bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of PKC-βI and -δ contributes to higher PKC activity in the proximal tubules of old Fischer 344 rats
Authors: Asghar Mohammad , Hussain Tahir , Lokhandwala Mustafa F.
Published: March 2015

Previously, we reported that natriuretic and diuretic response to dopamine is diminished in old Fischer 344 rats, which is due to higher basal protein kinase C (PKC) activity and hyperphosphorylation of Na-K-ATPase in the proximal tubules (PTs) of old rats. The present study was conducted to determine whether higher PKC activity could be due to altered expression of some of the PKC isoforms in the superficial cortex (rich in PTs) of old rats. Fluorimetric measurement showed almost twofold increase in the PKC activities in homogenates and membranes of old (24 mo) compared with adult (6 mo) rats. Interestingly, in the basal state PKC-βI was overexpressed in the membranes, whereas PKC-δ expression was increased in the cytosol of old compared with adult rats. Treatment of the cortical slices with either SKF-38393, a D1-like agonist, or PDBu, a direct activator of PKC, caused translocation of PKC-βI from cytosol to membranes in adult but not in old rats. Both of these drugs caused translocation of PKC-δ from membranes to cytosol in adult but not in old rats. These drugs had no effect on translocation of PKC-ζ in both adult and old rats. Both PKC-βI and -δ coimmunoprecipiated with α1-subunit of Na-K-ATPase in adult and old rats. These observations suggest that both SKF-38393 and PDBu differentially regulate PKC-βI and -δ in adult but not in old rats. Also, PKC-βI and -δ seem to interact with Na-K-ATPase in these animals. The overexpression of both PKC-βI and -δ in old rats could be responsible for a higher basal PKC activity, which causes the hyperphosphorylation of Na-K-ATPase and contributes to the diminished inhibition of Na-K-ATPase activity by dopamine in old rats.
Attenuated buffering of renal perfusion pressure variation in juxtamedullary cortex in SHR
Authors: Roald Anca B. , Ofstad Jarle , Iversen Bjarne M.
Published: March 2015

Renal tissue damage is substantially more pronounced in the juxtamedullary than in the superficial cortex in hypertensive rats, and the pathogenesis of the morphological changes are only partly understood. Glomerular capillary pressure (PGC) is increased, and steady-state autoregulation is normal in the deep renal cortex. We tested the hypothesis that the transient period from one pressure level to another may induce greater variation in local perfusion before stable autoregulation is established. An acute increase in local perfusion was compared in the superficial and juxtamedullary cortex of spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY) after an abrupt increase in perfusion pressure. Total renal blood flow (RBF) was measured by a Transonic flow probe and local renal perfusion by laser Doppler flowmetry. Renal perfusion pressure was lowered to 50% of initial values and released abruptly. The maximal RBF increased from 6.3 ± 0.4 to a maximal value of 7.6 ± 0.3 ml/min ( P < 0.001) in SHR and from 7.3 ± 0.3 to 8.2 ± 0.6 ml/min ( P < 0.001) in WKY. These changes were not significantly different from each other. The change in superficial cortical perfusion was also not different between SHR and WKY. Pressure release increased juxtamedullary perfusion in SHR from 146 ± 8 to a maximal value of 228 ± 17 units ( P < 0.001) and in WKY from 160 ± 13 to 179 ± 11 units ( P < 0.001). The results were significantly different from each other ( P < 0.001). The time for maximal flow response was shorter in the deep cortex of SHR, and the time for normalization was longer than in WKY. These data indicate that the buffering of perfusion pressure variation is significantly attenuated in the juxtamedullary cortex, and significantly more so in SHR than in WKY, assuming a covariation of RBF and PGC, and this finding may explain the extensive morphological damage in the juxtamedullary cortex of SHR.
Osmotically inactive skin Na+storage in rats
Authors: Titze Jens , Lang Rainer , Ilies Christoph , Schwind Karl H. , Kirsch Karl A. , Dietsch Peter , Luft Friedrich C. , Hilgers Karl F.
Published: March 2015

Compared with age-matched men, women are resistant to the hypertensive effects of dietary NaCl; however, after menopause, the incidence of salt-sensitive hypertension is similar in women and men. We recently suggested that osmotically inactive Na+storage contributes to the development of salt-sensitive hypertension. The connective tissues, including those immediately below the skin that may serve as a reservoir for osmotically inactive Na+storage, are affected by menopause. We tested the hypothesis that ovariectomy (OVX) might reduce osmotically inactive Na+storage capacity in the body, particularly in the skin. Male, female-fertile, and female OVX Sprague-Dawley (SD) rats were fed a high (8%)- or low (0.1%)-NaCl diet. The groups received the diet for 4 or 8 wk. At the end of the experiment, subgroups received 0.9% saline infusion and urinary Na+and K+excretion was measured. Wet and dry weight (DW), water content in the body and skin, total body Na+(rTBNa+) and skin Na+(rSKNa+) content were measured relative to DW by desiccation and dry ashing. There were no gender differences in osmotically inactive Na+storage in SD rats. All SD rats accumulated Na+if fed 8% NaCl, but rTBNa+was lower in OVX rats than in fertile rats on a low ( P < 0.001)- and a high ( P < 0.05)-salt diet. OVX decreased rSKNa+( P < 0.01) in the rats. A high-salt diet led to Na+accumulation (ΔSKNa+) in the skin in all SD rats. Osmotically inactive skin Na+accumulation was ∼66% of ΔSKNa+in female and 82% in male-fertile rats, but there was no osmotically inactive Na+accumulation in OVX rats fed 8% NaCl. We conclude that skin is an osmotically inactive Na+reservoir that accumulates Na+when dietary NaCl is excessive. OVX leads to an acquired reduction of osmotically inactive Na+storage in SD rats that predisposes the rats to volume excess despite a reduced Na+content relative to body weight.
ERK and p38 MAP kinase are involved in arachidonic acid release induced by H2O2and PDGF in mesangial cells
Authors: Hayama Misako , Inoue Risa , Akiba Satoshi , Sato Takashi
Published: March 2015

