bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction among nitric oxide, reactive oxygen species, and antioxidants during endotoxemia-related acute renal failure
Authors: Wang Wei , Jittikanont Suparoek , Falk Sandor A. , Li Ping , Feng Lili , Gengaro Patricia E. , Poole Brian D. , Bowler Russell P. , Day Brian J. , Crapo James D. , Schrier Robert W.
Published: March 2015

Acute renal failure (ARF) during sepsis is associated with increased nitric oxide (NO) and oxygen radicals, including superoxide (O[Formula: see text]). Because O[Formula: see text] reacts with NO in a rapid manner, it plays an important role in modulating NO levels. Therefore, scavenging of O[Formula: see text] by superoxide dismutase (SOD) may be critical for preserving NO bioavailability. In mice, substantial renal extracellular SOD (EC-SOD) expression implies its important role in scavenging O[Formula: see text] in the kidney. We hypothesized that during endotoxemic ARF, EC-SOD is decreased in the kidney, resulting in increased O[Formula: see text] and thus decreased vascular NO bioavailability with resultant renal vasoconstriction and ARF. In the present study, normotensive endotoxemic ARF was induced in mice using lipopolysaccharide (LPS; 5 mg/kg ip). Sixteen hours after LPS, glomerular filtration rate (GFR; 50 ± 16 vs. 229 ± 21 μl/min, n = 8, P < 0.01) and renal blood flow (RBF; 0.61 ± 0.10 vs. 0.86 ± 0.05 ml/min, n = 8, P < 0.05) were subsequently decreased. EC-SOD mRNA and protein expression in endotoxemic kidneys were decreased at 16 h compared with controls. A catalytic antioxidant, metalloporphyrin, reversed the deleterious effects of endotoxemia on renal function as GFR (182 ± 40 vs. 50 ± 16 μl/min, n = 6, P < 0.01) and RBF (1.08 ± 0.10 vs. 0.61 ± 0.10 ml/min, n = 6, P < 0.05) were preserved. Similar results were obtained with tempol, a chemically dissimilar antioxidant. Specific inhibition of inducible nitric oxide synthase (iNOS),l- N6-(1-iminoethyl)-lysine, reversed the renal protective effect on GFR and RBF observed with antioxidant treatment during endotoxemia. In summary, renal EC-SOD expression is decreased during endotoxemia. Antioxidant therapy preserved GFR and RBF during endotoxemia. The reversal of this protective effect by inhibition of iNOS suggests the importance of the bioavailability of NO for preservation of renal function during early endotoxemia.
Glucose stimulates O2consumption, NOS, and Na/H exchange in diabetic rat proximal tubules
Authors: Baines Andrew , Ho Patrick
Published: March 2015

Endothelial nitric oxide synthase (NOS) and neuronal NOS protein increased in proximal tubules of acidotic diabetic rats 3–5 wk after streptozotocin injection. NOS activity (citrulline production) was similar in nondiabetic and diabetic tubules incubated with low glucose (5 mM glucose + 20 mM mannitol); but after 30 min with high glucose (25 mM), Ca-sensitive citrulline production had increased 23% in diabetic tubules. Glucose concentration did not influence citrulline production in nondiabetic tubules. High glucose increased carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline 1-oxyl-3-oxide (cpt10)-scavenged NO sevenfold in a suspension of diabetic tubules but did not alter NO in nondiabetic tubules. Diabetes increased ouabain-sensitive86Rb uptake (141 ± 9 vs. 122 ± 6 nmol · min−1· mg−1) and oligomycin-sensitive O2consumption (Q˙o2; 16.0 ± 1.7 vs. 11.3 ± 0.7 nmol · min−1· mg−1). Ethylisopropyl amiloride-inhibitable Q˙o2(6.5 ± 0.6 vs. 2.4 ± 0.3 nmol · min−1· mg−1) accounted for increased oligomycin-sensitiveQ˙o2in diabetic tubules. NG-monomethyl-l-arginine methyl ester (l-NAME) inhibited most of the increase in86Rb uptake and Q˙o2in diabetic tubules. l-NAME had little effect on nondiabetic tubules. Inhibition of Q˙o2by ethylisopropyl amiloride and l-NAME was only 5–8% additive. Uncontrolled diabetes for 3–5 wk increases NOS protein in proximal tubules and makes NOS activity sensitive to glucose concentration. Under these conditions, NO stimulates Na-K-ATPase andQ˙o2in proximal tubules.
Functional analysis and molecular model of the human urate transporter/channel, hUAT
Authors: Leal-Pinto Edgar , Cohen B. Eleazar , Lipkowitz Michael S. , Abramson Ruth G.
Published: March 2015

