bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of HCO3- absorption by prostaglandin E2 and G proteins in rat medullary thick ascending limb
Authors: Good D. W. , George T.
Published: December 2017

Arginine vasopressin (AVP) inhibits HCO3- absorption (JHCO3) in the medullary thick ascending limb (MTAL) of the rat by increasing adenosine 3', 5'-cyclic monophosphate. Hyperosmolality also inhibits JHCO3 via a pathway additive to inhibition by AVP. To determine whether these regulatory effects are modulated by prostaglandin E2 (PGE2), MTAL were isolated and perfused in vitro with 25 mM HCO3- solutions (pH 7.4; 290 mosmol/kgH2O). PGE2 (10(-6) M in the bath) had no effect on JHCO3 in the absence of AVP. In contrast, with 10(-10) MAVP in the bath solution, addition of 10(-8) or 10(-6) M PGE2 to the bath increased JHCO3 from 9.7 +/- 0.8 to 14.3 +/- 1.1 pmol.min-1.mm-1 (P < 0.001). In the presence of AVP and hyperosmolality (75 mM NaCl added to perfusate and bath), PGE2 increased JHCO3 from 1.4 +/- 0.1 to 7.5 +/- 0.5 pmol.min-1.mm-1 (P < 0.005). PGE2 also stimulated JHCO3 in the presence of AVP and hypertonic urea. Cholera toxin (CTX, 10(-12)-10(-9) M in the bath) inhibited JHCO3 by 40%, and this inhibition was reversed by PGE2. PGE2 did not reverse inhibition of JHCO3 by forskolin. The stimulation of JHCO3 by PGE2 in the presence of AVP was blocked by pretreatment with pertusis toxin (PTX, 2 x 10(-11) or 10(-8) M). Neither CTX nor PTX affected inhibition of JHCO3 by hyperosmolality. These results demonstrate that PGE2 reverses inhibition of JHCO3 by AVP by acting via a PTX-sensitive G protein (presumably Gi) to inhibit AVP-stimulated adenosine 3', 5'-cyclic monophosphate production. PGE2 may act as a counterregulatory factor to maintain a stable rate of HCO3- absorption in the MTAL during antidiuresis when circulating AVP levels and medullary osmolality are elevated.
Differential regulation of rat glomerular and proximal tubular renin mRNA following uninephrectomy
Authors: Tank J. E. , Moe O. W. , Star R. A. , Henrich W. L.
Published: December 2017

Angiotensin II is thought to play a role in the renal adaptations to reduced renal mass, but earlier work has shown that plasma renin activity (PRA) does not increase in this setting. To examine this paradox, we studied the effect of uninephrectomy (UNX) on circulating, juxtaglomerular, glomerular, and proximal tubular (PT) renin. PRA was unchanged 2 wk following UNX and fell slightly at 6 wk. Single kidney renin secretory capacity and cortical renin mRNA, reflecting juxtaglomerular renin, were unchanged at 2 and 6 wk. With quantitative competitive reverse transcription-polymerase chain reaction, renin mRNA in microdissected glomeruli and PT were dramatically increased 2 wk post-UNIX (for glomeruli: sham, 1.2 +/- 0.3, vs. UNX, 8.8 +/- 1.9 x 10(5) copies/glomerulus; for PT: sham, 4.6 +/- 0.9, vs. UNX, 17.7 +/- 5.1 x 10(3) copies/mm). By 6 wk, glomerular renin was unchanged, and PT renin mRNA was markedly suppressed (for glomeruli; sham, 2.9 +/- 1.2, vs. UNX, 4.2 +/- 1.1 x 10(5) copies/glomerulus; for PT: sham, 7.5 +/- 2.1, vs. UNX, 1.0 +/- 0.3 x 10(3) copies/mm). These results demonstrate differential regulation of the circulating, juxtaglomerular, glomerular, and PT renin systems. Early activation of glomerular and PT renin may result in increased local generation of angiotensin II and thereby affect renal structural and functional adaptations following UNX.
Antioxidant PDTC induces stromelysin expression in mesangial cells via a tyrosine kinase-AP-1 pathway
Authors: Yokoo T. , Kitamura M.
Published: December 2017

We found that pyrrolidine dithiocarbamate (PDTC) induces the matrix metalloproteinase stromelysin in cultured glomerular mesangial cells. Although PDTC is a well-known inhibitor of nuclear factor-kappa B (NF-kappa B), this effect was independent of the NF-kappa B activity, since overexpression of a dominant negative mutant of p50 NF-kappa B subunit repressed activity of the kappa B site, whereas it failed to induce stromelysin. To elucidate the intracellular mechanisms involved, we focused on the role of activator protein 1 (AP-1), since its binding site, the 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE), is located in the 5'-flanking region of the stromelysin gene. Northern blot analysis revealed that PDTC upregulated expression of c-jun and c-fos before the expression of stromelysin. Transient transfection studies using a TRE-LacZ reporter plasmid elucidated that activity of AP-1 was significantly increased by PDTC. Stable transfection with a c-jun antisense cDNA or pretreatment with curcumin, a pharmacological inhibitor of c-Jun/AP-1, revealed that inactivation of AP-1 diminished the induction of stromelysin by PDTC. To identify the machinery involved upstream of AP-1 activation, the role of tyrosine kinases was investigated. Western blot analysis showed that PDTC induced phosphorylation of tyrosine kinases. Treatment of mesangial cells with tyrosine kinase inhibitors suppressed activation of AP-1 as well as induction of stromelysin by PDTC. These findings demonstrate that the antioxidant PDTC induces stromelysin expression via stimulation of the tyrosine kinase-AP-1 pathway independent of its suppressive action on NF-kappa B.
Medullary blood flow responses to changes in arterial pressure in canine kidney
Authors: Majid D. S. , Navar L. G.
Published: December 2017