Increased prostaglandin production is implicated in the pathogenesis of glomerular disease. With this consideration, we examined the combined effects of reactive oxygen species and platelet-derived growth factor (PDGF), which might initiate glomerular dysfunction, on arachidonic acid release and cytosolic phospholipase A2(cPLA2) activation in rat mesangial cells. H2O2-induced release of arachidonic acid was enhanced by PDGF, which by itself had little effect on the release, and the enhancement was completely inhibited by a cPLA2inhibitor. The phosphorylation of cPLA2, extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein (MAP) kinase was upregulated by H2O2or PDGF alone and except for ERK was enhanced further by the two in combination. The release of arachidonic acid induced by PDGF together with H2O2was inhibited partially by an inhibitor of ERK or p38 MAP kinase and completely when the two inhibitors were combined; the inhibitory pattern was similar to that for the phosphorylation of cPLA2. These results suggest that the ERK and p38 MAP kinase pathways are involved in the increase in cPLA2activation and arachidonic acid release induced by PDGF together with H2O2.
Neutralization of Western diet inhibits bone resorption independently of K intake and reduces cortisol secretion in humans
Authors: Maurer Marc , Riesen Walter , Muser Juergen , Hulter Henry N. , Krapf Reto
Published: March 2015

A Western-type diet is associated with osteoporosis and calcium nephrolithiasis. On the basis of observations that calcium retention and inhibition of bone resorption result from alkali administration, it is assumed that the acid load inherent in this diet is responsible for increased bone resorption and calcium loss from bone. However, it is not known whether the dietary acid load acts directly or indirectly (i.e., via endocrine changes) on bone metabolism. It is also unclear whether alkali administration affects bone resorption/calcium balance directly or whether alkali-induced calcium retention is dependent on the cation (i.e., potassium) supplied with administered base. The effects of neutralization of dietary acid load (equimolar amounts of NaHCO3and KHCO3substituted for NaCl and KCl) in nine healthy subjects (6 men, 3 women) under metabolic balance conditions on calcium balance, bone markers, and endocrine systems relevant to bone [glucocorticoid secretion, IGF-1, parathyroid hormone (PTH)/1,25(OH)2vitamin D and thyroid hormones] were studied. Neutralization for 7 days induced a significant cumulative calcium retention (10.7 ± 0.4 mmol) and significantly reduced the urinary excretion of deoxypyridinoline, pyridinoline, and n-telopeptide. Mean daily plasma cortisol decreased from 264 ± 45 to 232 ± 43 nmol/l ( P = 0.032), and urinary excretion of tetrahydrocortisol (THF) decreased from 2,410 ± 210 to 2,098 ± 190 μg/24 h ( P = 0.027). No significant effect was found on free IGF-1, PTH/1,25(OH)2vitamin D, or thyroid hormones. An acidogenic Western diet results in mild metabolic acidosis in association with a state of cortisol excess, altered divalent ion metabolism, and increased bone resorptive indices. Acidosis-induced increases in cortisol secretion and plasma concentration may play a role in mild acidosis-induced alterations in bone metabolism and possibly in osteoporosis associated with an acidogenic Western diet.
Impaired trafficking of distal renal tubular acidosis mutants of the human kidney anion exchanger kAE1
Authors: Quilty Janne A. , Li Jing , Reithmeier Reinhart A.
Published: March 2015

Distal renal tubular acidosis (dRTA) is an inherited disease characterized by the failure of the kidneys to appropriately acidify urine and is associated with mutations in the anion exchanger (AE)1 gene. The effect of the R589H dRTA mutation on the expression of the human erythroid AE1 and the truncated kidney form (kAE1) was examined in transfected human embryonic kidney 293 cells. AE1, AE1 R589H, and kAE1 were present at the cell surface, whereas kAE1 R589H was located primarily intracellularly as shown by immunofluorescence, cell surface biotinylation, N-glycosylation, and anion transport assays. Coexpression of kAE1 R589H reduced the cell surface expression of kAE1 and AE1 by a dominant-negative effect, due to heterodimer formation. The mutant AE1 and kAE1 bound to an inhibitor affinity resin, suggesting that they were not grossly misfolded. Other mutations at R589 also prevented the formation of the cell surface form of kAE1, indicating that this conserved arginine residue is important for proper trafficking. The R589H dRTA mutation creates a severe trafficking defect in kAE1 but not in erythroid AE1.
Differential regulation of glomerular arginine transporters (CAT-1 and CAT-2) in lipopolysaccharide-treated rats
Authors: Schwartz Doron , Schwartz Idit F. , Gnessin Ehud , Wollman Yoram , Chernichovsky Tamara , Blum Miriam , Iaina Adrian
Published: March 2015

