bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyol pathway and osmoregulation in JS1 Schwann cells grown in hyperglycemic and hyperosmotic conditions
Authors: Mizisin A. P. , Li L. , Perello M. , Freshwater J. D. , Kalichman M. W. , Roux L. , Calcutt N. A.
Published: December 2017

To test the osmoregulatory rules of Schwann cell aldose reductase (AR) and myo-inositol, JS1 Schwann cells were grown under control and hyperosmotic conditions with and without excess glucose or galactose. JS1 cells cultured in control conditions possessed AR protein and activity that were not altered by the inclusion of 25 mM glucose or galactose. Following culture with 100 mM NaCl, there was a decline in cell number accompanied by an increase in AR activity, both of which were attenuated by the addition of 25 mM glucose or galactose. Sorbitol was not detected in JS1 Schwann cells following culture in control, glucose-supplemented, or hyperosmotic medium, and dulcitol accumulated only following culture with galactose. However, both polyols were dramatically increased in JS1 cells cultured in hyperosmotic medium supplemented with 25 mM glucose or galactose. In contrast, myo-inositol levels were elevated only during hyperosmotic exposure but decreased when glucose or galactose was also present. These data are consistent with the use of polyol formation by JS1 Schwann cells as a means of responding to osmotic stress.
Absence of DA1/DA2 dopamine receptor interactions in proximal tubules of spontaneously hypertensive rats
Authors: Vachvanichsanong P. , Sela S. , Sidhu A.
Published: December 2017

The impact of defective DA1 dopamine receptors in proximal tubules (PT) of the spontaneously hypertensive rat (SHR) on DA1/DA2 receptor interactions was assessed with the DA1-selective photoaffinity ligand, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)- 2,3,4,5-tetrahydro-1H-3-benzazepine ([125I]MAB). In PT membranes from both normotensive (Wistar-Kyoto, WKY) and spontaneously hypertensive rats (SHR), [125I]MAB was specifically incorporated into a polypeptide with an M(r) of 74,000 Da, corresponding to the DA1 receptor. The labeling of this band by [125I]MAB in both SHR and WKY was not prevented by SKF-82526, a potent DA1-selective agonist. However, in the presence of the DA2 antagonist, (-)-sulpiride, but not DA2 agonist, LY-171555, SKF-82526 abolished photoincorporation of [125I]MAB into the 74,000-Da band in WKY. In SHR, (-)-sulpiride failed to enhance the ability of SKF-82526 to compete with [125I]MAB for binding to the 74,000-Da subunit. In competition binding studies with SKF-82526, (-)-sulpiride induced the formation of agonist high-affinity binding sites in WKY but not in SHR. These data suggest that in membranes of SHR, but not WKY, DA1/DA2 dopamine receptor interactions are lacking.
Studies of renal injury. I. Gentamicin toxicity and expression of basolateral transporters
Authors: Dominguez J. H. , Hale C. C. , Qulali M.
Published: December 2017

Gentamicin nephrotoxicity may arise in part from alterations in the expression of genes critical for renal proximal tubule metabolism. We tested the hypothesis that gentamicin suppressed the gene expression of the Na+/Ca2+ exchanger (NaCaX), glucose transporter 1 (GLUT1) and alpha 1-subunit of Na(+)-K(+)-ATPase (alpha 1-NKA) in renal tubules. The products of these genes mediate Na(+)-dependent Ca2+ efflux, glucose efflux and influx, and ATP-dependent Na+ efflux across tubular basolateral membranes, respectively. After 10 days of gentamicin intoxication (40 mg/kg ip, twice daily), levels of mRNAs encoding NaCaX and the cognate protein declined. GLUT1 mRNA levels increased, although GLUT1 protein levels were also reduced. Moreover, whereas alpha 1-NKA mRNA levels remained unchanged, alpha 1-NKA protein levels were also reduced. We suggest that the higher GLUT1 mRNA level is part of the stress response to tubular injury. However, regardless of the mRNA level, the most consistent effect of gentamicin was reduction of specific protein levels. We propose that failure to translate high levels of mRNA into proportionally high levels of protein, as in the case of GLUT1, may attenuate the expression of stress response gene products, and thus diminish the possibility of recovery in gentamicin intoxication.
Amylin binding in rat renal cortex, stimulation of adenylyl cyclase, and activation of plasma renin
Authors: Wookey P. J. , Tikellis C. , Du H. C. , Qin H. F. , Sexton P. M. , Cooper M. E.
Published: December 2017

125I-labeled rat amylin binds to specific sites in the cortex of rat kidney, which can be distinguished from those for 125I-labeled salmon calcitonin (sCT) and 125I-labeled rat alpha-calcitonin gene-related peptide (alpha-CGRP) on the basis of regional distribution. These sites have a high affinity (approximately 1 nM) for amylin, and 125I-amylin binding is potently inhibited by the peptide antagonists AC413 and sCT-(8-32), whereas CGRP-(8-37) is a poor inhibitor of binding. Furthermore, incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) inhibits 125I-amylin binding by > 90%, indicating that binding is dependent on coupling to G proteins. In renal cortex, amylin stimulated adenylyl cyclase activity three- to fourfold, and this was inhibited by AC413 and sCT-(8-32) but not by CGRP-(8-37). Amylin activated plasma renin twofold, and this was blunted by prior administration of AC413 but not CGRP-(8-37). We speculate that amylin may play an important role in renal physiology and that in states of hyperamylinemia, as found in obesity and the insulin resistance syndrome, this peptide may be involved in the genesis and development of hypertension.
Postprandial natriuresis in humans: further evidence that urodilatin, not ANP, modulates sodium excretion
Authors: Drummer C. , Franck W. , Heer M. , Forssmann W. G. , Gerzer R. , Goetz K.
Published: December 2017

