction, whereas the ETB receptor is largely responsible for the diuretic response during exogenous ET. This study also suggests that at low doses ET is natriuretic in vivo by decreasing proximal tubular reabsorption of sodium independent of ETA or ETB receptor activation.
Adhesion of calcium oxalate monohydrate crystals to renal epithelial cells is inhibited by specific anions
Authors: Lieske J. C. , Leonard R. , Toback F. G.
Published: December 2017

Adhesion of urinary crystals to the apical surface of renal tubular cells could be a critical step in the formation of kidney stones. The interaction between renal epithelial cells (BSC-1 line) and the most common crystal in kidney stones, calcium oxalate monohydrate (COM), was studied in a tissue culture model system. COM crystals bound to the cell surface within seconds in a concentration-dependent manner to a far greater extent than did brushite, another calcium-containing crystal found in urine. Adhesion of COM crystals to cells was blocked by the polyanion, heparin. Other glycosaminoglycans including chondroitin sulfate A or B, heparan sulfate, and hyaluronic acid, but not chondroitin sulfate C, prevented binding of COM crystals. Two nonsulfated polyanions, polyglutamic acid and polyaspartic acid, also blocked adherence of COM crystals. Three molecules found in urine, nephrocalcin, uropontin, and citrate, each inhibited binding of COM crystals, whereas Tamm-Horsfall glycoprotein (THP) did not. Prior exposure of crystals but not cells to inhibitory molecules blocked adhesion, suggesting that these agents exert their effect at the crystal surface. Inhibition of crystal binding followed a linear Langmuir adsorption isotherm for each inhibitor identified, suggesting that these molecules bind to a single class of sites on the crystal that are important for adhesion to the cell surface. Inhibition of crystal adhesion by heparin was rapidly overcome by the polycation protamine, suggesting that the glycosaminoglycan regulates cell-crystal interactions in a potentially reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Renal localization and actions of adrenomedullin: a natriuretic peptide
Authors: Jougasaki M. , Wei C. M. , Aarhus L. L. , Heublein D. M. , Sandberg S. M. , Burnett J. C.
Published: December 2017

Adrenomedullin (ADM) is a newly described 52-amino acid peptide originally isolated from extracts of human pheochromocytoma and, more recently, detected in human plasma. Based on the report that ADM mRNA and immunoreactivity are present in the kidney, the current study was designed to determine the renal distribution of ADM by immunohistochemistry and the renal biological actions of ADM. In the immunohistochemical studies, the present investigation demonstrated the localization of ADM in glomeruli, cortical distal tubules, and medullary collecting duct cells of the normal canine kidney. In the in vivo studies, ADM was administered (0.25 ng.kg-1.min-1 in group I and 1, 5, and 25 ng.kg-1.min-1 in group II) intrarenally in normal mongrel dogs with the contralateral kidney receiving only saline vehicle. Intrarenal infusion of ADM resulted in a marked diuretic and natriuretic response, whereas the contralateral kidney showed no renal effects. These significant natriuresis and diuresis in the ADM kidney were associated with increases in glomerular filtration rate and fractional sodium excretion and with a decrease in distal tubular sodium reabsorption. Intrarenal infusion of ADM also caused an increase in mean arterial blood pressure and a decrease in heart rate. Plasma concentrations of atrial natriuretic peptide, renin activity, aldosterone, and guanosine 3',5'-cyclic monophosphate were not changed during the infusion of ADM. The current study demonstrates that ADM is present in renal glomerular and tubular cells and is a potent natriuretic peptide that may play an important role in the regulation of sodium excretion.
Comparison of central core and radially separated models of renal inner medulla
Authors: Jen J. F. , Wang H. , Tewarson R. P. , Stephenson J. L.
Published: December 2017