The decrease in glomerular filtration rate (GFR) that is characteristic of sepsis has been shown to result from inhibition of glomerular endothelial nitric oxide synthase (eNOS) by nitric oxide (NO) generated from the inducible isoform of NOS (iNOS). Although l-arginine is the sole precursor for NO biosynthesis, its intracellular availability in glomeruli from septic animals has never been investigated. Arginine uptake was measured in freshly harvested glomeruli from the following experimental groups: 1) untreated rats; 2) rats pretreated with LPS (4 mg/kg body wt, 4 h before experiments); 3) rats treated with LPS as above with eitherl- N6-(1-iminoethyl)lysine hydrochloride (l-NIL), a selective iNOS antagonist, or 7-nitroindazole, a selective neuronal NOS antagonist; and 4) rats treated with l-NIL only. Both glomeular and mesangial arginine transport characteristics were found compatible with a y+system. Arginine uptake was augmented in glomeruli from LPS-treated rats. Treatment with l-NIL completely abolished this effect whereas l-NIL alone had no effect. Similar results were obtained when primary cultures of rat mesangial cells were preincubated with LPS (10 μg/ml for 24 h) with or withoutl-NIL. Using RT-PCR, we found that in vivo administration of LPS resulted in a significant increase in glomerular cationic amino acid transporter-2 (CAT-2) mRNA expression whereas CAT-1 mRNA was undetected. Northern blotting further confirmed a significant increase in glomerular CAT-2 by LPS. In mesangial cells, the expression of both CAT-1 and CAT-2 mRNA was augmented after incubation with LPS. In conclusion, in vivo administration of LPS augments glomerular arginine transport through upregulation of steady-state CAT-2 mRNA while downregulating CAT-1 mRNA. These results may correspond to the changes in glomerular iNOS and eNOS activity in sepsis.
Temporal decrease in renal sensory responses in rats after chronic ligation of the bile duct
Authors: Ma Ming-Chieh , Huang Ho-Shiang , Chien Chiang-Ting , Wu Ming-Shiou , Chen Chau-Fong
Published: March 2015

Renal responses to renal sensory receptor activation were examined in rats after 1 and 4 wk of common bile duct ligation (CBDL). Compared with sham-operated rats (Sham), urine and sodium excretion after acute saline loading was significantly reduced at both times after CBDL. The blunted excretory responses in CBDL rats, accompanied by less activation of afferent renal nerve activity (ARNA), were already apparent at 1 wk and became severe at 4 wk. The defect in ARNA activation in CBDL rats was further studied using specific stimuli to activate renal sensory receptors. Graded increases in intrapelvic pressure or renal pelvic perfusion of substance P (SP) elicited an increase in ARNA in Sham rats, these responses being temporally attenuated in CBDL rats. Despite no significant change in renal pelvic SP release, no renorenal reflex was demonstrable in 4-wk CBDL rats. Immunoblotting showed that expression of renal pelvic neurokinin 1 (NK-1) receptors was 32 and 47% lower in 1- and 4-wk CBDL rats, respectively, than in Sham rats, this decrease correlating well with plasma SP levels. The quantitative real-time RT-PCR showed similar levels of NK-1 receptor mRNA in the renal pelvis in the Sham and 4-wk CBDL groups. We conclude that impairment of renal excretory and sensory responses increases with the duration of cirrhosis. An impaired renorenal reflex in cirrhotic rats is involved in the defective activation of the renal sensory receptors could be due, in part, to the low expression of NK-1 receptors, which is dependent on the duration of CBDL. The decrease in NK-1 receptor protein levels is not due to a decrease in mRNA levels.
Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells
Authors: Nowak Grażyna , Price Peter M. , Schnellmann Rick G.
Published: March 2015

The lack of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) in mice increases renal proximal tubular cell death and enhances sensitivity to acute renal failure produced by the chemotherapeutic agent cisplatin. We used primary cultures of mouse renal proximal tubular cells (MPTC) grown in optimized culture conditions to investigate the cellular basis for increased apoptosis in p21 knockout mice. Cisplatin (15 μM) activated caspase-3 but not caspase-8 or caspase-9 and produced phosphatidylserine externalization, chromatin condensation, and nuclear fragmentation in wild-type [p21(+/+)] MPTC. Caspase-3 activation and apoptosis were accelerated in cisplatin-treated MPTC lacking p21 [p21(-/-) MPTC]. In contrast to p21(+/+) MPTC, cisplatin activated caspase-9 but not caspase-8 in p21(-/-) MPTC before caspase-3 activation. The caspase-3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone (DEVD-fmk) inhibited caspase-3 activity but did not abolish apoptosis in p21(+/+) and p21(-/-) MPTC. General caspase inhibitor Z-Val-Ala-Asp(OCH3)-fluoromethylketone (ZVAD-fmk) inhibited caspase activity and decreased chromatin condensation by 51% in p21(-/-) but not in p21(+/+) MPTC. However, cisplatin-induced phosphatidylserine externalization was not inhibited by ZVAD-fmk in p21(-/-) MPTC. We conclude that 1) in the presence of p21, cisplatin activates caspase-3 through a mechanism independent of caspase-8 or caspase-9; 2) in the absence of p21, caspase-9 activation precedes caspase-3 activation; 3) the lack of p21 accelerates caspase-3 activation and cisplatin-induced MPTC apoptosis; and 4) MPTC apoptosis is caspase independent in the presence of p21 but partially dependent on caspases in the absence of p21.
Axial heterogeneity in basolateral AQP2 localization in rat kidney: effect of vasopressin
Authors: Christensen Birgitte Mønster , Wang Weidong , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