Although it is well recognized that whole kidney and cortical blood flow exhibit efficient autoregulation in response to alterations in renal arterial pressure (RAP), the autoregulatory behavior of medullary blood flow (MBF) has remained uncertain. We have evaluated MBF responses to stepwise reductions in RAP for both short-term (2 min, n = 6) and longer periods (15 min, n = 7) using single-fiber laser-Doppler flowmetry with needle probes inserted into the mid-medullary region in denervated kidneys of 13 anesthetized dogs. The changes in cortical blood flow (CBF) were assessed with either a surface probe or a needle probe inserted into the cortex. Control total renal blood flow (RBF), assessed by electromagnetic flow probe in these dogs, was 5.2 +/- 0.3 ml.min-1.g-1, and glomerular filtration rate was 0.97 +/- 0.05 ml.min-1.g-1 (n = 7). RBF, MBF, and CBF all exhibited efficient autoregulatory behavior during changes in RAP from 150 to 75 mmHg. The slopes of RAP vs. RBF, CBF, as well as MBF, were not significantly different from zero within this range of RAP. Below RAP of 75 mmHg, all indexes of blood flow showed linear decreases with reductions in pressure. The data indicate that blood flow in the renal medulla of dogs exhibits efficient autoregulatory behavior, similar to that in the cortex.
Effects of systemic NO synthesis inhibition on RPF, GFR, UNa, and vasoactive hormones in healthy humans
Authors: Bech J. N. , Nielsen C. B. , Pedersen E. B.
Published: December 2017

Animal studies have implicated an important role of nitric oxide (NO) in the regulation of blood pressure, renal hemodynamics, and renal excretion of sodium. NG-monomethyl-L-arginine (L-NMMA) is a specific, competitive inhibitor of NO synthesis interfering with NO synthase. The purpose of the present study was to investigate the effect of L-NMMA on renal plasma flow (RPF), glomerular filtration rate (GFR), urinary sodium excretion (UNa), fractional sodium excretion (FENa), fractional lithium excretion (FELi), mean arterial blood pressure (MAP), and heart rate (HR) in healthy humans. In a randomized placebo-controlled study, 23 healthy subjects were randomized to receive either bolus injection of L-NMMA (3 mg/kg in 10 ml saline, n = 12 subjects) or placebo (10 ml saline, n = 11). GFR and RPF were measured using the renal clearances of 51Cr-labeled EDTA and 125I-labeled hippuran by the constant infusion technique. L-NMMA treatment induced 60 min after injection a 14.6% decrease in RPF, a 5.8% decrease in GFR, a 9.8% increase in filtration fraction, a 34.7% decrease in UNa a 28.6% decrease in FENa, and a 12.1% decrease in FELi. These changes were still evident 120 min after injection. None of the effect parameters were changed after placebo, except FENa, which increased 9.9% 60 min after injection. Ten minutes after L-NMMA injection, MAP increased significantly (80 vs. 88 mmHg), and HR decreased (58 vs. 47 beats/min). The changes in HR and MAP normalized within 30 min. L-NMMA significantly reduced the plasma level of cGMP 60 min (3.0 vs. 3.7 pmol/l) and 120 min after injection (2.5 vs. 3.7 pmol/l). It is concluded that, in healthy humans, NO is a regulator of renal hemodynamics as a tonic vasodilator and a regulator of sodium excretion, due at least in part to a proximal tubular effect.
Role of H(+)-K(+)-ATPase in pHi regulation in inner medullary collecting duct cells in culture
Authors: Ono S. , Guntupalli J. , DuBose T. D.
Published: December 2017

Studies in inner medullary collecting duct (IMCD) cells in primary culture have proposed two mechanisms for Na(+)-independent hydrogen ion transport: an H(+)-adenosinetriphosphatase (H(+)-ATPase) and an H(+)-K(+)-ATPase. In the present study, we have employed two sources of IMCD cells, cells in primary culture derived from the terminal papilla of the Munich-Wistar rat (IMCDp) and an established murine cell line (mIMCD-3), to define the predominant mechanism(s) of Na(+)-independent intracellular pH (pHi) recovery in the IMCD. In confluent monolayers of IMCDp and mIMCD-3 cells, pHi was measured using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) following addition and withdrawal of NH4Cl. Removal of K+ completely abolished Na(+)-independent pHi recovery in both IMCDp (delta pHi/min = 0.039 +/- 0.006 to 0.005 +/- 0.003; P < 0.001) and in mIMCD-3 (delta pHi/min = 0.055 +/- 0.009 to -0.003 +/- 0.002; P < 0.001) cells, respectively. In mIMCD-3 cells, K(+)-dependent pHi recovery was abolished by either of two specific inhibitors of the H(+)-K(+)-ATPase, Sch-28080 (5 or 10 microM) or A-80915A (10 microM). In contrast, bafilomycin A1 (2.5 and 10 nM), an inhibitor of the H(+)-ATPase, failed to attenuate K(+)-dependent pHi recovery. Moreover, sequence verified mouse gastric and colonic alpha-H(+)-K(+)-ATPase probes hybridized to total RNA from mIMCD-3 cells. Based on these findings, we conclude that Na(+)-independent pHi recovery from an acid load in both IMCDp and mIMCD-3 cells in critically dependent on extracellular K(+)-That K(+)-dependent pHi recovery was inhibited by both Sch-28080 and A-80915A but not by bafilomycin A1 suggests that the predominant mechanism by which Na(+)-independent pHi recovery is accomplished in IMCD is through the H(+)-K(+)-ATPase. Expression of both gastric and colonic alpha-H(+)-K(+)-ATPase mRNA in mIMCD-3 cells suggests that one or both of these H(+)-K(+)-ATPases may be responsible for proton secretion in the IMCD.
Tissue remodeling during tumor necrosis factor-induced apoptosis in LLC-PK1 renal epithelial cells
Authors: Peralta Soler A. , Mullin J. M. , Knudsen K. A. , Marano C. W.
Published: December 2017