We examined the effects of a high-salt (100 mmol NaCl) and a low-salt (5 mmol NaCl) meal on the renal excretion of sodium and chloride in 12 healthy male upright subjects. We also measured the urinary excretion of urodilatin [ANP-(95-126)], and the plasma or serum concentrations of atrial natriuretic peptide [ANP-(99-126)], aldosterone, and renin. The high-salt meal produced a postprandial natriuresis (urinary sodium excretion from 59.0 to a peak rate of 204.6 mumol/min in 3rd h after ingestion of meal) and chloride excretion. In parallel, the urinary excretion of urodilatin increased from 35.7 to a peak rate of 105 fmol/min. The effect of high-salt intake on urinary sodium, chloride, and urodilatin excretion was significant (analysis of variance, P < 0.01), and close significant correlations were observed between urodilatin and sodium excretion (mean R = 0.702) as well as between urodilatin and chloride excretion (mean R = 0.776). In contrast, plasma ANP, which was acutely elevated 15 min after high-salt intake, was already back to low-salt values 1 h later. It did not parallel the postprandial natriuretic profile, and no positive correlation between plasma ANP and sodium excretion was observed. These results provide further evidence that urodilatin, not ANP, is the member of this peptide family primarily involved in the regulation of the excretion of sodium and chloride.
Transient expression of oatp organic anion transporter in mammalian cells: identification of candidate substrates
Authors: Kanai N. , Lu R. , Bao Y. , Wolkoff A. W. , Schuster V. L.
Published: December 2017

The cDNA for the rat liver organic anion-transporting polypeptide "oatp" has been shown to encode transport of bromosulfophthalein (BSP) and bile salts in Xenopus oocytes (E. Jacquemin, B. Hagenbuch, B. Stieger, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133-137, 1994). Because oatp mRNA is expressed strongly in the kidney, we sought to determine whether renal oatp might play a role in the known secretion of a large variety of organic anions by the kidney. We transiently expressed a full-length oatp cDNA, cloned in pSPORT, in HeLa cell monolayers using the recombinant vaccinia virus vtf7-3. We tested an array of organic anions as candidate substrates by determining their ability to compete with tracer BSP for transport. HeLa cell monolayers transfected with the oatp cDNA transported tracer BSP and taurocholate at rates substantially higher than monolayers transfected with a control plasmid. Thus good expression can be obtained with the vaccinia-HeLa system using a standard plasmid cloning vector. BSP transport varied as a function of the medium albumin, ionic conditions, and pH in a fashion similar to that in Xenopus oocytes. Several organic anions known to be secreted by the classic secretory pathway, including p-aminohippurate (PAH), phenol red, and indigo carmine (10 microM) failed to inhibit oatp-mediated BSP transport. Direct testing using tracers revealed no oatp-mediated transport of sulfate, urate, PAH, several eicosanoids, or unconjugated or conjugated bilirubin. On the other hand, BSP transport was inhibited by approximately 50% by 10 microM corticosterone sulfate, spironolactone, and several other steroids. We conclude that the functional properties of oatp expressed in the HeLa cell/vaccinia transient expression system are comparable to those following expression in Xenopus oocytes and that steroids are likely to represent high-affinity endogenous oatp substrates. The latter hypothesis is addressed in greater detail in a companion paper.
Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C
Authors: Frindt G. , Palmer L. G. , Windhager E. E.
Published: December 2017

The hypothesis that feedback inhibition of the apical Na+ channels in the cortical collecting tubule (CCT) is mediated by activation of a Ca(2+)-dependent protein kinase was tested using the patch-clamp technique. Na+ channel activity was monitored in cell-attached patches in principal cells of split-open rat tubules. Mean number of open channels (NPo) and single-channel current (i) were measured at 37 degrees C during continuous tubule superfusion. Phorbol 12-myristate 13-acetate (PMA; 50 nM), an activator of protein kinase C (PKC), decreased NPo to 33% of the control value. Staurosporine (200 nM), an inhibitor of PKC and of Ca(2+)-calmodulin kinase II, practically abolished the effect of PMA. Ouabain (1 mM), reduced NPo to 29% of control values and decreased i. Ouabain did not downregulate the channels in tubules exposed to staurosporine, although it still reduced i. Incubation of the tubules with 10 microM KN-62, a specific cell membrane-permeable inhibitor of Ca(2+)-calmodulin kinase II, did not interfere with the ouabain-dependent downregulation of the channels. The results support the view that the downregulation caused by ouabain involves the Ca(2+)-dependent phosphorylation of the channel itself or of proteins regulating the channel.
Angiotensin II: a humoral mediator for the gastric sodium monitor
Authors: Duggan K. A. , Ye V. Z. , Jones D. M. , Macdonald G. J.
Published: December 2017

Natriuresis in direct response to a gastric sodium stimulus (upper-gut sodium monitor) has paradoxically only been demonstrated in humans and animals on a low-sodium diet preceding each study. It is possible that the low-sodium diet itself induces or suppresses systems that mediate or oppose the ensuing natriuresis. In this study, we sought to determine whether a system activated by this diet, the renin-angiotensin system, mediates the natriuretic response. Specifically, we sought to show whether changes in the circulating concentration of angiotensin II (ANG II) may mediate the renal response to stimulation of the gastric sodium monitor. Male New Zealand White rabbits were randomly assigned to low- (0.008%) or normal (2.2%) sodium diets. After 1 wk on the experimental diet, they received a sodium load intragastrically or intravenously, and plasma ANG II was measured at 0, 5, 10, 30, 60, and 120 min. Urine was collected for 4 h after the sodium load, and plasma sodium was measured at 0, 2, and 4 h. Urinary sodium excretion was greater in the 4 h after gastric than after intravenous sodium administration (P < 0.025) in the rabbits on the low-sodium diet. No significant difference was noted in the rabbits on the normal sodium. In rabbits on the low-sodium diet, there was an immediate and significant decline in plasma ANG II after sodium was administered both intragastrically (P < 0.025) and intravenously (P < 0.05). This decrease was greater after intragastric than intravenous sodium (P < 0.0025), and the difference was still evident at 120 min (P < 0.05). No significant difference in plasma ANG II was found in the normal diet group. We conclude, therefore, that a prolonged decrease in ANG II concentration may play a role in mediating the natriuretic response to the gastric sodium monitor.
Dexamethasone upregulates ANP C-receptor protein in human mesangial cells without affecting mRNA
Authors: Ardaillou N. , Blaise V. , Placier S. , Amestoy F. , Ardaillou R.
Published: December 2017