The purpose of the present study was to examine whether there is axial heterogeneity in the basolateral plasma membrane (BLM) localization of AQP2 and whether altered vasopressin action or medullary tonicity affects the BLM localization of AQP2. Immunocytochemistry and immunoelectron microscopy revealed AQP2 labeling of the BLM in connecting tubule (CNT) cells and inner medullary collecting duct (IMCD) principal cells in normal rats and vasopressin-deficient Brattleboro rats. In contrast there was little basolateral AQP2 labeling in cortical (CCD) and outer medullary collecting duct principal cells. Short-term desamino-Cys1,D-Arg8vasopressin (dDAVP) treatment (2 h) of Brattleboro rats caused no increase in AQP2 labeling of the BLM. In contrast, long-term dDAVP treatment (6 days) of Brattleboro rats caused an increased BLM labeling in CNT, CCD, and IMCD. Treatment of normal rats with V2-receptor antagonist for 60 min caused retrieval of AQP2 from the apical plasma membrane. Moreover, AQP2 labeling of the BLM was unchanged in CNT and IMCD but increased in CCD. In conclusion, there is an axial heterogeneity in the subcellular localization of AQP2 with prominent AQP2 labeling of the BLM in CNT and IMCD. There was no increase in AQP2 labeling of the BLM in response to short-term dDAVP. Moreover, acute V2-receptor antagonist treatment did not cause retrieval of AQP2 from the BLM. In contrast, long-term dDAVP treatment caused a major increase in AQP2 expression in the BLM in CCD.
Increase in renal glutathione in cholestatic liver disease is due to a direct effect of bile acids
Authors: Purucker Edmund , Marschall Hanns-Ulrich , Geier Andreas , Gartung Carsten , Matern Siegfried
Published: March 2015

Hepatic synthesis and plasma levels of glutathione are markedly decreased in chronic liver disease. Because glutathione turnover is highest in kidneys, we examined whether changes in kidney glutathione occur in chronic cholestasis and whether they are related to kidney dysfunction in liver disease. Kidney and plasma GSH and GSSG were measured 1) in bile duct-ligated (BDL) rats; 2) in healthy rats after bile acid loading to mimic cholestasis; and 3) after irreversible inhibition of glutathione synthetase with buthionine-sulfoximine (BSO), where glutathione consumption, urinary volume, and sodium excretion were also estimated. In addition, γ-glutamylcysteine synthetase (γ-GCS) mRNA, protein, and enzymatic specific activity were measured in kidney tissue after BDL. After BDL, kidney GSH and GSSG increased within hours by 67 and 66%, respectively. The increases were not related to plasma glutathione, which decreased below control values. Intravenous bile acid loading caused identical increases in GSH and GSSG as occurred after BDL, when glycine- or taurine-conjugated dihydroxy bile acids were administered. Glutathione consumption, as estimated after blocking of de novo synthesis with BSO, was significantly increased after BDL (127 vs. 44 nmol · g−1· min−1). γ-GCS mRNA and enzymatic specific activity were significantly reduced 5 days after BDL, whereas protein concentrations did not change. The urinary sodium concentration was 70% lower in BDL than in control rats. Depletion of renal glutathione normalized sodium excretion by increasing urinary sodium concentration and urinary volume. The increase in kidney glutathione after BDL seems to be mediated by an increase in plasma bile acids and is critically related to sodium retention. The increase in GSH consumption despite reduced γ-GCS activity indicates a decreased GSH turnover tentatively due to reduced renal GSH efflux by competition with organic anions at membrane transport proteins.
D2-like receptor-mediated inhibition of Na+-K+-ATPase activity is dependent on the opening of K+channels
Authors: Gomes Pedro , Soares-da-Silva P.
Published: March 2015

This study examined the effects of D2-like dopamine receptor activation on Na+-K+-ATPase activity while apical-to-basal, ouabain-sensitive, amphotericin B-induced increases in short-circuit current and basolateral K+( IK) currents in opossum kidney cells were measured. The inhibitory effect of dopamine on Na+-K+-ATPase activity was completely abolished by either D1- or D2-like receptor antagonists and mimicked by D1- and D2-like receptor agonists SKF-38393 and quinerolane, respectively. Blockade of basolateral K+channels with BaCl2(1 mM) or glibenclamide (10 μM), but not apamin (1 μM), totally prevented the inhibitory effects of quinerolane. The K+channel opener pinacidil decreased Na+-K+-ATPase activity. The inhibitory effect of quinerolane on Na+-K+- ATPase activity was abolished by pretreatment of opossum kidney cells with pertussis toxin (PTX). Quinerolane increased IKacross the basolateral membrane in a concentration-dependent manner; this effect was abolished by pretreatment with PTX, S-sulpiride, and glibenclamide. SKF-38393 did not change IK. Both H-89 (protein kinase A inhibitor) and chelerythrine (protein kinase C inhibitor) failed to prevent the stimulatory effect of quinerolane on IK. The stimulation of the D2-like receptor was associated with a rapid hyperpolarizing effect, whereas D1-like receptor activation was accompanied by increases in cell membrane potential. It is concluded that stimulation of D2-like receptors leads to inhibition of Na+-K+-ATPase activity and hyperpolarization; both effects are associated with the opening of K+channels.
Angiotensin II and renal medullary blood flow in Lyon rats
Authors: Sarkis Albert , Liu Kiao Ling , Lo Ming , Benzoni Daniel
Published: March 2015