Recombinant protein, designated hUAT, the human homologue of the rat urate transporter/channel (UAT), functions as a highly selective urate channel in lipid bilayers. Functional analysis indicates that hUAT activity, like UAT, is selectively blocked by oxonate from its cytosolic side, whereas pyrazinoate and adenosine selectively block from the channel's extracellular face. Importantly, hUAT is a galectin, a protein with two β-galactoside binding domains that bind lactose. Lactose significantly increased hUAT open probability but only when added to the channel's extracellular side. This effect on open probability was mimicked by glucose, but not ribose, suggesting a role for extracellular glucose in regulating hUAT channel activity. These functional observations support a four-transmembrane-domain structural model of hUAT, as previously predicted from the primary structure of UAT. hUAT and UAT, however, are not functionally identical: hUAT has a significantly lower single-channel conductance and open probability is voltage independent. These differences suggest that evolutionary changes in specific amino acids in these highly homologous proteins are functionally relevant in defining these biophysical properties.
Angiotensin II AT1 receptor antagonism prevents detrimental renal actions of acute diuretic therapy in human heart failure
Authors: Chen Horng H. , Redfield Margaret M. , Nordstrom Linda J. , Cataliotti Alessandro , Burnett John C.
Published: March 2015

Although effective in relieving symptoms of edema in congestive heart failure (CHF), diuretic-induced natriuresis may be associated with reductions in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), which subsequently may reduce the duration of natriuresis. Moreover, recent studies have reported that the preservation of GFR is an important predictor of survival in human CHF. We hypothesized that the acute detrimental renal hemodynamic and tubular responses to furosemide in symptomatic human CHF will be attenuated by AT1 receptor blockade with losartan. We defined the renal hemodynamic and tubular actions and aldosterone responses to furosemide (40 mg, orally) in the presence of acute AT1 receptor antagonism (losartan, MSD, 50 mg orally) vs. placebo in 10 subjects with CHF (New York Heart Association II-III) in a double-blind, placebo-controlled crossover study. Furosemide with placebo increased sodium excretion and reduced ERPF and GFR ( P < 0.05 vs. baseline). After 4 h, sodium excretion compared with baseline was decreased ( P < 0.05). In contrast, furosemide with losartan resulted in a greater increase in sodium excretion but without reductions in ERPF and GFR ( P < 0.05 vs. placebo). After 4 h, sodium excretion was greater compared with the placebo group. Importantly, plasma aldosterone tended to increase in the placebo group, whereas it was decreased ( P < 0.05 vs. baseline) only in the losartan group. These studies underscore the pathophysiological role of the AT1 receptor in mediating detrimental renal and adrenal properties of diuretics in human CHF. AT1 receptor antagonism preserves GFR and renal blood flow and enhances sodium excretion during acute diuretic therapy in addition to inhibiting aldosterone secretion. These findings support the use of AT1 receptor blockade for human CHF requiring acute diuretics to improve renal hemodynamic and tubular function and to suppress aldosterone.
Calcium-sensing receptor regulation of PTH-dependent calcium absorption by mouse cortical ascending limbs
Authors: Motoyama Hiroki I. , Friedman Peter A.
Published: March 2015