The objective of this study was to examine the role of dexamethasone on the expression of natriuretic peptide B-type and C-type receptors (ANPR-B and ANPR-C) in cultured human mesangial cells, which only possess these two subtypes. Dexamethasone caused concentration- and time-dependent increases in 125I-labeled ANP binding, which were prevented by glucocorticoid receptor inhibition with RU-38486. A lag time of 24 h and a concentration of dexamethasone of at least 1 nmol/l were necessary for this effect to occur. Dexamethasone-induced upregulation of 125I-ANP binding resulted from increased receptor density. No change in dissociation constant (Kd) was observed. Only ANPR-C were affected by dexamethasone. Indeed, dexamethasone did not modify C-type natriuretic peptide (i.e., CNP)-dependent cGMP production by mesangial cells. Moreover, dexamethasone upregulated ANPR-C protein expression as shown by Western blot analysis and by an increase in ANPR-C immunoreactivity at the cell surface. In contrast, dexamethasone did not modify ANPR-C mRNA expression. In conclusion, glucocorticoids increase ANPR-C density on mesangial cells through a mechanism implying, successively, interaction with the glucocorticoid receptor and increase of ANPR-C protein synthesis at a posttranscriptional stage. Thus dexamethasone may influence availability of natriuretic peptides at their glomerular target sites.
Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney
Authors: Brosius F. C. , Nguyen K. , Stuart-Tilley A. K. , Haller C. , Briggs J. P. , Alper S. L.
Published: December 2017

Pages F461–F468: F. C. Brosius III, K. Nguyen, A. K. Stuart-Tilley, C. Haller, J. P. Briggs, and S. L. Alper. “Regional and segmental localization of AE2 anion exchanger mRNA and protein in rat kidney.” The last portion of the legend to Fig. 6 (p. F465) should read as follows: “ B: immunoblot of mouse kidney microsomes prepared with high concentrations of protease inhibitors. All lanes were loaded with 25 μg protein from medulla ( lanes 1 and 4), cortex ( lanes 2 and 5), and whole kidney ( lanes 3 and 6). Blots were visualized with ECL.” Also, in the accompanying section of text in the results (p. 465, column 2), at the paragraph beginning “Membranes prepared...”, the second sentence should read as follows: “Maximal levels of 170-kDa AE2 polypeptide were present in medulla (Fig. 6B, lane 1), whereas cortex (Fig. 6B, lane 2) contained lower levels of AE2.”
F-actin disorganization in apoptotic cell death of cultured rat renal proximal tubular cells
Authors: Van de Water B. , Kruidering M. , Nagelkerke J. F.
Published: December 2017

The mechanism of nephrotoxin-induced apoptosis was studied in rat renal proximal tubular cells (PTC) exposed to the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC). After a 6-h incubation, DCVC caused a condensation of heterochromatin and a fragmentation of the nucleus in 84 and 16% of the cells, respectively, which is indicative of apoptosis. This was confirmed biochemically by agarose gel electrophoresis demonstrating the formation of DNA fragments with multiples of 200 bp. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented neither the fragmentation of the nucleus nor the formation of DNA fragments, but it did prevent lactate dehydrogenase release and bleb formation by DCVC. Apoptosis induced by DCVC was closely associated with F-actin disorganization: every cell with a fragmented nucleus displayed completely disorganized F-actin, while cells with a normal nucleus still possessed at least some intact F-actin also induced apoptosis in PTC. Similarly, dithiothreitol, which damages F-actin in PTC, caused apoptosis of PTC. These data suggest a causal relationship between F-actin disorganization and apoptosis of PTC.
Bilateral ureteral obstruction downregulates expression of vasopressin-sensitive AQP-2 water channel in rat kidney
Authors: Frokiaer J. , Marples D. , Knepper M. A. , Nielsen S.
Published: December 2017

Polyuria after release of bilateral ureteral obstruction (BUO) is frequently seen in patients with urological disorders. In this study, we examined the effect of BUO and release of BUO on the expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in rat kidney. Ureters were obstructed for 24 h in all experiments, and BUO was either not released or released for 24 or 48 h or 7 days. Each group of experimental rats were matched with sham-operated controls. One kidney was used for membrane fractionation and immunoblotting, whereas the contralateral was fixed for immunocytochemistry. Immunoblotting demonstrated a significant reduction in AQP-2 expression in inner medullar during 24 h of BUO to 26 +/- 8% (P < 0.001). Release of BUO was associated with immediate onset of a predominant osmotic-dependent polyuria. Forty-eight hours after release of BUO, the reduction in AQP-2 expression persisted (19 +/- 8%, P < 0.001), concurrent with a marked nonosmotic postobstructive polyuria, as determined by a significant reduction in free-water clearance (-50 +/- 7 vs. -85 +/- 10 microliters.min-1.kg-1, P < 0.05). Immunofluorescence and immunoelectron microscopy confirmed the reduced levels of AQP-2 in collecting duct principal cells. Seven days after release, the renal excretion of water and electrolytes had almost normalized. However, the downregulation of AQP-2 was not partly reversed (49 +/- 14%, P < 0.001), and, consistent with this, the urinary concentrating capacity was significantly reduced 7 days after release to a 18-h period of thirst. This strongly suggests that the persistent downregulation of AQP-2 is the cause of the slow recovery in concentration capacity. In conclusion, BUO and release of BUO were associated with a marked reduction in expression of AQP-2, coincident with the development and maintenance of postobstructive polyuria. Thus reduced AQP-2 levels may represent an important factor in the slow recovery from postobstructive diuresis.
RT-PCR microlocalization of mRNAs for calbindin D28k and vitamin D receptor in the murine nephron
Authors: Liu L. , Khastgir A. , McCauley J. M. , Dunn S. T. , Morrissey J. H. , Christakos S. , Hughes M. R. , Bourdeau J. E.
Published: December 2017