The cytokine tumor necrosis factor-alpha (TNF) increases the frequency of apoptosis in confluent renal epithelial LLC-PK1 cells, an effect that can be blocked by an anti-TNFR1 monoclonal antibody. However, there were no visible "holes" in the cell sheet as a result of TNF-induced apoptosis. Instead a striking tissue remodeling occurred in response to the TNF-induced apoptosis. Apoptotic cells became surrounded and engulfed by repositioned neighboring cells distributed in a distinct "rosette" pattern. The cadherin-catenin cell-cell adhesion molecules, the tight junction-associated protein ZO-1, and actin accumulated at the sites of contact between apoptotic and neighboring cells. Pretreatment with cytochalasin B prevented the accumulation of cadherins-catenins and ZO-1 at the sites of apoptosis and resulted in microscopic holes in the TNF-treated cell sheet. Our results indicate that a renal epithelium can accommodate an increased frequency of apoptosis and still maintain its integrity by mechanisms of tissue remodeling involving the cadherin-catenin adhesion molecules, tight junctional proteins, and actin filaments.
Progesterone inhibition of water permeability in Bufo arenarum oocytes and urinary bladder
Authors: Ford P. , Amodeo G. , Capurro C. , Ibarra C. , Dorr R. , Ripoche P. , Parisi M.
Published: December 2017

The ovarian oocytes from Bufo arenarum (BAO) but not those from Xenopus laevis (XLO) would have water channels (WC). We now report that the injection of the mRNA from BAO into the oocytes from XLO increased their water osmotic permeability (Pi) (reduced by 0.3 mM HgCl2 and reversed by 5 mM beta-mercaptoethanol). A 30-min challenge with progesterone induced, 18 h later, a reduction of the mercury-sensitive fraction of Pf in the BAO (but not in XLO). The mRNA from BAO pretreated with progesterone lost its capacity to induce WC in the XLO, but the hormone did not affect the expression of the WC in XLO previously injected with the mRNA from BAO. Pf was also measured in urinary bladders of BAO. Eighteen hours after a challenge with progesterone, a reduction in the hydrosmotic response to oxytocin was observed. Finally, the mRNA from the urinary bladder of BAO was injected into XLO. An increase in Pf was observed. This was not the case if, before the mRNA extraction, the bladders were treated with progesterone. We conclude that the BAO WC share progesterone sensitivity with the oxytocin-regulated water channel present in the toad urinary bladder.
The effects of collecting duct active NaCl reabsorption and inner medulla anatomy on renal concentrating mechanism
Authors: Wang X. , Wexler A. S.
Published: December 2017

First, the representation of the inner medulla incorporates an exaggerated radial separation between tubules, vessels, and collecting ducts; and, second, the hydraulic permeability in the upper portion of the inner medullary collecting ducts was erroneously set to zero. In the current work, we explore the role of collecting duct hydraulic permeability and anatomical heterogeneity via mathematical modeling. The model predicts concentrated urine for measured values of the hydraulic permeability and homogeneous lower inner medulla as long as net active NaCl reabsorption is incorporated in the upper inner medullary collecting duct epithelium. This new three-dimensional model results in two recycling paths. The upper portion of the inner medulla recycles NaCl, whereas the lower portion recycles urea.
Alpha 2B-adrenergic receptors: immunolocalization and regulation by potassium depletion in rat kidney
Authors: Huang L. , Wei Y. Y. , Momose-Hotokezaka A. , Dickey J. , Okusa M. D.
Published: December 2017

Regulation of transport processes in renal epithelial cells in response to alpha 2-adrenergic receptor (alpha 2-AR) stimulation is likely due to specific effects of the A-, B-, or C-subtype alpha 2-AR. The purpose of the present study was 1) to examine the precise localization, using subtype-specific antibodies, the B-subtype alpha 2-AR protein, the most abundant alpha 2-AR in rat kidney; and 2) to determine the effect of dietary potassium restriction, a factor known to increase alpha 2B-ARs, on the distribution of receptors. We employed a method to generate a specific antibody to the third intracellular loop of the alpha 2B-AR and demonstrated the specificity of this antibody. Our results demonstrated, for the first time, evidence for the alpha 2B-receptor protein in the basolateral membrane of proximal convoluted and straight tubules. No specific immunoreactivity was detected in other nephron segments. Finally, the increase in alpha 2B-AR expression observed previously was due to the recruitment of alpha 2B-ARs to the basolateral membrane of proximal tubules.
The inner ear produces a natriuretic hormone
Authors: Qvortrup K. , Rostgaard J. , Holstein-Rathlou N. H.
Published: December 2017

Cytoplasmic granules have been demonstrated in epithelial cells from the endolymphatic sac, an extraosseus part of the inner ear located in the posterior cranial fossa. Intravenously infused extracts from endolymphatic sacs in anesthetized rats elicited a potent natriuresis and diuresis without effects on blood pressure, glomerular filtration rate, or lithium clearance. Only a minor kaliuresis was observed. Extracts of dural tissue adjacent to the endolymphatic sacs had no effect. It is concluded that the endolymphatic sac contains as endogenous inhibitor of sodium reabsorption and could be the sensory organ/mediator of "cerebral" natriuresis. Furthermore, this substance, tentatively named saccin, may influence the homeostasis of the inner ear fluids and accordingly play a significant role in the pathogenesis of Meniere's disease.
Na-Pi cotransport in flounder: same transport system in kidney and intestine
Authors: Kohl B. , Herter P. , Hulseweh B. , Elger M. , Hentschel H. , Kinne R. K. , Werner A.
Published: December 2017