Resting Ca2+absorption by cortical thick ascending limbs (CALs) is passive and proceeds through the paracellular pathway. In contrast, parathyroid hormone (PTH) stimulates active, transcellular Ca2+absorption ( JCa). The Ca2+-sensing receptor (CaSR) is expressed on serosal membranes of CALs. In the present study, we tested the hypothesis that activation of the CAL CaSR indirectly inhibits passive Ca2+transport and directly suppresses PTH-induced cellular JCa. To test this theory, we measured JCaand Na absorption ( JNa) by single perfused mouse CALs. Net absorption was measured microfluorimetrically in samples collected from tubules perfused and bathed in symmetrical HEPES-buffered solutions or those in which luminal Na+was reduced from 150 to 50 mM. We first confirmed that Gd3+activated the CaSR by measuring intracellular Ca2+concentration ([Ca2+]i) in CALs loaded with fura 2. On stepwise addition of Gd3+to the bath, [Ca2+]iincreased, with a half-maximal rise at 30 μM Gd3+. JCaand transepithelial voltage ( Ve,) were measured in symmetrical Na+-containing solutions. PTH increased JCaby 100%, and 30 μM Gd3+inhibited this effect. Vewas unchanged by either PTH or Gd3+. Similarly, NPS R-467, an organic CaSR agonist, inhibited PTH-stimulated JCawithout altering Ve. Neither PTH nor Gd3+affected JNa. Addition of bumetanide to the luminal perfusate abolished JNaand Ve. These results show that CaSR activation directly inhibited PTH-induced transcellular JCaand that cellular Ca2+and Na+transport can be dissociated. To test the effect of CaSR activation on passive paracellular Ca2+transport, JCawas measured under asymmetrical Na conditions, in which passive Ca2+transport dominates transepithelial absorption. PTH stimulated JCaby 24% and was suppressed by Gd3+. In this setting, Gd3+reduced Veby 32%, indicating that CaSR activation inhibited both transcellular and paracellular Ca2+transport. We conclude that the CaSR regulates both active transcellular and passive paracellular Ca2+reabsorption but has no effect on JNaby CALs.
ACE inhibition increases expression of the ETBreceptor in kidneys of mice with unilateral obstruction
Authors: Moridaira Kazuaki , Morrissey Jeremiah , Fitzgerald Melanie , Guo Guangjie , McCracken Ruth , Tolley Timothy , Klahr Saulo
Published: March 2015

Unilateral ureteral obstruction (UUO) is a well-established model for the study of interstitial fibrosis in the kidney. It has been shown that the renin-angiotensin system plays a central role in the progression of interstitial fibrosis. Recent studies indicate that endothelin, a powerful vasoconstrictive peptide, may play an important role in some types of renal disease. To investigate the effects of angiotensin II on endothelin and its receptors in the kidney, mice were subjected to UUO and treated with or without enalapril, an orally active angiotensin-converting enzyme inhibitor, in their drinking water (100 mg/l). The animals were killed 5 days later. Using RT coupled with PCR, we measured the levels of endothelin-1, endothelin A, and endothelin B (ETB) along with transforming growth factor-β, TNF-α, and collagen type IV mRNA expression in the kidney with UUO and the contralateral kidney along with interstitial expansion in the kidney cortex by a standard point counting method. We found that enalapril administration ameliorated the increased expression of ET-1 mRNA in the obstructed kidney by 44% ( P < 0.02). Although the level of endothelin A mRNA expression was significantly increased in the obstructed kidney, it was not affected by enalapril. We found that enalapril treatment increased ETBmRNA expression by 115% ( P < 0.05) and protein expression (measured by Western blot) in the kidney with an obstructed ureter. Enalapril treatment alone inhibited the expansion of interstitial volume due to UUO by 52%. Cotreatment with enalapril and the ETBreceptor antagonist BQ-788 inhibited the expression of interstitial volume by only 19%. This study confirms that enalapril inhibits the interstitial fibrosis in UUO kidneys. It also suggests a beneficial and unforeseen effect of enalapril on the obstructed kidney by potentially stimulating the production of nitric oxide through an increased expression of the ETBreceptor.
Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus
Authors: Peti-Peterdi János , Morishima Shigeru , Bell P. Darwin , Okada Yasunobu
Published: March 2015