The spatial relationship between vitamin D receptor (VDR) and calbindin D28k [calcium binding protein D28k (CaBP-D28k)] gene expression within the murine kidney was studied by localizing their mRNAs in discrete nephron structures using reverse transcription-polymerase chain reaction (RT-PCR). Primers for beta-actin mRNA were used as a control for the presence of tissue during RT-PCR for CaBP-D28k mRNA. mRNA for CaBP-D28k was found only in distal convoluted tubules (DCTs), connecting tubules (CNTs), and cortical collecting ducts (CCDs). In contrast, VDR mRNA was detected in glomeruli, S2 proximal convoluted tubules, cortical thick ascending limbs of Henle's loop, DCTs, CNTs, and initial CCDs. The presence of both VDR and CaBP-D28k mRNA in DCTs, CNTs, and CCDs is consistent with the hypothesis that cacitriol acts via the VDR to stimulate CaBP-D28k synthesis. Conversely, the presence of VDR mRNA in other parts of the nephron suggests that calcitriol has genomically mediated actions within the kidney in addition to stimulation of CaBP-D28k synthesis.
Annexin II inhibition of G protein-regulated inositol trisphosphate formation in rat aortic smooth muscle
Authors: Schelling J. R. , Gentry D. J. , Dubyak G. R.
Published: December 2017

Vasoconstrictor hormones contribute to the pathogenesis of hypertension through intracellular signals that stimulate vascular smooth muscle (VSMC) contraction and/or growth. We previously showed that the glucocorticoid dexamethasone (DEX) inhibited angiotensin II-stimulated inositol trisphosphate (IP3) formation in VSMC, but the mechanism of inhibition is not known. Because glucocorticoids stimulate the expression of annexins and annexin II potently binds phosphoinositides, the role of DEX and annexin II in VSMC G protein-coupled phosphoinositide hydrolysis was investigated. DEX incubation blunted increases in guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-stimulated IP3 generation and angiotensin II-induced intracellular Ca2+ mobilization but stimulated elevations in VSMC annexin II content. VSMC incubation with exogenous purified annexin II resulted in concentration-dependent decreases in GTP gamma S-stimulated IP3 formation. In DEX-treated cells, exogenous annexin II did not further diminish GTP gamma S-stimulated IP3 formation, suggesting that endogenous annexin II may be a mediator of DEX-induced inhibition of G protein-coupled IP3 generation. These data represent the first direct evidence of G protein-dependent phosphoinositide hydrolysis regulation by glucocorticoids or annexins. We speculate that annexin II may play a role in the pathogenesis of hypertension through stimulation of VSMC growth.
Blockade of chloride channels by DIDS stimulates renin release and inhibits contraction of afferent arterioles
Authors: Jensen B. L. , Skott O.
Published: December 2017

Calcium-activated chloride channels have been proposed to control renin release from juxtaglomerular cells and to be involved in the excitation-contraction coupling of the renal afferent arteriole. The hypothesis was tested on renin release from rat glomeruli and in microperfused rabbit afferent arterioles with the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Renin secretion was equally enhanced by omission of extracellular calcium and by addition of 0.5 mM DIDS. The inhibitory effect of calcium was blocked by DIDS. The stimulatory effects of low calcium [with or without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] and DIDS were not additive. In the absence of chloride, basal renin release was suppressed and the stimulatory effect of DIDS was abolished. The DIDS-induced enhancement of renin release was not dependent on bicarbonate. Norepinephrine (5 x 10(-7)-1 x 10(-6) M) and angiotensin II (1 x 10(-8)-10(-6) M) evoked reversible and dose-dependent contractions of microperfused rabbit afferent arterioles. DIDS (0.5 mM) did not affect the basal diameter of the arterioles but strongly inhibited the response to angiotensin II and attenuated the duration of the contractile response to norepinephrine. The results support the hypothesis that DIDS-sensitive calcium-activated chloride channels are involved in regulation of renin release and in the afferent arteriolar contraction after angiotensin II but do not play a pivotal role in the response to norepinephrine.
Cyclosporin A treatment induces overexpression of P-glycoprotein in the kidney and other tissues
Authors: Jette L. , Beaulieu E. , Leclerc J. M. , Beliveau R.
Published: December 2017

To see whether P-glycoprotein (PGP) expressed in renal brush-border membranes (BBM) could interact with compounds known as modulators of multidrug resistance (MDR), photoaffinity-labeling experiments were performed. A 145k-Da protein was photolabeled with [125I] iodoarylazidoprazosin, and this labeling was reduced in the presence of cyclosporin A (CsA) and PSC-833 (PSC). Interaction of CsA with PGP was further investigated by treating rats with daily subcutaneous injections of CsA (10 mg.kg-1.day-1). After this treatment, PGP expression levels were dramatically increased in renal BBM, intestine, liver, and many other tissues except the brain. This induction was a reversible process, since after cessation of CsA administration PGP levels declined to reach values similar to those of the control groups. The increase in PGP expression in the kidney was also detected in photolabeling experiments, suggesting the induction of a functional PGP. A higher dose of CsA (50 mg/kg) given as a bolus injection did not modify PGP expression] in renal BBM. These results demonstrate that CsA induces reversible overexpression of PGP in the rat. This may present significant relevance in the design of clinical trials using CsA as a chemosensitizing agent.
High glucose-induced TGF-beta 1 regulates mesangial production of heparan sulfate proteoglycan
Authors: Kolm V. , Sauer U. , Olgemooller B. , Schleicher E. D.
Published: December 2017