The cloning of a renal Na-Pi contransport in system from winter flounder (P eudopleuronectes americanus) has recently been reported. We used this information to answer the questions 1) what is the distribution of the transport protein along the nephron? and 2) how are renal and intestinal transporters related? The distribution of the flounder NaPi-II protein was tested using two antisera raised against partial sequences (amino acids 1-14 and 388-441) of the transporter. Antibody-specific fluorescence was detected at the basolateral membrane of epithelial cells in the proximal tubular segment PII. Two clones corresponding to the renal Na-Pi cotransporter were isolated from a flounder intestinal cDNA library. Their functional properties were determined using Xenopus laevis oocytes. The apparent affinities for Pi [Michaelis constant (K(m)) = 0.063 mM] and Na (K(m) = 45.3 mM), as well as the pH dependency (increasing transport activity with increasing pH), showed the same characteristics in both intestinal and the renal systems. Sequence analysis revealed that the two intestinal clones were 100% homologous to the renal cDNA, Flounder NaPi-II-specific immunofluorescence was observed predominantly at the apical membrane on intestinal cross sections. We report the cloning and expression of the first intestinal Na-Pi cotransport system. This transporter belongs to the small group of proteins that exhibit the same function in the apical and the basolateral membranes of different cells.
Role of protein kinase C in angiotensin II-induced renal vasoconstriction in genetically hypertensive rats
Authors: Ruan X. , Arendshorst W. J.
Published: December 2017

The renal vasculature of young spontaneously hypertensive rats (SHR) responds to angiotensin II (ANG II) with exaggerated vasoconstriction, due in part to defective buffering by the adenosine 3',5'-cyclic monophosphate (cAMP) pathway. In vitro studies suggest greater activation of phospholipase C and protein kinase C (PKC) in cultured mesangial cells and vascular smooth muscle cells. The present studies evaluated the role of PKC activation in renal vascular responses to ANG II receptor activation and the relative contributions in SHR vs. Wistar-Kyoto control rats (WKY). Renal blood flow was measured in 8-wk-old anesthetized SHR and WKY pretreated with indomethacin. ANG II (2 ng) injection into the renal artery produced a transient 45-50% maximum reduction of renal blood flow in both rat strains. Intrarenal infusion of either staurosporine or chelerythrine into the renal artery effectively attenuated the vasoconstriction elicited by ANG II in a dose-dependent manner, with maximum inhibition of 60-70%. The PKC inhibitory effects were significant and independent of strain. Coadministration of the PKC inhibitors produced maximal inhibition similar to that observed with one agent, suggesting action via a common pathway. In other studies, the linkage of the PKC pathway to the AT1 receptor was evaluated using sub and maximal doses of losartan to antagonize 50-80% of ANG II-induced vasoconstriction. The same degree of inhibition was observed when a PKC inhibitor was coadministered with losartan. These findings support the views that the PKC system is a major intracellular signaling pathway coupled to the AT1 receptor in renal resistance vessels and that PKC activation is involved to similar degrees in the renal vasoconstriction elicited by ANG II in young WKY and SHR. Exaggerated vascular reactivity to vasoconstrictor agents in genetically hypertensive animals is probably due to a defect in cAMP generation in the presence of a normally operating PKC pathway.
PGE2 reverses AVP inhibition of HCO3- absorption in rat MTAL by activation of protein kinase C
Authors: Good D. W.
Published: December 2017

In the medullary thick ascending limb (MTAL) of the rat, prostaglandin E2 (PGE2) reverses inhibition of HCO3- absorption (JHCO3) by arginine vasopressin (AVP) by inhibiting AVP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production. To determine whether this regulation by PGE2 involves protein kinase C (PKC), MTAL segments were perfused in vitro with physiological solutions containing 25 mM HCO3- (pH 7.4). With 10(-10) MAVP in the bath, addition of 10(-6) M PGE2 to the bath increased JHCO3 from 7.8 +/- 0.4 to 13.0 +/- 1.1 pmol.min-1.mm-1 (P < 0.01). This effect was blocked completely by pretreatment with the PKC inhibitors staurosporine or chelerythrine chloride (10(-7) M in the bath). With both AVP and PGE2 in the bath, addition of staurosporine or chelerythrine to the bath decreased JHCO3 from 12.2 +/- 1.1 to 7.3 +/- 0.6 pmol.min-1.mm-1 (P < 0.005). Neither staurosporine nor chelerythrine affected JHCO3 under basal conditions or in the presence of AVP alone. With AVP in the bath, addition of phorbol 12-myristate 13-acetate (PMA, 10(-6) M) to the bath increased JHCO3 from 5.0 +/- 0.5 to 9.1 +/- 1.0 pmol.min-1.mm-1 (P < 0.01). Similar to PGE2, PMA had no effect on JHCO3 in the absence of AVP or in the presence of 10(-6) M bath forskolin. The effect of PMA to stimulate JHCO3 in the presence of AVP was abolished by pretreatment with pertussis toxin (2 x 10(-11) M). We conclude that 1) PGE2 reverses AVP inhibition of HCO3- absorption by activation of PKC, 2) PKC likely increases JHCO3 by inhibiting AVP-stimulated cAMP production via a Gi-dependent mechanism, and 3) PKC activity has no influence on basal HCO3- absorption rate.
PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells
Authors: Ganz M. B. , Saksa B. , Saxena R. , Hawkins K. , Sedor J. R.
Published: December 2017

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.
The 3'-nontranslated region of rat renal glutaminase mRNA contains a pH-responsive stability element
Authors: Hansen W. R. , Barsic-Tress N. , Taylor L. , Curthoys N. P.
Published: December 2017