Recently, multiphoton excitation fluorescence microscopy has been developed that offers important advantages over confocal imaging, particularly for in vivo visualization of thick tissue samples. We used this state-of-the-art technique to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney. This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal hemodynamics. We report, for the first time, on high-resolution three-dimensional morphology and Z-sectioning through isolated, perfused kidney glomeruli, tubules, and JGA. Time-series images show how alterations in tubular fluid composition cause striking changes in single-cell volume of the unique macula densa tubular epithelium in situ and how they also affect glomerular filtration through alterations in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. This imaging approach to the study of isolated, perfused live tissue with multiphoton microscopy may be applied to other biological systems in which multiple cell types form a functionally integrated syncytium.
Characterization of a type IIb sodium-phosphate cotransporter from zebrafish(Danio rerio)kidney
Authors: Graham C. , Nalbant P. , Schölermann B. , Hentschel H. , Kinne R. K. H. , Werner A.
Published: March 2015

Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent Picotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3′-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5′ end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number AW423104 ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.
Upregulation of renal BSC1 and TSC in prenatally programmed hypertension
Authors: Manning Jennifer , Beutler Kathleen , Knepper Mark A. , Vehaskari V. Matti
Published: March 2015

Prenatal factors, especially intrauterine growth retardation, have been shown to correlate with the risk of essential hypertension in adult life, but the mechanisms are unknown. An experimental model of prenatal programming of hypertension in the rat, induced by a maternal low-protein diet during pregnancy, was employed to study the role of renal Na reabsorption in the pathogenesis. The abundance of the apical Na transporter type III Na/H exchanger (NHE3), bumetanide-sensitive Na-K-2Cl cotransporter (BSC1), thiazide-sensitive Na-Cl cotransporter (TSC), and the amiloride-sensitive epithelial Na channel (ENaC) was determined by semiquantitative immunoblotting in kidneys from the offspring at 4 wk of age, before hypertension became manifest. There were no significant differences between the experimental and control rats in the abundance of NHE3 or any of the ENaC subunits. In contrast, the quantity of BSC1 in the experimental group was increased to 302% of control ( P < 0.001) and that of TSC to 157% of control ( P < 0.05). Determination of specific mRNA levels by ELISA-linked RT-PCR revealed a significantly increased BSC1 mRNA at 1 day ( P < 0.01), 4 wk ( P < 0.01), and 8 wk ( P < 0.001) of age, and a significantly increased TSC mRNA at 4 wk of age ( P < 0.05) in the experimental group. The results suggest that prenatal programming of hypertension involves transcriptional upregulation of Na transport in thick ascending limb and distal convoluted tubule.
UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney
Authors: Trinh-Trang-Tan M.-M. , Lasbennes F. , Gane P . , Roudier N. , Ripoche P. , Cartron J.-P. , Bailly P.
Published: March 2015

UT-B1 is the facilitated urea transporter of red blood cells (RBCs) and endothelial cells of descending vasa recta in the kidney. Immunoblotting with a polyclonal antibody against the C-ter sequence of rat UT-B1 revealed UT-B1 as both nonglycosylated (29 kDa) and N-glycosylated (47.5 and 33 kDa) proteins in RBC membranes, kidney medulla, brain, and bladder in rat. In testis, UT-B1 was expressed only as a nonglycosylated protein of 47.5 kDa. Immunocytochemistry confirmed that the location of UT-B1 is restricted to descending vasa recta. In brain, UT-B1 protein was found in astrocytes and ependymal cells. Cell bodies and perivascular end feet of astrocytes were labeled in brain cortex, whereas astrocyte cell processes were labeled in corpus callosum. Flow cytometry analysis of RBCs revealed a good cross-reactivity of the antibody with mouse and human UT-B1. UT-B1 protein expression in rat kidney medulla was downregulated greatly by long-term [deamino-Cys1,d-Arg8]vasopressin infusion and moderately by furosemide treatment. This study discloses an uneven distribution of UT-B1 protein within astrocytes and the regulation of renal UT-B1 protein by antidiuretic hormone.
Obstructive nephropathy and renal fibrosis
Authors: Klahr Saulo , Morrissey Jeremiah
Published: March 2015