Previous investigations have demonstrated that growing mesangial cells in high glucose concentration stimulates extracellular matrix synthesis and also increases the expression of transforming growth factor-beta (TGF-beta). We examined the effects of hyperglycemia on mesangial proliferation and heparan sulfate proteoglycan (HSPG) and fibronectin production. Prolonged exposure of mesangial cells to increasing glucose concentrations resulted in dose-dependent effects on growth inhibition and stimulation of matrix production. Treatment of mesangial cells with high glucose-conditioned medium or with TGF-beta 1 mimicked the effects of high-glucose incubation. Furthermore, TGF-beta 1 caused a dose-dependent increase in HSPG mRNA levels. The high-glucose effects on mesangial cells were preceded by an increase in total TGF-beta 1 protein. The presence of TGF-beta 1 antisense oligonucleotide attenuated the glucose-mediated effects on mesangial proliferation and matrix production. The data show that even moderately elevated glucose concentrations appear to affect the mesangial cells. The results indicate that 1) TGF-beta 1 protein production is necessary to obtain the high glucose-induced effects and 2) TGF-beta 1 stimulates mesangial HSPG expression and production. Because these effects may be attenuated by oligonucleotides antisense to TGF-beta 1, the results suggest a possible way for effective intervention in TGF-beta-mediated glomerulosclerosis.
Correction of anemia by erythropoietin reverses insulin resistance and hyperinsulinemia in uremia
Authors: Mak R. H.
Published: December 2017

Ten patients (18 +/- 1 yr) on chronic hemodialysis (HD) with anemia were studied before and after treatment with erythropoietin (EPO) for 9 mo. Six patients had evidence of iron overload (serum ferritin over 300 ng/ml; group I) and the other four patients (serum ferritin below 300 ng/ml; group II) did not. Before treatment, both groups of patients were glucose tolerant but insulin resistant and hyperinsulinemic. There was equal correction of anemia but no significant changes in serum biochemistry (apart from iron studies) or anthropometric measurements in both groups. With amelioration of anemia and iron overload in group I, insulin sensitivity increased by 53% to within normal values. Insulin secretion also normalized. With amelioration of anemia but no change in iron status in group II, insulin sensitivity (increased by 60%) and insulin secretion also normalized. Thus correction of anemia by EPO reversed insulin resistance and hyperinsulinemia in HD patients with or without iron overload. The effects of correction of anemia rather than iron overload may be more important in the pathogenesis of insulin abnormalities in end-stage renal disease.
Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys
Authors: Hyink D. P. , Tucker D. C. , St John P. L. , Leardkamolkarn V. , Accavitti M. A. , Abrass C. K. , Abrahamson D. R.
Published: December 2017

To address origins of glomerular endothelial and mesangial cells in embryonic mammalian kidneys, we established interspecies grafts between rats and mice, in which fetal kidneys were implanted into the anterior eye chamber of adult hosts. After 5-7 days, hosts bearing grafts received intravenous injections with species-specific monoclonal antibodies (MAbs) to matrix components. In all cases, glomerular basement membranes and mesangial matrices labeled solely for donor-derived matrix. Additionally, microvessel extracellular matrices within grafts were usually of donor origin. To examine directly the origin of glomerular endothelial and mesangial cells, we grafted embryonic gestational days 11-12 (E11-12) kidneys from normal mice into anterior eye chambers of host reverse-orientation splice acceptor 26 mice, which are transgenic animals that express beta-galactosidase in every cell. When grafts were developed for beta-galactosidase activity, host cells were seen in peripheral vessels, but the majority of glomerular endothelial cells were of donor, not host, origin. Where host-derived-endothelial cells were found in glomeruli, donor endothelial cells were present as well. Mesangial cells were always of donor origin. When E11 mouse kidneys were labeled with the endothelial cell-specific Bandeiraea simplicifolia isolectin B4, we determined that endothelial cells are present from the inception of metanephrogenesis. Together, the evidence shows that cells of endogenous kidney origin were almost entirely responsible for development of the glomerular microvasculature in oculo. External vessels from the host, although important for graft maintenance, were not major contributors to the glomerulus.
Alpha 1-adrenergic control of chloride transport in the rat S1 proximal tubule
Authors: Wong K. R. , Berry C. A. , Cogan M. G.
Published: December 2017

To identify in vivo the specific alpha-adrenergic receptor mediating direct neural control of chloride transport in the rat S1 proximal convoluted tubule (PCT), the major effector site of neural regulation, microperfusion was employed in conjunction with the alpha 1- and alpha 2-adrenergic receptor antagonists, prazosin and rauwolscine. Using a glomerular ultrafiltrate-like perfusate, prazosin markedly inhibited chloride transport by -42% (302 +/- 10 to 176 +/- 5 peq.mm-1.min-1, P < 0.0001). Using a sodium chloride perfusate, which measures the active component of chloride absorption (J(Cl)act) (control, 153 peq.mm-1.min-1) plus a constant passive (479 peq.mm-1.min-1) component, both prazosin and acute renal denervation reduced J(Cl)act by -38% and -44% (-58 and -67 peq.mm-1.min-1, each P < 0.05). In contrast, rauwolscine caused no significant change in J(Cl)act using either perfusate. Prazosin regulates chloride transport via protein kinase C (PKC), since preactivation of PKC by phorbol abolished inhibitory impact of prazosin. Inhibition of J(Cl)act by prazosin (-58 peq.mm-1.min-1) was fully additive to either the stimulation or inhibition (losartan) of angiotensin II (55 or -49 peq.mm-1.min-1), which uses the adenosine 3',5'-cyclic monophosphate (cAMP) second messenger system [observed changes, not significantly different from 0 and -99 peq.mm-1.min-1; expected changes, not significantly different from 0 and -107 peq.mm-1.min-1]. In conclusion, neural control of S1 PCT chloride absorption in vivo is mediated by alpha 1-adrenergic receptors, which can selectively regulate J(Cl)act by altering PKC activity, independently of the cAMP second messenger system.
Modulation of Na+/Ca2+ exchange in epithelial cells of porcine thick ascending limb
Authors: Dai L. J. , Ritchie G. , Bapty B. , Auger V. , Quamme G. A.
Published: December 2017