Rat kidney expresses two forms of glutaminase (GA) mRNA which probably result from the use of alternative polyadenylation signals. The two mRNAs are increased coordinately in response to metabolic acidosis via a mechanism that apparently does not involve transcriptional or translational regulation. A 956-bp fragment that contains the 3'-nontranslated sequence of the smaller GA cDNA was cloned into an expression vector (p beta G) that encodes a chimeric beta-globin growth hormone mRNA. Both the parent and the derived construct (p beta G-GA) were transfected into LLC-PK1-F+ cells. Stable transfectants express sixfold lower levels of beta G-GA mRNA than that of the parent beta G mRNA. However, only the beta G-GA mRNA is increased 2.5-fold by growth in acidic medium (pH 6.9, 10 mM HCO3-). The apparent half-life of the beta G mRNA (> 24 h) is unaffected by the pH of the growth media. In contrast, the apparent half-life of the beta G-GA mRNA is increased from 4.5 h to approximately 24 h when cells are transferred to acidic medium for 8 h. The observed pH response is not reproduced when the beta G-GA construct is stably transfected into COS-7 cells or when a beta-globin-phosphoenolpyruvate carboxykinase chimeric gene is expressed in LLC-PK1-F+ cells. Thus the 3'-nontranslated region of the GA mRNA contains a pH-responsive stability element.
Response of hepatic amino acid consumption to chronic metabolic acidosis
Authors: Boon L. , Blommaart P. J. , Meijer A. J. , Lamers W. H. , Schoolwerth A. C.
Published: December 2017

In a previous paper, we showed that an inhibition of amino acid transport across the liver plasma membrane is responsible for the decrease in urea synthesis in acute metabolic acidosis. We have now studied the mechanism responsible for the decline in urea synthesis in chronic acidosis. Chronic metabolic acidosis and alkalosis were induced by feeding three groups of rats HCl, NH4Cl, and NaHCO3 (8 mmol/day) for 7 days. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences were calculated. The rates of urinary urea and NH4+ excretion were also determined. Hepatic amino acid consumption was lower in both HCl and NH4Cl acidosis compared with NaHCO3-fed rats. Glutamine release was not different in the three conditions. Because intrahepatic concentrations of amino acids and intracellular protein degradation were similar under these conditions, it can be concluded that at low blood pH amino acid catabolism may be inhibited and might explain the observed decrease in urea excretion in HCl, but not NH4Cl, acidosis; urea excretion was comparable in the NH4Cl and NaHCO3 groups presumably because the increased NH4+ load in the former group was processed, uninhibited, to urea. Amino acids not used by the liver in acidosis could account for the 25-fold increase in NH4+ excretion in HCl and NH4Cl compared with alkalosis (P < 0.05). These findings indicate that urea synthesis is decreased in chronic HCl acidosis. They show that urea synthesis is controlled in chronic, as in acute, acidosis by amino acid uptake by the liver and/or intrahepatic degradation and that the ornithine cycle per se has only minor control of acid-base homeostasis.
Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells
Authors: Hagar H. , Ueda N. , Shah S. V.
Published: December 2017

Hypoxia is considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activation, generally considered a feature of apoptosis, to be almost entirely responsible for DNA damage in hypoxic injury to renal tubular epithelial cells. The role of reactive oxygen metabolites in endonuclease-induced DNA damage and cell death in chemical hypoxic injury has not been previously examined. LLC-PK1 cells exposed to chemical hypoxia with antimycin A resulted in enhanced generation of intracellular reactive oxygen species as measured by oxidation of a sensitive fluorescent probe, 2',7'-dichlorofluorescin diacetate. Superoxide dismutase, a scavenger of superoxide radical, significantly reduced the fluorescence induced by antimycin A and provided significant protection against chemical hypoxia-induced DNA strand breaks (as measured by the alkaline unwinding assay). Pyruvate, a scavenger of hydrogen peroxide, provided significant protection against chemical hypoxia-induced DNA strand breaks and DNA fragmentation (as measured by agarose gel electrophoresis). The interaction between superoxide anion and hydrogen peroxide in the presence of a metal catalyst leads to generation of other oxidant species such as hydroxyl radical. Hydroxyl radical scavengers, dimethylthiourea, salicylate, and sodium benzoate, and two metal chelators, deferoxamine and 1,10-phenanthroline, also provided marked protection against DNA strand breaks and DNA fragmentation. These scavengers of reactive oxygen metabolites and metal chelators provided significant protection against cell death as measured by trypan blue exclusion and lactate dehydrogenase release. Taken together, these data indicate that reactive oxygen species play an important role in the endonuclease activation and consequent DNA damage, as well as cell death in chemical hypoxic injury to renal tubular epithelial cells.
Organic osmolyte distribution and levels in the mammalian urinary bladder in diuresis and antidiuresis
Authors: Kwon E. D. , Dooley J. A. , Jung K. Y. , Andrews P. M. , Garcia-Perez A. , Burg M. B.
Published: December 2017

Inositol, sorbitol, glycerophosphocholine (GPC), and betaine are organic osmolytes that are accumulated by renal medullary cells in response to hyperosmotic stress. Previous screening studies, using nuclear magnetic resonance spectroscopy, have shown some of these same compounds to be present in extracts of whole urinary bladder from rabbits and rats. In the present study, we used high-performance liquid chromatography to quantify levels of these compounds in the separated epithelium and muscle of bladders taken from normal rabbits as well as diuretic and thirsted rats. We find that 1) high concentrations of organic osmolytes, namely inositol, GPC, and sorbitol, are present in urinary bladder; 2) levels of these osmolytes in the bladder epithelium are higher than in the muscle; 3) increased urinary osmolality due to antidiuresis is associated with a 2.4-fold increase in total osmolyte levels in rat bladder epithelium and a lesser (1.5-fold) increase in the muscle, compared with corresponding levels in tissues from diuretic animals; and 4) these increases in total osmolyte amounts in the epithelium are due to increased levels of GPC, sorbitol, and perhaps inositol (P = 0.07), whereas only GPC increases in the bladder muscle.
Differential regulation of rat glomerular and proximal tubular renin mRNA following uninephrectomy
Authors: Tank J. E. , Moe O. W. , Star R. A. , Henrich W. L.
Published: December 2017

Pages F776–F783: J. E. Tank, O. W. Moe, R. A. Star, and W. L. Henrich. “Differential regulation of rat glomerular and proximal tubular renin mRNA following uninephrectomy.” On p. F779, a registration error during printing caused the Southern blots of Fig. 2 to be misaligned with respect to the line graphs. The correct image of Fig. 2 is shown below.