Interstitial fibrosis has a major role in the progression of renal diseases. Several animal models are available for the study of renal fibrosis. The models of aminonucleoside-induced nephrotic syndrome, cyclosporin nephrotoxicity, and passive Heyman nephritis are characterized by molecular and cellular events similar to those that occur in obstructive nephropathy. Additionally, inhibition of angiotensin-converting enzyme exerts salutary effects on the progression of renal fibrosis in obstructive nephropathy. Unilateral ureteral obstruction (UUO) has emerged as an important model for the study of the mechanisms of renal fibrosis and also for the evaluation of the impact of potential therapeutic approaches to ameliorate renal disease. Many quantifiable pathophysiological events occur over the span of 1 wk of UUO, making this an attractive model for study. This paper reviews some of the ongoing studies that utilized a rodent model of UUO. Some of the findings of the animal model have been compared with observations made in patients with obstructive nephropathy. Most of the evidence suggests that the rodent model of UUO is reflective of human renal disease processes.
Insulin activates Na+/H+exchanger 3: biphasic response and glucocorticoid dependence
Authors: Klisic Jelena , Hu Ming Chang , Nief Vera , Reyes Livia , Fuster Daniel , Moe Orson W. , Ambühl Patrice M.
Published: March 2015

Insulin is an important regulator of renal salt and water excretion, and hyperinsulinemia has been implicated to play a role in hypertension. One of the target proteins of insulin action in the kidney is Na+/H+exchanger 3 (NHE3), a principal Na+transporter responsible for salt absorption in the mammalian proximal tubule. The molecular mechanisms involved in activation of NHE3 by insulin have not been studied so far. In opossum kidney (OK) cells, insulin increased Na+/H+exchange activity in a time- and concentration-dependent manner. This effect is due to activation of NHE3 as it persisted after pharmacological inhibition of NHE1 and NHE2. In the early phase of stimulation (2–12 h), NHE3 activity was increased without changes in NHE3 protein and mRNA. At 24 h, enhanced NHE3 activity was accompanied by an increase in total and cell surface NHE3 protein and NHE3 mRNA abundance. All the effects of insulin on NHE3 activity, protein, and mRNA were amplified in the presence of hydrocortisone. These results suggest that insulin stimulates renal tubular NHE3 activity via a biphasic mechanism involving posttranslational factors and an increase in NHE3 gene expression and the effects are dependent on the permissive action of hydrocortisone.
CNP production in the kidney and effects of protein intake restriction in nephrotic syndrome
Authors: Cataliotti Alessandro , Giordano Mauro , De Pascale Emanuela , Giordano Gelsomina , Castellino Pietro , Jougasaki Michihisa , Costello Lisa C. , Boerrigter Guido , Tsuruda Toshihiro , Belluardo Paola , Lee Shang-Chiun , Huntley Brenda , Sandberg Sharon , Malatino Lorenzo S. , Burnett John C.
Published: March 2015

C-type natriuretic peptide (CNP) possesses well-established cardiovascular properties. Although present in the mammalian kidney, CNP production in human kidney and its modulation in human renal disease remain less defined. We investigated the presence of CNP in normal human kidney and in patients with nephrotic syndrome (NS). We also addressed whether or not a low-protein diet (LPD) alters plasma CNP and urinary CNP excretion in NS. In situ hybridization studies demonstrated CNP mRNA expression in tubular cells and glomeruli of normal human kidneys. CNP immunoreactivity was positive in proximal, distal, and medullary collecting duct tubular cells in both controls and patients with NS. The ratios of plasma CNP and urinary CNP to creatinine were significantly higher in patients with NS compared with controls. Urinary CNP, but not plasma CNP, was significantly lowered in patients with NS after an LPD. Similarly, the ratios of urinary protein to creatinine and urinary albumin to creatinine, but not urinary guanosine 3′,5′-cyclic monophosphate to creatinine, decreased significantly with an LPD. These data confirm and extend previous reports and demonstrate for the first time the presence of CNP in human kidney with NS. We also report increased plasma CNP concentration and urinary CNP excretion in NS patients and a significant reduction of CNP excretion with an LPD. Our findings demonstrate that CNP metabolism is altered in patients with NS and support the hypothesis that activation of renal CNP can be partially offset by an LPD. These results underscore that the beneficial effect of an LPD on protein excretion is paralleled by a substantial reduction in intrarenal CNP release.
Glutamatergic input in the PVN is important in renal nerve response to elevations in osmolality
Authors: Badoer Emilio , Ng Chi-Wai , De Matteo Robert
Published: March 2015