We have provided functional and molecular evidence for the presence of Na+/Ca2+ exchange in isolated porcine cortical thick ascending limb (CTAL) cells. The present studies were designed to show that this exchange activity may be modulated by phosphorylative processes. Control of intracellular Ca2+ concentration ([Ca2+]i) was determined in isolated CTAL cells with microfluorescence. CTAL cells were pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) from 10 to 20 mM. These cells had normal basal [Ca2+]i (79 +/- 3 nM). Substitution of extracellular NaCl (50 mM) with KCl resulted in the rapid elevation of [Ca2+]i to maximal levels of 795 +/- 60 nM (n = 17). The increments of [Ca2+]i were associated with [Na+]i. We next determined the modulation of Na+/Ca2+ exchange activity with phosphorylative inhibitors. Pretreatment of cells with calmidazolium, a Ca(2+)-calmodulin inhibitor, resulted in a shift of the [Na+]i dependence curve to the right. Pretreatment with okadaic acid, a phosphatase 1 and 2A inhibitor, increased the Na+/Ca2+ exchanger activity resulting in half-maximal [Ca2+]i increase near normal [Na+]i of 12 mM. Furthermore, in the presence of okadaic acid in normal CTAL cells, pretreatment with ouabain and the elevation of [Na+]i was not required to elicit increments in [Ca2+]i. These data indicate that Na+/Ca2+ exchange is present in CTAL cells and the exchange activity appears to be modulated, directly or indirectly, by phosphorylation events.
Phosphodiesterases (PDEs) hydrolyze the 3' phosphoester bond of the purine 3',5'-cyclic monophosphates, cAMP and cGMP
Authors: Zeidel M. L.
Published: December 2017

Resistance to the natriuretic action of atrial natriuretic peptide (ANP) is a hallmark of states of pathological sodium retention including congestive heart failure, cirrhosis of the liver, and nephrotic syndrome. A variety of mechanisms including reduced delivery of filtrate to ANP-sensitive sites in the inner medullary collecting duct and diminished receptor density in this tubular segment have been offered to account for this resistance. Recent studies in experimental nephrotic syndrome and in liver disease produced by ligation of the common bile duct in rats suggest that increased activity of cyclic guanosine 3',5'-monophosphate (cGMP) phosphodiesterase may be an important mediator of renal resistance to ANP. Such increased enzyme activity rapidly catabolizes the second messenger cGMP, normally formed when ANP interacts with its biologically active natriuretic peptide. A receptors, thereby leading to blunted ANP responsiveness. This increased phosphodiesterase activity offers a novel approach to the management of clinical conditions associated with sodium retention and edema formation.
Dietary HCO3 reduces distal tubule acidification by increasing cellular HCO3 secretion
Authors: Wesson D. E.
Published: December 2017

We examined the components of net HCO3 reabsorption (H+/HCO3 secretion and transepithelial HCO3 permeability) in in vivo perfused distal tubules of anesthetized rats to determine the mechanisms by which dietary HCO3 reduces acidification in this nephron segment. Animals eating a minimum electrolyte diet drank either (in mM) 80 NaHCO3, 80 NaCl, or 40 Na2SO4 for 7-10 days and were compared with controls drinking distilled H2O. On perfusion with a HCO3- and Cl- -containing solution, net HCO3 reabsorption was lower than control in only the NaHCO3 animals (14.4 +/- 1.3 vs. 4.1 +/- 0.5 pmol.mm-1.min-1, P < 0.001). On perfusion with a 0 HCO3-0 Cl- solution, distal tubule luminal HCO3 accumulation (JHCO3) was higher in NaHCO3 animals than control (-13.7 +/- 1.3 vs. -4.7 +/- 0.7 pmol.mm-1.min-1, P < 0.002). Despite a higher JHCO3, estimated transepithelial HCO3 permeability in the NaHCO3 animals was similar to control [0.52 +/- 0.06 vs. 0.36 +/- 0.04 x 10(-7) cm2/s, P = not significant (NS)]. Luminal acetazolamide (Az) reduced JHCO3 in NaHCO3 animals to a level similar to control (-6.2 +/- 0.6 vs. -4.0 +/- 0.5 pmol.mm-1.min-1, P = NS) in this nephron segment containing cells with cytoplasmic but no luminal carbonic anhydrase activity. Including Cl- in the initial perfusate increased JHCO3 in NaHCO3 animals only (-20.8 +/- 1.9 vs. -13.7 +/- 1.3 pmol.mm-1.min-1, P < 0.02), and this increase was inhibited by luminal Az. Calculated H+ secretion was similar among groups. Together, the data indicate that dietary HCO3 reduces distal tubule acidification by increasing Az-sensitive generation of HCO3 by distal tubule cells that enters the lumen by a mechanism augmented by luminal Cl-.
Effect of vasopressin on renal lithium reabsorption: a micropuncture and microperfusion study
Authors: Walter S. J. , Shirley D. G. , Unwin R. J.
Published: December 2017