The present study evaluated the acute effects of ANG II (5–480 ng/kg iv) and phenylephrine (PE; 0.2–146 μg/kg iv) on total renal (RBF) and medullary blood flow (MBF) in anesthetized Lyon hypertensive (LH) and low-blood-pressure (LL) rats. ANG II and PE induced dose-dependent decreases in both RBF and MBF, which were greater in LH than in LL rats. Interestingly, after ANG II, but not after PE, the initial medullary vasoconstriction was followed by a long-lasting and dose-dependent vasodilation that was significantly blunted in LH compared with LL rats. The mechanisms of the MBF effects of ANG II were studied in LL rats only. Blockade of AT1 receptors with losartan (10 mg/kg) abolished all the effects of ANG II, whereas AT2 receptor blockade with PD-123319 (50 μg · kg−1 · min−1iv) did not change these effects. Indomethacin (5 mg/kg) decreased by ∼90% the medullary vasodilation induced by the lowest doses of ANG II (from 15 ng/kg). In contrast, N G-nitro-l-arginine methyl ester (10 mg/kg and 0.1 mg · kg−1 · min−1iv) and the bradykinin B2-receptor antagonist HOE-140 (20 μg/kg and 10 μg · kg−1 · min−1iv) markedly lowered the medullary vasodilation at the highest doses of ANG II only. In conclusion, this study shows that LH rats exhibit an altered MBF response to ANG II compared with LL rats and indicates that the AT1 receptor-mediated medullary vasodilator response to low doses of ANG II is mainly due to the release of PGs, whereas the dilator response to high doses of ANG II has additional nitric oxide- and kinin-dependent components.
Peroxisome proliferator-activated receptor-γ activity is associated with renal microvasculature
Authors: Guan Youfei , Zhang Yahua , Schneider André , Davis Linda , Breyer Richard M. , Breyer Matthew D.
Published: March 2015

First published July 12, 2001; 10.1152/ajprenal.00025.2001.—Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear transcription factor and the pharmacological target for antidiabetic thiazolidinediones (TZDs). TZDs ameliorate diabetic nephropathy and have direct effects on cultured mesangial cells (MCs); however, in situ hybridization failed to detect expression of PPARγ in glomeruli in vivo. The purpose of this study was to determine whether PPARγ is expressed in renal glomeruli. Two rabbit PPARγ isoforms were cloned. Nuclease protection assays demonstrate that both PPARγ isoforms are expressed in freshly isolated glomeruli. Treatment of rabbits with the TZD troglitazone selectively induced expression of an endogenous PPARγ target gene, adipocyte fatty acid-binding protein (A-FABP), in renal glomerular cells and renal medullary microvascular endothelial cells, demonstrated by both in situ hybridization and immunostain. Troglitazone also dramatically increased A-FABP expression in cultured MCs. Constitutive PPARγ expression was detected in cultured rabbit MCs. Endogenous MC PPARγ can also drive PPARγ reporter. Troglitazone and 15-deoxy-Δ12,14 prostaglandin J2at low concentrations reduced mesangial cell [3H]thymidine incorporation without affecting viability. These data suggest that constitutive PPARγ activity exists in renal glomeruli in vivo and could provide a pharmacological target to directly modulate glomerular injury.
TGF-β-induced Ca2+influx involves the type III IP3receptor and regulates actin cytoskeleton
Authors: McGowan Tracy A. , Madesh Muniswamy , Zhu Yanqing , Wang Lewei , Russo Mark , Deelman Leo , Henning Rob , Joseph Suresh , Hajnoczky Gyorgy , Sharma Kumar
Published: March 2015

Ca2+influx has been postulated to modulate the signaling pathway of transforming growth factor-β (TGF-β); however, the underlying mechanism and functional significance of TGF-β-induced stimulation of Ca2+influx are unclear. We show here that TGF-β stimulates Ca2+influx in mesangial cells without Ca2+release. The influx of Ca2+is prevented by pharmacological inhibitors of inositol 1,4,5-trisphosphate receptors (IP3R) as well as specific antibodies to type III IP3R (IP3RIII) but not to type I IP3R (IP3RI). TGF-β enhances plasma membrane localization of IP3RIII, whereas the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) preferentially translocates to the nucleus. Untreated mesangial cells exhibit actin filamentous protrusions on the cell surface, and treatment with TGF-β dramatically reduces this pattern. The alterations in the actin cytoskeleton by TGF-β are dependent on TGF-β-induced Ca2+influx. These studies identify a novel pathway by which TGF-β regulates Ca2+influx and induces cytoskeletal alterations.
Effects of AT1A receptor deletion on blood pressure and sodium excretion during altered dietary salt intake
Authors: Mangrum Amy J. , Gomez R. Ariel , Norwood Victoria F.
Published: March 2015

The present study was performed to investigate the role of type 1A ANG II (AT1A) receptors in regulating sodium balance and blood pressure maintenance during chronic dietary sodium variations in AT1A receptor-deficient (−/−) mice. Groups of AT1A (−/−) and wild-type mice were placed on a low (LS)-, normal (NS)-, or high-salt (HS) diet for 3 wk. AT1A(−/−) mice on an LS diet had high urinary volume and low blood pressure despite increased renin and aldosterone levels. On an HS diet, (−/−) mice demonstrated significant diuresis, yet blood pressure increased to levels greater than control littermates. There was no effect of dietary sodium intake on systolic blood pressures in wild-type animals. The pressure-natriuresis relationship in AT1A (−/−) mice demonstrated a shift to the left and a decreased slope compared with wild-type littermates. These studies demonstrate that mice lacking the AT1A receptor have blood pressures sensitive to changes in dietary sodium, marked alterations of the pressure-natriuresis relationship, and compensatory mechanisms capable of maintaining normal sodium balance across a wide range of sodium intakes.
CytochromeP-450 4A isoform expression and 20-HETE synthesis in renal preglomerular arteries
Authors: Marji Jackleen S. , Wang Mong-Heng , Laniado-Schwartzman Michal
Published: March 2015