Elevations in plasma osmolality elicit reflex humoral and neural responses. The hypothalamic paraventricular nucleus (PVN) is important in humoral responses. We have investigated whether the PVN contributed to the renal nerve reduction that is normally elicited by increased plasma osmolality in the conscious rabbit. Renal sympathetic nerve activity (RSNA) was monitored after an intravenous infusion of hypertonic saline (1.7 M NaCl, 2 ml/min for 7 min). The responses were examined in animals microinjected with muscimol (10 nmol) into, and outside, the PVN to acutely inhibit neuronal function or with kynurenate (25 nmol) to block glutamate receptors. Compared with vehicle, the maximum reduction in RSNA elicited by hypertonic saline was significantly less with muscimol or kynurenate pretreatment into the PVN. A similar study with kynurenate was also performed in sinoaortically denervated rabbits, and similar effects were observed. The effect was specific to the PVN because microinjections of the drugs outside the PVN had no effect on the response. The findings suggest that excitatory inputs into the PVN may be important in the neural responses elicited by elevations in plasma osmolality.
Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+gradient
Authors: Sweet Douglas H. , Chan Lauretta M. S. , Walden Ramsey , Yang Xiao-Ping , Miller David S. , Pritchard John B.
Published: March 2015

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na+gradient through Na+-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na+-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li+or methylsuccinate (MS) or when Na+was absent. All these treatments inhibit the Na+-dicarboxylate cotransporter, but not rat Oat3. Li+, MS, and Na+removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na+sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li+, MS, taurocholate, and removal of Na+. Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na+gradient.
TGF-β1-mediated alterations of renal proximal tubular epithelial cell phenotype
Authors: Tian Ya-Chung , Fraser Donald , Attisano Liliana , Phillips Aled O.
Published: March 2015

The aim of this study was to characterize the mechanism of transforming growth factor (TGF)-β1-mediated alteration of renal proximal tubular cell phenotype. TGF-β1altered cell phenotype, with cells appearing elongated and spindle shaped. This was associated with loss of cell-cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibers, and focal adhesions. Addition of the tyrosine phosphatase inhibitor sodium orthovanadate also led to rapid but transient loss of cell-cell contact, but it did not lead to a change of phenotype comparable to that seen following addition of TGF-β1. There was, however, no change in the formation of focal adhesions and no associated reorganization of the Factin cytoskeleton. Disruption of the actin cytoskeleton with cytochalasin D prevented phenotypic alterations following addition of TGF-β1. Transient transfection with Smad2/4 or Smad3/4 expression vectors did not alter cell phenotype. Previously, we demonstrated β-catenin translocation to proximal tubule cell nuclei and its association with Smad proteins following addition of TGF-β1, suggesting the possibility that TGF-β1may modulate Wnt signaling. The Wnt-responsive Xtwn-reporter construct was, however, silent in response to TGF-β1. Similarly, a second Wnt/LEF-1-regulated element, Toplflash, which does not contain Smad binding sites, was insensitive to TGF-β1signaling. In contrast, phenotypic changes in response to TGF-β1were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell-cell contact and adherens junction disassembly.
Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney
Authors: Helou Claudia M. B. , Imbert-Teboul Martine , Doucet Alain , Rajerison Rabary , Chollet Catherine , Alhenc-Gelas François , Marchetti Jeannine
Published: March 2015