In this paper we describe the effect of partitioning exchange of ascending thin limb (ATL) and collecting duct (CD) between a central vascular space (CORE) and a radially separated capillary node (NODE) in a mathematical model of the concentrating mechanism of the renal inner medulla. A detailed description of the model has been provided [J. L. Stephenson, J. F. Jen, H. Wang, and R. P. Tewarson. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F680–F692, 1995]. We define a partition coefficient theta, which denotes the fractional exchange of CD and ATL with the NODE. Thus with theta = 0 we have a central core model, in which the ATL and CD exchange with the CORE only, and with theta = 1 we have a totally radially separated model, in which the ATL and CD exchange with the NODE only. Decreasing the partition coefficient from 1 to 0 effects a continuous transition from a totally radially separated model to a central core model. As this transition progresses with increasing exchange with the CORE, the osmolalities in all structures become nearly the same at the papilla, and the ability to transport salt uphill is lost. This is true even with no radial diffusion. However, radial diffusion and direct exchange with the CORE act synergistically in decreasing osmolality differences at the papilla and the capacity for convective uphill transport. These are lost in a more or less parallel way. There is, however, no significant concomitant change in concentrating ability. These results indicate that models with radial mixing of the interstitial vascular space are probably reasonably good approximations for the inner medulla.
Effect of vasa recta flow on concentrating ability of models of renal inner medulla
Authors: Stephenson J. L. , Wang H. , Tewarson R. P.
Published: December 2017

In this study we extend the analysis of the preceding two studies [J. L. Stephenson, J. F. Jen, H. Wang, and R. P. Tewarson. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F698–F709, 1995; and J. F. Jen, H. Wang, R. P. Tewarson, and J. L. Stephenson. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F000–F000, 1995] to a model that includes vasa recta. Distribution of nephron and vasa recta lengths is represented by shunting from descending to ascending flow. It is found that the effect of radial separation of structures on concentrating ability is closely linked to vasa recta flow. With minimal or no vasa recta flow the extent of radial mixing has little effect on concentrating ability. As vasa recta flow increases, concentrating ability is decreased by radial mixing. Convective uphill transport of NaCl is again observed, but concentrating ability appears to depend primarily on urea delivery to the inner medulla from the collecting duct rather than on the mechanism of salt transport out of thin ascending limb. Central core models give an upper bound on concentrating ability but do not attain the maximum urine osmolality of the rat with experimental values of tubular permeabilities.
Effect of peptide YY on human renal function
Authors: Playford R. J. , Mehta S. , Upton P. , Rentch R. , Moss S. , Calam J. , Bloom S. , Payne N. , Ghatei M. , Edwards R. , et al.
Published: December 2017

Peptide YY (PYY) is released from the intestine into the circulation in response to ingestion of food. As PYY/neuropeptide Y (NPY) receptors have recently been identified in the human kidney, we examined the effect of PYY on human renal function. Male subjects (6 per group) received control infusate or PYY at 0.4 or 1.2 pmol.kg-1.min-1 intravenously for 90 min with control periods before and after. Infusion of PYY at the lower dose reproduced normal postprandial plasma concentrations and caused significant decreases in glomerular filtration rate (10% reduction), plasma renin activity (30% reduction), and aldosterone levels while increasing sodium excretion by approximately 30% (all P < 0.01). Infusion of PYY at the higher dose, which reproduced plasma levels seen during a diarrheal illness, resulted in similar changes in renal function and also reduced renal plasma flow by approximately 10% (P < 0.05). Therefore, PYY may be an important mediator of the normal postprandial natriuretic response, and PYY agonists could provide a novel approach to the treatment of patients with sodium overload.
Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells
Authors: Mohaupt M. G. , Schwobel J. , Elzie J. L. , Kannan G. S. , Kone B. C.
Published: December 2017

The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.
Effect of barium on potassium diffusion across the proximal convoluted tubule of the anesthetized rat
Authors: Kibble J. D. , Wareing M. , Wilson R. W. , Green R.
Published: December 2017

The role of diffusion in transepithelial potassium flux and the importance of potassium channels in the luminal cell membrane to this process were examined by applying a luminal microperfusion technique to surface tubules in kidneys of anesthetized rats. Potassium concentration gradients were applied by altering the concentration of KCl in perfusates. To some perfusates, 2 mmol/l BaCl2 was added to block potassium channels in the luminal cell membrane. The mean applied potassium concentration gradient was highly predictive of net potassium transport in the absence of any change in fluid reabsorption, with an apparent potassium permeability of 22 x 10(-5) cm/s. Thus potassium transport in the proximal tubule may have an important diffusive component. Luminal barium significantly reduced the concentration of potassium in collected fluid under conditions of net potassium secretion, although a substantial barium-insensitive potassium permeability was also observed. However, the site of action of luminally applied barium is uncertain in proximal tubule, since barium was reabsorbed by the tubule at a rate of 13.6 pmol.mm-1.min-1. We conclude that diffusion is a significant driving force for potassium reabsorption in proximal tubule and that most diffusive potassium transport occurs via a barium-insensitive route, possibly the paracellular pathway.
Parathyroid hormone action on phosphate transporter mRNA and protein in rat renal proximal tubules
Authors: Kempson S. A. , Lotscher M. , Kaissling B. , Biber J. , Murer H. , Levi M.
Published: December 2017