20-Hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor and mediator of the myogenic response, is a major arachidonic acid metabolite in the microvasculature of the rat kidney formed primarily by the cytochrome P-450 (CYP) 4A isoforms, CYP4A1, CYP4A2, and CYP4A3. We examined CYP4A isoform expression and 20-HETE synthesis in microdissected interlobar, arcuate, and interlobular arteries; mRNA for all CYP4A isoforms was identified by RT-PCR. Western blot analysis indicated that the levels of CYP4A2/4A3-immunoreactive protein increased with decreased arterial diameter, whereas those of CYP4A1-immunoreactive protein remained unchanged. 20-HETE synthesis was the highest in the interlobular arteries (17 ± 1.62 nmol · mg−1· h−1) and, like CYP4A2/4A3-immunoreactive protein, decreased with increasing vessel diameter (4.5 ± 1.21, 2.65 ± 0.58, and 0.81 ± 0.14 nmol · mg−1· h−1in the arcuate, interlobar, and segmental arteries, respectively). 20-HETE synthesis in the renal artery and the abdominal aorta was undetectable. The observed decreased immunoreactivity of NADPH-cytochrome P-450 ( c) oxidoreductase with increased arterial diameter provided a possible explanation for the decreased capacity to generate 20-HETE in the large arteries. The increase in CYP4A isoform expression and 20-HETE synthesis with decreasing diameter along the preglomerular arteries and the potent biological activity of 20-HETE underscore the significance of 20-HETE as a modulator of renal hemodynamics.
Genomic organization of the 5′ end of human β-ENaC and preliminary characterization of its promoter
Authors: Thomas Christie P. , Loftus Randy W. , Liu Kang Z. , Itani Omar A.
Published: March 2015

The mRNA for the β-subunit of the epithelial Na+channel (β-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human β-ENaC gene, we characterized the 5′ end of the gene and its 5′-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5′ rapid amplification of cDNA ends, and transcription start sites of two 5′ variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5′ end of the gene were isolated, and analysis of these clones indicated that alternate first exons ∼1.5 kb apart and ∼ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5′-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate β-ENaC expression.
Protective effect of renal denervation on normotensive endotoxemia-induced acute renal failure in mice
Authors: Wang Wei , Falk Sandor A. , Jittikanont Suparoek , Gengaro Patricia E. , Edelstein Charles L. , Schrier Robert W.
Published: March 2015

Acute renal failure (ARF) contributes substantially to the high morbidity and mortality observed during endotoxemia. We hypothesized that selective blockade of the renal nerves would be protective against ARF during the early (16 h) stage of endotoxemia [5 mg lipopolysaccharide (LPS)/kg ip in mice]. At 16 h after LPS, there was no change in mean arterial pressure, but plasma epinephrine (4,604 ± 719 vs. 490 ± 152 pg/ml, P < 0.001), norepinephrine (2,176 ± 306 vs. 1,224 ± 218 pg/ml, P < 0.05), and plasma renin activity (40 ± 5 vs. 27 ± 2 ng · ml−1· h−1, P < 0.05) were higher in the LPS-treated vs. control mice. The high plasma renin activity level decreased to the control level with renal denervation in endotoxemic mice. After intravenous injection of phentolamine (200 μg/kg), the decrement in mean arterial pressure was significantly greater in LPS-treated vs. control mice (19.4 ± 3.5 vs. 8.1 ± 1.5 mmHg, P < 0.01). Sixteen hours after LPS administration, there were significant decreases in glomerular filtration rate (52 ± 18 vs. 212 ± 23 μl/min, P < 0.01) and renal blood flow (0.58 ± 0.08 vs. 0.85 ± 0.06 ml/min, P < 0.01) in sham-operated mice. The decrement in glomerular filtration rate during endotoxemia was significantly attenuated in mice with denervated kidneys (32 vs. 79%). Moreover, there was no change in renal blood flow during endotoxemia in mice with renal denervation. The present results therefore demonstrate a protective role of renal denervation during normotensive endotoxemia-related ARF in mice, an effect that may be, at least in part, due to a diminished activation of the renin-angiotensin system.
A3adenosine receptor knockout mice are protected against ischemia- and myoglobinuria-induced renal failure
Authors: Lee H. Thomas , Ota-Setlik Ayuko , Xu Hua , D'Agati Vivette D. , Jacobson Marlene A. , Emala Charles W.
Published: March 2015