ANG II controls the vascular tone of pre- and postglomerular arterioles, and thereby glomerular filtration, through binding to either AT1A, AT1B, or AT2receptors. AT1receptors, which are coupled to intracellular Ca2+signaling, have vasoconstricting effects, whereas AT2receptors, whose signaling mechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types of receptors, but not in efferent arterioles. Two subpopulations of juxtamedullary efferent arterioles, muscular ones which terminate as vasa rectae and thin ones which terminate as peritubular capillaries, have been described. They display functional heterogeneity with regard to the ANG II response. To evaluate whether these differences are associated with differential expression of ANG II receptors, we examined the expression pattern of AT1A, AT1B, and AT2receptor mRNAs by RT-PCR in these arterioles and studied the effect of valsartan, a specific AT1-receptor antagonist. Results indicate that muscular arterioles express AT1A, AT1B, and AT2receptors, whereas thin arterioles only express the AT1Aand AT2types, and at a much lower level. Valsartan fully inhibited ANG II-induced increases in intracellular Ca2+in both arteriolar types, but with different kinetics. In muscular arterioles, inhibition was monoexponential, whereas it displayed a marked positive cooperativity in thin arterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documents the differences between muscular and thin efferent arterioles with regard to ANG II signalization in the rat kidney.
Immunolocalization of a microsomal prostaglandin E synthase in rabbit kidney
Authors: Fuson Amanda L. , Komlosi Peter , Unlap Tino M. , Bell P. Darwin , Peti-Peterdi János
Published: March 2015

PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2synthase (mPGES), the terminal enzyme of PGE2synthesis, which converts COX-derived PGH2to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2in descending thin limb and collecting tubule and a paracrine effect of PGE2on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.
Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats
Authors: Kim Dongun , Sands Jeff M. , Klein Janet D.
Published: March 2015

We tested whether the abundance of transport proteins involved in the urinary concentrating mechanism was altered in rats with uncontrolled diabetes mellitus (DM). Rats were injected with streptozotocin and killed 5, 10, 14, or 20 days later. Blood glucose in DM rats was 300–450 mg/dl (control: 70–130 mg/dl). Urine volume increased in DM rats from 41 ± 7 ml/100 g body wt (BW) at 5 days to 69 ± 3 ml/100 g BW at 20 days (control: 9 ± 1). Urine osmolality of DM rats decreased at 5 days DM and remained low at 20 days. UT-A1 urea transporter protein in the inner medullary (IM) tip was 55% of control in 5-day DM rats but increased to 170, 220, and 280% at 10, 14, and 20 days DM, respectively, due to an increase in the 117-kDa glycoprotein form. UT-A1 in the IM base was increased to 325% of control at 5 days DM with no further increase at 20 days. Aquaporin-2 (AQP2) increased to 290% in the IM base at 5 days DM and 150% in the IM tip at 10 days; both showed no further increase at 20 days. NKCC2/BSC1 increased to 240% in outer medulla at 20 days DM, but not at 5 or 10 days. UT-B and ROMK were unchanged at any time point. The increases in UT-A1, AQP2, and NKCC2/BSC1 proteins during uncontrolled DM would tend to limit the loss of fluid and solute during uncontrolled diabetes.
Role of Na-K-ATPase in the assembly of tight junctions
Authors: Rajasekaran Ayyappan K. , Rajasekaran Sigrid A.
Published: March 2015