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Expression and localization of prostaglandin EP3 receptor mRNA in the immature rabbit kidney
Authors: Bonilla-Felix M. , Jiang W.
Published: December 2017

Arginine vasopressin (AVP)-stimulated cAMP generation is decreased in the immature collecting duct (CD). This is the result of prostaglandin antagonism, most likely via the inhibitory guanine nucleotide-binding protein (Gi). The EP3-subtype prostaglandin E2 (PGE2) receptor, which is coupled to Gi, could mediate this effect. We studied the developmental expression of EP3 receptor in the rabbit kidney. Higher levels of EP3 mRNA were observed in the immature kidney using three different assays: 1) reverse transcription-polymerase chain reaction (RT-PCR) with internal standard, 2) competitive PCR, and 3) ribonuclease protection assay. The highest levels were observed at 2 wk of age. RT-PCR from isolated nephron segments detected EP3 mRNA in the medullary thick ascending limb, cortical CD (CCD), and inner medullary CD (IMCD) of adult and immature kidneys. We conclude that 1) renal expression of EP3 mRNA is increased in immature kidneys and 2) EP3 mRNA is localized in the distal nephron. This suggests that EP3 receptor may play a role in the regulation of distal tubular transport during development.
Quantitation of aquaporin-2 abundance in microdissected collecting ducts: axial distribution and control by AVP
Authors: Kishore B. K. , Terris J. M. , Knepper M. A.
Published: December 2017

Aquaporin-2 (AQP-2) is the arginine vasopressin-regulated water channel of the renal collecting ducts. Using an improved version of a fluorescence-based enzyme-linked immunosorbent assay (Y. Maeda, B. L. Smith, P. Agre, and M. A. Knepper. J. Clin. Invest. 95: 422-428, 1995), we quantified AQP-2 protein abundance in microdissected renal collecting ducts from normal Sprague-Dawley (SD) rats and vasopressin-deficient Brattleboro rats. Standard curves were linear in the range of 0-200 fmol/well and were highly reproducible from day to day (lower limit of detection 2.3 fmol; coefficient of variation 6-9%). In SD rats thirsted for 24 h, the measured quantities of AQP-2 were as follows (x 10(9) molecules/mm): cortical collecting ducts (CCD), 4.3 +/- 0.5; outer medullary collecting ducts (OMCD), 10.1 +/- 1.7; initial one-third of inner medullary collecting duct (IMCD-1), 9.2 +/- 1.1; middle one-third of the IMCD (IMCD-2), 7.5 +/- 0.8; terminal one-third of the IMCD (IMCD-3), 3.3 +/- 0.6; n = 7-12. In IMCD-2 this corresponds to 11.8 +/- 1.3 x 10(6) AQP-2 molecules per cell. Thus AQP-2 is extremely abundant in collecting duct cells. AQP-2 levels were decreased in untreated Brattleboro rats relative to the parent strain Long-Evans (LE) by 68% in IMCD-2 and 44% in CCD. Following vasopressin infusion by osmotic minipumps, AQP-2 levels in IMCD-2 of Brattleboro rats rose gradually, reaching levels equivalent to those seen in LE rats after 5 days. A similar rise was seen in the CCD, indicating that the vasopressin-induced increase was not dependent on a large increase in the interstitial tonicity. Thus a rise in circulating vasopressin concentration increases the level of AQP-2 protein expression in collecting ducts, presumably via a direct action of vasopressin.
Aldosterone-dependent regulation of Na-K-ATPase subunit mRNA in the rat CCD: competitive PCR analysis
Authors: Tsuchiya K. , Giebisch G. , Welling P. A.
Published: December 2017

In the cortical collecting duct (CCD), aldosterone increases the number of functionally active Na-K-adenosin-etriphosphatase (Na-K-ATPase) molecules by a mechanism involving an isoform-specific increase in the abundance of the Na-K-ATPase alpha 1- and beta 1-subunit protein. However, the molecular basis for the response, particularly in the mammalian CCD in vivo, has remained unclear. To resolve this issue, reverse transcription (RT) and a competitive polymerase chain reaction (PCR) were employed to study mineralocorticoid-dependent regulation of alpha 1- and beta 1-subunit mRNA in the rat CCD. Na-K-ATPase subunit-specific oligonucleotides primers were used in the PCR to amplify reverse-transcribed subunit mRNA (RT-mRNA) from single microdissected CCD. Control templates were constructed (84-bp deletion mutation of the rat Na-K-ATPase alpha 1-subunit cDNA and 70-bp deletion of the beta 1-subunit cDNA), serially diluted, and coamplified with the wild-type Na-K-ATPase subunit RT-mRNA from single CCD. PCR products of predicted size were observed by ethidium bromide staining. Southern blots with an internal subunit-specific oligonucleotide confirmed Na-K-ATPase alpha 1- and beta 1-subunit identity. The ratio of the amplified wild-type to mutant PCR products was found to be linear over the range of input control cDNA tested so that the amount of subunit mRNA could be determined. A chronic reduction in corticosteroid levels by bilateral adrenalectomy (7 days) reduced the apparent level of alpha 1-subunit transcript by 54.0 +/- 6.3% but not the beta 1-subunit. Administering aldosterone to physiological levels is sufficient to restore CCD alpha 1-subunit mRNA abundance toward control levels within 6 h. We conclude the following: 1) regulation of Na-K-ATPase of CCD in vivo can be attributed, at least in part, to mineralocorticoid-dependent control of Na-K-ATPase alpha 1-subunit mRNA abundance; and 2) competitive PCR may provide a sensitive and quantitative tool for determining hormone-dependent regulation of mRNA abundance in nephron segments.
Differential regulation of kallikrein, kininogen, and kallikrein-binding protein in arterial hypertensive rats
Authors: Chao C. , Madeddu P. , Wang C. , Liang Y. , Chao L. , Chao J.
Published: December 2017