A3adenosine receptor (AR) activation and inhibition worsen and improve, respectively, renal function after ischemia-reperfusion (I/R) injury in rats. We sought to further characterize the role of A3ARs in modulating renal function after either I/R or myoglobinuric renal injury. A3knockout mice had significantly lower plasma creatinines compared with C57 controls 24 h after I/R or myoglobinuric renal injury. C57 control mice pretreated with the A3AR antagonist [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5 dicarboxylate] or agonist [0.125 mg/kg N6-(3-iodobenzyl)- N-methyl-5′-carbamoyladenosine (IB-MECA)] demonstrated improved or worsened renal function, respectively, after I/R or myoglobinuric renal injury. Higher doses of IB-MECA were lethal in C57 mice subjected to renal ischemia. H1but not H2histamine receptor antagonist prevented death in mice pretreated with IB-MECA before renal ischemia. Improvement in renal function was associated with significantly improved renal histology. In conclusion, preischemic A3AR activation (0.125 mg/kg IB-MECA) exacerbated renal I/R injury in mice. Mice lacking A3ARs or blocking A3ARs in wild-type mice resulted in significant renal protection from ischemic or myoglobinuric renal failure.
Polaris, a protein disrupted inorpkmutant mice, is required for assembly of renal cilium
Authors: Yoder Bradley K. , Tousson Albert , Millican Leigh , Wu John H. , Bugg Charles E. , Schafer James A. , Balkovetz Daniel F. ,   (With the Technical Assistance of Li Li and Mary Lou Watkins)
Published: March 2015

Cilia are organelles that play diverse roles, from fluid movement to sensory reception. Polaris, a protein associated with cystic kidney disease in Tg737°rpkmice, functions in a ciliogenic pathway. Here, we explore the role of polaris in primary cilia on Madin-Darby canine kidney cells. The results indicate that polaris localization and solubility change dramatically during cilia formation. These changes correlate with the formation of basal bodies and large protein rafts at the apical surface of the epithelia. A cortical collecting duct cell line has been derived from mice with a mutation in the Tg737 gene. These cells do not develop normal cilia, which can be corrected by reexpression of the wild-type Tg737 gene. These data suggest that the primary cilia are important for normal renal function and/or development and that the ciliary defect may be a contributing factor to the cystic disease in Tg737°rpkmice. Further characterization of these cells will be important in elucidating the physiological role of renal cilia and in determining their relationship to cystic disease.
Functional evidence that vascular endothelial growth factor may act as an autocrine factor on human podocytes
Authors: Foster Rebecca R. , Hole Rachel , Anderson Karen , Satchell Simon C. , Coward Richard J. , Mathieson Peter W. , Gillatt David A. , Saleem Moin A. , Bates David O. , Harper Steven J.
Published: March 2015

Vascular endothelial growth factor (VEGF) is expressed by renal glomerular epithelial cells (podocytes) and is thought to be protective against nephrotoxic agents. VEGF has been shown to be an autocrine survival factor in neuropilin-1-positive, VEGF receptor-negative breast carcinoma cells. Normal human podocytes are also known to express neuropilin-1, VEGF, and are VEGF-R2 negative. Here, we investigated whether a similar VEGF autocrine loop may exist in podocytes. Podocyte cytosolic calcium concentration ([Ca2+]i) was analyzed in primary cultured and conditionally immortalized podocytes using ratiometric fluorescence measurement. Cytotoxicity was determined by lactate dehydrogenase assay, proliferation by [3H]-thymidine incorporation, and cell counts by hemocytometric assay. VEGF decreased [Ca2+]iin primary podocytes (from 179 ± 36 to 121 ± 25 nM, P < 0.05) and conditionally immortalized podocytes (from 95 ± 10 to 66 ± 8 nM, P < 0.02) in the absence of extracellular calcium. The type III receptor tyrosine-kinase inhibitor PTK787/ZK222584 abolished this reduction. VEGF increased podocyte [3H]-thymidine incorporation (3,349 ± 283 cpm, control 2,364 ± 301 cpm, P < 0.05) and cell number (4.5 ± 0.7 × 104/ml, control 2.6 ± 0.5 × 104/ml, P < 0.05) and decreased cytotoxicity (5.9 ± 0.7%, control 12 ± 3%, P < 0.05), whereas a monoclonal antibody to VEGF increased cytotoxicity. Electron microscopy of normal human glomeruli demonstrated that the glomerular VEGF is mostly podocyte cell membrane associated. These results indicate that one of the functions of VEGF secreted from podocytes may be to act as an autocrine factor on calcium homeostasis and cell survival.
Mitochondrial dysfunction is an early event in high-NaCl-induced apoptosis of mIMCD3 cells
Authors: Michea Luis , Combs Christian , Andrews Peter , Dmitrieva Natalia , Burg Maurice B.
Published: March 2015

Raising osmolality to 700 mosmol/kgH2O by the addition of NaCl rapidly kills most murine inner renal medullary collecting duct cells (mIMCD3), but they survive at 500 mosmol/kgH2O. At 300 and 500 mosmol/kgH2O, NADH autofluorescence is present in a mitochondria-associated, punctate perinuclear pattern. Within 45 s to 30 min at 700 mosmol/kgH2O, the autofluorescence spreads diffusely throughout the cell. This correlates with mitochondrial membrane depolarization, measured as decreased tetramethylrhodamine methyl ester perchlorate (TMRM) fluorescence. Mitochondrial dysfunction should increase the cellular ADP/ATP ratio. In agreement, this ratio increases within 1–6 h. Mitochondrial morphology (transmission electron microscopy) is unaffected, but nuclear hypercondensation becomes evident. Progressive apoptosis occurs beginning 1 h after osmolality is raised to 700, but not to 500, mosmol/kgH2O. General caspase activity and caspase-9 activity increase only after 6 h at 700 mosmol/kgH2O. The mitochondrial Bcl-2/Bax ratio decreases within 1–3 h, but no cytochrome c release is evident. The mitochondria contain little p53 at any osmolality. Adding urea to 700 mosmol/kgH2O does not change NADH or TMRM fluorescence. We conclude that extreme acute hypertonicity causes a mitochondrial dysfunction involved in the initiation of apoptosis.
PTH increases renal 25(OH)D3-1α-hydroxylase (CYP1α) mRNA but not renal 1,25(OH)2D3production in adult rats
Authors: Armbrecht H. J. , Boltz M. A. , Hodam T. L.
Published: March 2015