Micropuncture techniques were used to investigate the nephron site(s) responsible for the vasopressin-induced reductions in lithium clearance and fractional lithium excretion (FELi) in anesthetized Brattleboro rats lacking endogenous vasopressin. In rats treated intravenously with the vasopressin analogue 1-desamino-8-D-arginine vasopressin (DDAVP; 40 pg/min), FELi was significantly lower than in untreated animals (0.23 +/- 0.01 vs. 0.28 +/- 0.02, P < 0.05). Free-flow micropuncture showed that fractional lithium delivery (FDLi) to late proximal convolutions was identical in the two groups, whereas at the early distal tubule both FDLi (0.28 +/- 0.02 vs. 0.33 +/- 0.01, P < 0.05) and the tubular fluid-to-plasma lithium concentration ratio (1.18 +/- 0.04 vs. 1.36 +/- 0.06, P < 0.05) were lower in the DDAVP-treated group. No differences between the groups with respect to lithium handling beyond the early distal tubule were observed. In rats subjected to in vivo microperfusion of loops of Henle, intravenous infusion of DDAVP (40 pg/min) increased fractional lithium reabsorption in the loop from 0.56 +/- 0.03 to 0.66 +/- 0.04 (P < 0.05) and from 0.39 +/- 0.02 to 0.45 +/- 0.02 (P < 0.05) at perfusion rates of 10 and 15 nl/min, respectively. We conclude that DDAVP stimulates lithium reabsorption in the loop of Henle and suggest that this results from an increased transepithelial potential difference in the thick ascending limb.
Tubulogenesis from isolated single cells of adult mammalian kidney: clonal analysis with a recombinant retrovirus
Authors: Humes H. D. , Krauss J. C. , Cieslinski D. A. , Funke A. J.
Published: December 2017

The adult mammalian kidney tubule epithelium exists in a relatively dormant, slowly replicative state but has a large potential for regenerative morphogenesis following severe ischemic or toxic injury. Under selective serum-free growth conditions, which included epidermal growth factor and retinoic acid, a subpopulation of renal proximal tubule cells isolated from adult rabbit kidney were grown in cell culture. These cells possessed two important characteristics: 1) an ability to differentiate morphogenically into tubule structures when grown in three-dimensional collagen gels and 2) a high capacity for self-renewal, since cell lineage analysis with a recombinant retrovirus demonstrated that in vitro tubulogenesis arose from clonal expansion of a single cell. Thus individual cells in the adult kidney have retained the ability for kidney tubulogenesis in vitro.
Beta 2-adrenergic function in cultured rat proximal tubule epithelial cells
Authors: Singh H. , Linas S.
Published: December 2017

We conducted studies to determine whether functional beta 2-adrenoceptors are present in cultured rat proximal tubule epithelial cells. To determine whether cultured cells maintain polarity with respect to sodium transport, cells were acid loaded. Acid loading resulted in stimulation of sodium transport. Exposure of acid-loaded cells to alkaline extracellular pH further enhanced sodium transport (22Na flux at pH 7.50 was 68.1 +/- 44% above pH 7.00, P < 0.05). Cultured proximal tubules also exhibited basolateral 86Rb uptake, 65% of which was ouabain sensitive. Thus cultured cells maintain apical Na/H antiport and basolateral Na-K-adenosinetriphosphatase (Na-K-ATPase). Metaproterenol (10(-6) M), a selective beta 2-agonist, stimulated Na-K-ATPase activity by 36 +/- 6% above control (P < 0.05). The stimulatory effect was blocked by ICI-118551, a selective beta 2-antagonist. To determine whether metaproterenol-dependent increases in Na-K-ATPase were dependent on apical sodium entry, apical entry was blocked with dimethylamiloride or maximized with monensin. Both dimethylamiloride and monensin prevented metaproterenol activation of Na-K-ATPase. Metaproterenol-mediated increases in Na-K-ATPase activity were associated with increases in sodium transport (27 +/- 10% above control, P < 0.05), which was prevented by dimethylamiloride. In contrast to isoproterenol, metaproterenol did not stimulate cAMP production. In summary, we have shown that functional beta 2-adrenoceptors are present on cultured rat proximal tubules. beta 2-Adrenoceptor activation results in increases in Na-K-ATPase and Na transport as a consequence of increased apical sodium entry.
Enhanced renal vasoconstriction induced by vasopressin in SHR is mediated by V1 receptors
Authors: Feng J. J. , Arendshorst W. J.
Published: December 2017

The development of hypertension in the spontaneously hypertensive rat (SHR) is associated with renal dysfunction; the observed renal vasoconstriction may reflect an imbalance of constrictor and dilator systems. The present studies evaluated renal vascular reactivity to arginine vasopressin (AVP) and mediation by V1 and/or V2 receptors. Renal blood flow (electromagnetic flowmetry) was measured in water-loaded, 8-wk-old SHR, Wistar-Kyoto rats (WKY), and Munich-Wistar rats. Injection of AVP (2 and 5 ng) into the renal artery caused dose-dependent renal vasoconstriction. The maximum blood flow response was approximately twofold larger in SHR than both normotensive strains. The strain difference was largely unaffected by indomethacin administration, although the reduction in blood flow produced by 5 ng AVP was 4-6% larger in both SHR and WKY during cyclooxygenase inhibition. The V1 receptor antagonist, [D-(CH2)5,Tyr(Me)2,Tyr(NH2)9]Arg8-vasopressin, blocked up to 90% of the renal vasoconstriction elicited by AVP. Intrarenal injection of the V1-receptor agonist [Phe2,Ile3,Org8]vasopressin produced renal hemodynamic effects similar to AVP; this agonist reduced renal blood flow, with twofold larger responses in SHR (-40 vs. -18% for 10 ng). In contrast, similar doses of the V2-receptor agonist 1-desamino-8-D-arginine vasopressin had no effect. These results indicate that AVP-induced vasoconstriction is mediated predominantly by the V1 receptor in the rat kidney. The enhanced vascular reactivity in 8-wk-old SHR may reflect an increased V1 receptor density and/or affinity or postreceptor signaling pathways, largely independent of buffering by the vascular V2 receptor or vasodilator prostaglandin activity. The strain difference in the vascular response to AVP may contribute to the renal vasoconstriction observed during the development of genetic hypertension.
Effects of intravenous adenosine on renal function in healthy human subjects
Authors: Balakrishnan V. S. , Coles G. A. , Williams J. D.
Published: December 2017