This study was designed to determine whether the kallikrein-kinin system exerts a protective action in hypertension induced by chronic inhibition of nitric oxide synthase. N omega-nitro-L-arginine methyl ester (L-NAME, 40 mg/100 ml water) was given orally to Sprague-Dawley rats, while controls received regular tap water. Hepatic kininogen mRNA levels in the L-NAME-treated group were 2.9- and 2.5-fold higher at 3 and 4 wk, respectively, compared with control rats, whereas kallikrein-binding protein (KBP) mRNA levels were 82% and 45% of the values found in control rats at 3 and 4 wk, respectively. There was no significant change in hepatic alpha 1-antitrypsin mRNA levels under the same conditions. At 3 and 4 wk post L-NAME treatment, renal kallikrein mRNA levels were 2.5- and 3.4-fold higher than in controls, whereas renal beta-actin mRNA levels were similar between groups. Changes in the transcript levels of renal kallikrein, kininogen, and KBP were consistent with their protein levels. Immunoreactive total kininogen and low-Mr kininogen levels in sera and tissue kallikrein levels in kidney were significantly higher in the L-NAME-treated group, whereas KBP levels in the circulation were lower compared with controls. Systolic blood pressure was increased by 58 +/- 4 mmHg after 4 wk of L-NAME treatment. This effect was enhanced in rats given L-NAME in combination with HOE-140, a bradykinin B2-receptor antagonist, at the dose of 100 micrograms/day ip (79 +/- 5 vs. 58 +/- 4 mmHg, P < 0.05). This difference was confirmed by direct measurement of mean blood pressure (MBP). An intra-arterial bolus injection of 200 ng bradykinin significantly decreased MBP of L-NAME-treated rats, and this effect was blunted in the group treated with the bradykinin antagonist (-29 +/- 3 vs. -9 +/- 2 mmHg, P < 0.01). These results suggest that enhanced kallikrein and kininogen synthesis may have a protective role against the cardiovascular effects induced by chronic inhibition of nitric oxide synthesis.
Afferent arteriolar response to arachidonic acid: involvement of metabolic pathways
Authors: Imig J. D. , Navar L. G.
Published: December 2017

Arachidonic acid (AA) metabolites have been implicated in the control of renal hemodynamics, but the nature of the metabolites produced by renal cells when AA is released has remained uncertain. Experiments were performed using the in vitro perfused juxtamedullary nephron preparation to examine the effects of perfusion and superfusion of AA on the renal microvasculature. Extraluminal exposure of the vessels by superfusion with solutions containing 0.1, 1.0, and 10 microM AA decreased afferent arteriolar diameter by 8 +/- 2, 16 +/- 3, and 20 +/- 3%, respectively. The same doses of AA added to the perfusate produced a similar afferent arteriolar vasoconstriction. Inhibition of the major enzymatic pathways unmasked differential responses of AA that were dependent on the direction from which the vasculature was exposed to AA. 17-Octadecynoic acid (1 microM), an inhibitor of the cytochrome P-450 pathway, eliminated the vasoconstrictor response to superfused AA but had little effect on the response to perfused AA. Lipoxygenase inhibition with baicalein (0.5 microM) did not alter the afferent arteriolar vasoconstriction during superfusion with AA but did attenuate the vasoconstrictor response to perfused AA by 34%. Cyclooxygenase inhibition with 10 microM indomethacin reduced the afferent arteriolar response to superfusion with 10 microM AA by 46%, but the responses to perfusion with AA were reversed, leading to the unmasking of a 17% afferent arteriolar dilation. The AA-induced vasorelaxation observed during cyclooxygenase inhibition was prevented by the subsequent addition of a P-450 inhibitor. Additionally, after endothelial removal with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the vasodilatory response reverted to a vasoconstriction. The results of this study demonstrate that in the rat, AA metabolites exert predominant actions on afferent arterioles, but differential responses are mediated via different enzymatic pathways depending on the origin of AA. Increased AA availability of intraluminal origin leads to production of cyclooxygenase-derived vasoconstrictor metabolites and also to endothelial-derived cytochrome P-450 vasodilatory metabolites. In contrast, increased AA availability of interstitial origin leads to production of vasoconstrictor cytochrome P-450 metabolites.
Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2)
Authors: McNicholas C. M. , Yang Y. , Giebisch G. , Hebert S. C.
Published: December 2017