The inhibitory action of parathyroid hormone (PTH) on Pi reabsorption in the renal proximal tubule is accompanied by a specific decrease in Na-Pi cotransport at the apical brush-border membrane (BBM). It is not known whether this decrease represents decreased activity of Na-Pi cotransporters already present in the BBM or whether the number of cotransporters is decreased. The present study of the molecular mechanism of PTH action made use of a specific cDNA probe and antiserum to a rat renal Na-Pi cotransporter (NaPi-2). Three groups of rats were used: intact controls, chronically parathyroidectomized (PTX), and PTX rats treated acutely (2 h) with bovine PTH-(1--34). Na-Pi cotransport by isolated renal BBM vesicles was increased to 1,315 +/- 44 in PTX rats, compared with 721 +/- 94 pmol.mg-1.10 s-1 in controls (P < 0.002), and was returned to control levels by PTH. Western blots of these BBM showed that PTX caused a 2.8-fold increase in NaPi-2 protein content, which was reduced to control levels by PTH. Immunohistochemistry of perfusion-fixed kidneys showed NaPi-2-specific immunofluorescence exclusively in apical BBM of proximal tubules. Expression of NaPi-2 protein at these sites was increased in PTX rats and decreased after PTH treatment. Northern analysis of total RNA showed that the abundance of NaPi-2-specific mRNA was not changed by PTX but there was a small decrease in response to PTH. The data indicate that PTH regulation of renal Na-Pi cotransport is determined by changes in expression of NaPi-2 protein in the renal BBM.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of nitric oxide on renin secretion. I. Studies in isolated juxtaglomerular granular cells
Authors: Greenberg S. G. , He X. R. , Schnermann J. B. , Briggs J. P.
Published: December 2017

Experiments were performed on juxtaglomerular granular cells (JGC) in short-term primary culture to determine the direct immediate effect of NO on renin secretion and to test whether JGC are able to generate NO. Renin secretion was measured repeatedly over short time intervals in a cell superfusion system. Renin release did not significantly decrease over a 40-min observation period in untreated JGC. Addition of sodium nitroprusside (SNP) caused a reduction in renin release (measured in nano-Goldblatt hog units vs. time, i.e., nGU/min) from 479 +/- 25, 423 +/- 70, and 388 +/- 54 nGU/min to 295 +/- 19 (n = 5), 102 +/- 21 (n = 7), and 71 +/- 9 nGU/min (n = 6) with 10(-5), 10(-4), and 10(-3) M SNP, respectively. In the presence of the guanylate cyclase inhibitor methylene blue at 10(-4) M, SNP at 10(-4) M had no significant effect on renin secretion. 8-Bromoguanosine 3',5'-cyclic monophosphate at 10(-4) M in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (10(-3) M) caused a reduction of renin secretion to 50.1 +/- 3.6% of control. To examine the possibility that renin secretion is affected by NO release from JGC, we assessed the effect of the NO synthase (NOS) substrate L-arginine (10(-3) M) and the NOS blocker N omega-nitro-L-arginine (10(-4) M) on renin secretion. Renin release was not significantly altered by either stimulation or inhibition of NOS activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Differences in photoaffinity labeling of DA1 receptors in renal proximal tubules from normotensive rat and SHR
Authors: Vachvanichsanong P. , Kimura K. , Sidhu A.
Published: December 2017

Renal DA1 dopamine receptors in proximal tubule membranes of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were characterized with the novel D1 dopamine receptor-selective photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5- tetrahydro-1H-3-benzazepine ([125I]MAB). Under nonphotolyzing conditions, saturation studies showed that [125I]MAB bound with similar affinity to DA1 dopamine receptors in both WKY [dissociation constant (Kd) = 16.3 nM] and SHR (Kd = 19.5 nM). At photolysis, [125I]MAB was irreversibly incorporated into a single major protein of 74,000 Da in both WKY and SHR. DA1-selective antagonists blocked photolabeling of DA1 sites with similar efficiency and specificity in SHR and WKY. However, under identical assay conditions, dopaminergic agonists were unable to block photoincorporation of [125I]MAB in SHR but not in WKY. This pattern of labeling of DA1 sites by [125I]MAB may suggest the presence of defective agonist, but not antagonist, binding domains on the receptor in SHR but not in WKY rats.