The vasoactive nucleoside adenosine has an important regulatory influence on most aspects of renal function in experimental animals. In this study, we evaluated the effects of intravenous adenosine on systemic and renal hemodynamics, tubular function, and plasma renin concentration in 10 healthy male subjects. Each of the subjects received two intravenous infusions of adenosine (70 micrograms.kg-1.min-1) and saline on three separate study days. There was no significant change in systemic blood pressure in response to adenosine, although there was a significant rise in heart rate postcommencement of adenosine (61.5 +/- 2.9 to 78.0 +/- 7.9 beats/min, 1 h postcommencement of adenosine on day 1, P < 0.01 vs. saline). There was a significant decline in 51Cr-EDTA clearance (glomerular filtration rate) (118.5 +/- 13.2 to 88.0 +/- 8.3 ml/min, P < 0.05 vs. saline) and filtration fraction (19.4 +/- 1.01 to 16.0 +/- 1.03%, P < 0.01 vs. saline) 1 h postcommencement of adenosine, although there was no significant change in 125I-hippuran clearance (effective renal plasma flow). Urine flow rate and osmolar and free water clearance decreased significantly in response to adenosine (particularly on study day 1). There was, in addition, a significant reduction in absolute and fractional excretion rates of sodium, lithium, phosphate, uric acid, chloride, and urea in response to adenosine. There was a rise in plasma renin concentration in response to adenosine, reaching levels of statistical significance on study day 1 (15.0 +/- 2.02 to 22.2 +/- 2.00 microU/ml, 1 h postcommencement of adenosine; P < 0.05 vs. saline). These data are consistent with observations in experimental animals and complement the results of previous studies in man using a selective adenosine A1-receptor antagonist, thereby confirming that adenosine has a significant regulatory influence on human renal function.
Renal ischemic-reperfusion injury in L-selectin-deficient mice
Authors: Rabb H. , Ramirez G. , Saba S. R. , Reynolds D. , Xu J. , Flavell R. , Antonia S.
Published: December 2017

L-selectin on leukocyte surfaces mediates cell rolling on endothelium. L-selectin blockade with antibodies attenuated ischemic-reperfusion injury (IRI) in heart and skeletal muscle, but its role in renal IRI is unknown. We evaluated the role of L-selectin in renal IRI using L-selectin-deficient mice. Neutrophil migration to chemically inflamed peritoneum was reduced by 47% (P < 0.01) in L-selectin-deficient mice. Ischemia was induced by bilateral renal pedicle clamping for 30 min. Control and L-selectin groups had similar elevations of serum creatinine (1.8 +/- 0.3 vs. 1.7 +/- 0.2 mg/dl) and blood urea nitrogen (111 +/- 17 vs. 128 +/- 12 mg/dl) 24 h postischemia. Pathological assessment showed comparable degrees of tubular necrosis at 24 h. The postischemic increase in peritubular neutrophils per 10 high-power field was similar in control and L-selectin-deficient groups at 4 (28 +/- 10 vs. 22 +/- 5), 12 (245 +/- 80 vs. 236 +/- 78), and 24 h (130 +/- 12 vs. 156 +/- 18). These data argue against a significant role for L-selectin in renal IRI. Patho-physiological roles of L-selectin in vivo appear to be more complex than in vitro data would suggest.
Insulin-like growth factor-I action on glucose transport in acute renal failure
Authors: Mak R. H. , Mehls O.
Published: December 2017

Insulin resistance is common in acute renal failure (ARF). Insulin-like growth factor (IGF)-I has insulin-like actions. The present study was undertaken to determine whether effects of IGF-I on glucose transport are maintained in rats with ARF and to compare these actions of IGF-I with those of insulin. ARF was induced in male Sprague-Dawley rats by bilateral ureteric ligation, and vascular catheters were inserted at the same time in all animals. Sham-operated controls were pair fed, and metabolic studies were performed 48 h later. ARF rats had higher serum creatinine (1.3 +/- 0.2 mg/dl vs. 0.3 +/- 0.1), lower arterial pH (7.15 +/- 0.03 vs. 7.35 +/- 0.02), and higher fasting serum glucose (150 +/- 13 vs. 123 +/- 12 mg/dl) compared with control rats (P < 0.001 in all cases). The hypoglycemic effects of insulin and IGF-I were compared using euglycemic clamps. In control rats, rates of total body glucose uptake were not different during euglycemic insulin clamps at 2 mU.kg-1.min-1 and euglycemic IGF-I clamps at 5 micrograms.kg-1.min-1. In rats with ARF, total body glucose uptake during euglycemic insulin clamps at 2 mU.kg-1.min-1 was significantly reduced (6.5 +/- 0.5 mg.kg-1.min-1) compared with controls (10.5 +/- 1.5 mg.kg-1.min-1, P < 0.01). In contrast, total body glucose uptake during euglycemic IGF-I clamps at 5 micrograms.kg-1.min-1 in ARF rats (10.1 +/- 1.2 mg.kg-1.min-1) was not different from the corresponding value in control animals (10.3 +/- 1.3 mg.kg-1.min-1). Hepatic glucose production was suppressed by insulin equally but not suppressed by IGF-I in both groups. In a second experiment, ARF was induced by bilateral renal ischemia. The following groups of animals were studied: ARF + NaCl, ARF + NaHCO3, and control + NaCl. Insulin and IGF-I clamps were performed as above. Correction of acidosis partially corrected insulin resistance but did not affect IGF-I sensitivity. Thus the capacity of IGF-I infusion to stimulate glucose uptake is maintained in ARF rats, which are insulin resistant.