ATP-sensitive, inwardly rectifying K+ channels are present in apical membranes of the distal nephron and play a major role in K+ recycling and secretion. The cloned renal K+ channel, ROMK1, is a candidate for the renal epithelial K+ channel, since it shares many functional characteristics with the native channel. Additionally, ROMK1 contains a putative carboxy-terminal ATP-binding site. Although ROMK1 channel activity could be reactivated by cytosolic Mg-ATP after rundown, the role of nucleotides in channel gating was less certain. We now show that an alternatively spliced transcript of the ROMK channel gene, ROMK2, which encodes a K+ channel with a truncated amino terminus, expresses an ATP-regulated and ATP-sensitive K+ channel (IKATP). Differences in the amino terminus of ROMK isoforms alters the sensitivity of the channel-gating mechanism to ATP. To test whether ATP sensitivity of renal IKATP is mediated by direct interaction of nucleotide, point mutation of specific residues within the ROMK2 phosphate loop (P-loop) were investigated. These either enhanced or attenuated the sensitivity to both activation and inhibition by Mg-ATP, thus demonstrating a direct interaction of nucleotide with the channel-forming polypeptide.
Chronic hypokalemia enhances expression of the H(+)-K(+)-ATPase alpha 2-subunit gene in renal medulla
Authors: Ahn K. Y. , Park K. Y. , Kim K. K. , Kone B. C.
Published: December 2017

Recent molecular and physiological studies suggested that at least two H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) isozymes are expressed in the rat kidney and that these ion pumps respond to changes in dietary potassium balance. We used Northern analysis and in situ hybridization to analyze the expression of mRNA encoding the "colonic" isoform of the H(+)-K(+)-ATPase alpha-subunit (HK alpha 2) in normal and potassium-deprived (2 wk) rats. Control rats exhibited low levels of HK alpha 2 mRNA in the cortical and medullary thick ascending limb, distal convoluted tubule, connecting segment, and the entire collecting duct. The potassium-deprived rats expressed approximately fivefold higher levels of HK alpha 2 mRNA in the outer and inner medulla compared with controls, as well as hypertrophy and increased in situ hybridization signal in the intercalated cells of the inner stripe of the outer medullary collecting duct and the proximal inner medullary collecting duct. In contrast, renal cortical expression of HK alpha 2 mRNA was low and comparable in the two groups. Our results suggest that enhanced expression of the HK alpha 2 subunit gene in the renal medulla contributes to potassium conservation during chronic hypokalemia.
Aldosterone induction and epithelial distribution of CHIF
Authors: Wald H. , Goldstein O. , Asher C. , Yagil Y. , Garty H.
Published: December 2017

CHIF is a recently cloned, corticosteroid-induced gene which evokes K+ channel activity in oocytes (B. Attali, H. Latter, N. Rachamim, and H. Garty. Proc. Natl. Acad. Sci. USA 92: 6092-6096, 1995). To further characterize the possible role of this gene in epithelial ion transport, we have studied its epithelial distribution and hormonal induction. Northern hybridizations indicate that the zonal distribution of CHIF mRNA in kidney is: papilla >>medulla>> cortex. High levels of CHIF were also detected in a primary culture from inner medullary collecting duct (IMCD). Perfusing rats with < 20 nM aldosterone through osmotic minipumps evoked a 22.4 +/- 1.9-fold increase in colonic CHIF. A significant increase was observed 3 h after administrating the corticosteroid, but maximal response was detected only after a 72-h incubation. This response appears to be mineralocorticoid specific; perfusing or injecting rats with maximal doses of dexamethasone did not evoke a further increase in CHIF mRNA. In contrast, high levels of CHIF are expressed in kidney papilla and IMCD primary culture, irrespective of corticosteroid treatment. Thus, like the apical Na+ channel and the H(+)-K(+)-adenosinetriphosphatase, CHIF is mineralocorticoid induced in the colon but constitutively expressed in kidney.
Synthesis and secretion of endothelin in a cortical collecting duct cell line
Authors: Todd-Turla K. M. , Zhu X. L. , Shu X. , Chen M. , Yu T. , Smart A. , Killen P. D. , Fejes-Toth G. , Briggs J. P. , Schnermann J. B.
Published: December 2017

Previous experiments have shown that epithelial cells in the renal medulla produce endothelin-1 (ET-1) and possess ETB receptors. It has been suggested that medullary ET-1 may affect water and sodium absorption along the collecting ducts in an autocrine fashion. To study possible mechanisms responsible for the regulation of medullary ET-1 production, experiments were performed in M-1 cells and mIMCD-K2 cells, cell lines derived from cortical and inner medullary collecting ducts of SV40 transgenic mice, grown to confluence on collagen-coated filter inserts. Both cell lines were found to express ET-1 mRNA and to secrete ET almost exclusively into the basolateral medium as long as the transepithelial resistance was high. Inhibition of transcription with actinomycin D was followed by a decline in both ET mRNA [halftime (t1/2) = 30 min] and ET secretion (t1/2 = approximately 90 min). The addition of arginine vasopressin (AVP, 10(-8) M; 2- or 4-h exposure) or incubation of M-1 cells in hypertonic media (+50 mM NaCl, 4- or 6-h exposure) did not significantly alter ET secretion or ET-1 mRNA expression. In contrast, simultaneously increasing AVP(10(-8) M in the basolateral medium) and tonicity (+50 mM NaCl) for 4 h increased ET secretion (from 28.9 +/- 3.9 to 41.8 +/- 3.8 pg.h-1.mg protein-1; P = 0.029, n = 10) and ET-1 mRNA (control = 2,138 cpm/microliter, log of 3.33 +/- 0.048, n = 4; AVP + NaCl = 3,548.1 cpm/microliter, log of 3.55 +/- 0.09; P = 0.045, n = 5). Exposure of M-1 cells to hypertonic media (+50 mM NaCl or 100 mM mannitol) for 24 h was associated with a marked reduction of ET secretion (-83.9% with NaCl and -78.4% with mannitol; P < 0.0001). This reduction was attenuated, but not prevented, by the presence of AVP in the basolateral medium (-40%). ET-1 mRNA, in contrast, did not change with 24-h exposure to hypertonic media and increased when AVP was present. Results are compatible with the concept that generation of ET by collecting duct cells may contribute in a complex and time-dependent fashion to the paracrine control of collecting duct cell function.