The capacity of parathyroid hormone (PTH) to stimulate renal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] production declines with age in the rat. The purpose of these studies was to determine whether this decline is due to a decreased capacity of PTH to increase the mRNA levels of CYP1α, the cytochrome P-450 component of the 25(OH)D3-1α-hydroxylase. Young (2 mo) and adult (12 mo) male Fischer 344 rats were parathyroidectomized (PTX). After 72 h, PTX rats were injected with PTH or vehicle at 24, 6, and 3 h before death, and renal CYP1α mRNA levels were measured by ribonuclease protection assay. In young rats, PTH markedly increased plasma 1,25(OH)2D3 and renal 1,25(OH)2D3 production. However, in adult rats, the response to PTH was less than 30% of that seen in young rats. Renal CYP1α mRNA levels, on the other hand, were increased over fivefold by PTH in both young and adult rats. In in vitro studies, PTH/forskolin increased CYP1α mRNA levels over twofold in renal slices from both young and adult PTX rats. These studies demonstrate that the decreased capacity of PTH to increase 1,25(OH)2D3 production in adult rats is not due to decreased induction of CYP1α mRNA.
Bladder permeability barrier: recovery from selective injury of surface epithelial cells
Authors: Lavelle John , Meyers Susan , Ramage Richard , Bastacky Sheldon , Doty Debra , Apodaca Gerard , Zeidel Mark L.
Published: March 2015

The mammalian bladder maintains high electrochemical gradients between urine and blood, permitting the kidney to modify body chemistries through urinary excretion. To perform this function, the urothelium maintains a tight permeability barrier. When this barrier is damaged, leakage of urine components into the underlying bladder layers results, with symptoms of cystitis. In these studies, we develop a model of selective urothelial injury using protamine sulfate (PS) and define the process by which this epithelium recovers from damage. Exposure to PS (10 mg/ml), but not vehicle, caused a rapid fall in transepithelial resistance as well as striking increases in water and urea permeabilities. These changes were accompanied by necrosis and sloughing of sheets of umbrella cells, as seen by scanning and transmission electron microscopy. Over the 72 h after PS exposure, barrier function recovered, with transepithelial resistance and water and urea permeabilities returning to normal values. After loss of umbrella cells, the underlying intermediate cells underwent rapid maturation, as evidenced by increased expression of uroplakins and gradual formation of well-defined tight junctions. At day 5 after PS exposure, barrier function was restored and the surface cells exhibited normal-appearing tight junctions and normal labeling for uroplakins and zonula occludens 1. However, the cells remained smaller than umbrella cells until day 10 after exposure, when normal size was restored. These studies develop for the first time a controlled model of selective urothelial damage and demonstrate a characteristic process by which barrier function is restored and underlying intermediate cells develop into mature umbrella cells. This model will be useful in defining the mechanisms that regulate repair of urothelial damage.
Immortalized epithelial cells from human autosomal dominant polycystic kidney cysts
Authors: Loghman-Adham Mahmoud , Nauli Surya M. , Soto Carlos E. , Kariuki Barbara , Zhou Jing
Published: March 2015

Autosomal dominant polycystic kidney disease (ADPKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by “second hit” somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with ADPKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37°C in the WT cell lines and at 33°C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39°C. The majority ( 29 ) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD.
Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na+/H+exchanger
Authors: Wu Karen L. , Khan Shenaz , Lakhe-Reddy Sujata , Wang Liming , Jarad George , Miller R. Tyler , Konieczkowski Martha , Brown Arthur M. , Sedor John R. , Schelling Jeffrey R.
Published: March 2015

Renal tubular epithelial cell (RTC) apoptosis causes tubular atrophy, a hallmark of renal disease progression. Apoptosis is generally characterized by reduced cell volume and cytosolic pH, but epithelial cells are relatively resistant to shrinkage due to regulatory volume increase, which is mediated by Na+/H+exchanger (NHE) 1. We investigated whether RTC apoptosis requires caspase cleavage of NHE1. Staurosporine- and hypertonic NaCl-induced RTC apoptosis was associated with cell shrinkage and diminished cytosolic pH, and apoptosis was potentiated by amiloride analogs, suggesting NHE1 activity opposes apoptosis. NHE1-deficient fibroblasts demonstrated increased susceptibility to apoptosis, which was reversed by NHE1 reconstitution. NHE1 expression was markedly decreased in apoptotic RTC due to degradation, and preincubation with peptide caspase antagonists restored NHE1 expression, indicating that NHE1 is degraded by caspases. Recombinant caspase-3 cleaved the in vitro-translated NHE1 cytoplasmic domain into five distinct peptides, identical in molecular weight to NHE1 degradation products derived from staurosporine-stimulated RTC lysates. In vivo, NHE1 loss-of-function C57BL/6.SJL- swe/swemice with adriamycin-induced nephropathy demonstrated increased RTC apoptosis compared with adriamycin-treated wild-type controls, thereby implicating NHE1 inactivation as a potential mechanism of tubular atrophy. We conclude that NHE1 activity is critical for RTC survival after injury and that caspase cleavage of RTC NHE1 may promote apoptosis and tubular atrophy by preventing compensatory intracellular volume and pH regulation.