Na-K-ATPase, also known as the sodium pump, is a crucial enzyme that regulates intracellular sodium homeostasis in mammalian cells. In epithelial cells Na-K-ATPase function is also involved in the formation of tight junctions through RhoA GTPase and stress fibers. In this review, a new two-step model for the assembly of tight junctions is proposed: step 1, an E-cadherin-dependent formation of partial tight junction strands and of the circumferential actin ring; and step 2, active actin polymerization-dependent tethering of tight junction strands to form functional tight junctions, an event requiring normal function of Na-K-ATPase in epithelial cells. A new role for stress fibers in the assembly of tight junctions is proposed. Also, implications of Na-K-ATPase function on tight junction assembly in diseases such as cancer, ischemia, hypomagnesemia, and polycystic kidney disease are discussed.
Mesangial cell filamentous actin disassembly and hypocontractility in high glucose are mediated by PKC-ζ
Authors: Dlugosz John A. , Munk Snezana , Ispanovic Eric , Goldberg Howard J. , Whiteside Catharine I.
Published: March 2015

In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (MLC20) phosphorylation, or Ca2+signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 ± 135 vs. 3,952 ± 225 (SE) μm2in NG, P < 0.01, n = 31] with no contractile response to 0.1 μM ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced Ca2+signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC20showed that HG increased mono- and decreased unphosphorylated MLC20(42 ± 16 and 49 ± 15 vs. 13 ± 3 and 80 ± 4% of total in NG, P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-δ and -ε from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-ζ translocation. In HG, membrane accumulation of PKC-ζ was observed. Membrane PKC-ζ activity measured by immunoprecipitation and32P phosphorylation of PKC-ε pseudosubstrate peptide was 190 ± 18% of NG ( P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal Ca2+signaling and normal MLC20phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-ζ.
NO decreases thick ascending limb chloride absorption by reducing Na+-K+-2Cl−cotransporter activity
Authors: Ortiz Pablo A. , Hong Nancy J. , Garvin Jeffrey L.
Published: March 2015

First published August 9, 2001; 10.1152/ajprenal.0075.2001.—We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption ( JCl−). NaCl transport in the THAL depends on apical Na+-K+-2Cl−cotransporters, apical K+channels, and basolateral Na+-K+-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL JCl−by inhibiting the Na+-K+-2Cl−cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na+]i) and chloride concentrations ([Cl−]i) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na+]ifrom 13.5 ± 1.2 to 9.6 ± 1.6 mM ( P < 0.05) and also decreased [Cl−]i( P < 0.01). We next tested whether NO decreases Na+-K+-2Cl−cotransporter activity by measuring the initial rate of Na+transport. In the presence of SPM in the bath, initial rates of Na+entry were 49.6 ± 6.0% slower compared with control rates ( P < 0.05). To determine whether NO inhibits apical K+channel activity, we measured the change in membrane potential caused by an increase in luminal K+from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K+concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K+permeability could affect NO-induced inhibition of THAL JCl−. In the presence of luminal valinomycin, which increases K+permeability, addition of SPM decreased THAL JCl−by 41.2 ± 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na+-K+-ATPase by measuring oxygen consumption rate (Qo2) in THAL suspensions in the presence of nystatin in varying concentrations of Na+. In the presence of 10.5 mM Na+, nystatin increased Qo2to 119.1 ± 19.2 and 125.6 ± 23.4 nmol O2· mg protein−1· min−1in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na+, nystatin increased Qo2by 104 ± 7 and 94 ± 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL JCl−by inhibiting Na+-K+-2Cl−cotransport rather than inhibiting apical K+channels or the sodium pump.
Endothelin resets renal blood flow autoregulatory efficiency during acute blockade of NO in the rat
Authors: Kramp R. , Fourmanoir P. , Caron N.
Published: March 2015

First published August 15, 2001; 10.1152/ajprenal.00078.2001.—Renal blood flow (RBF) autoregulatory efficiency may be enhanced during NO inhibition in the rat, as recently reported. Under these conditions, endothelin (ET) synthesis and release may be increased. Our purpose was therefore to determine the role of ET in RBF autoregulatory changes induced by NO inhibition. To address this point, ETA/Breceptors were blocked in anesthetized rats with bosentan, or selectively with BQ-610 or BQ-788. NO synthesis was inhibited with NG-nitro-l-arginine methyl ester (l-NAME). Mean arterial pressure (MAP) was decreased after bosentan (−10 mmHg; P < 0.01) or increased after l-NAME (25 mmHg; P < 0.001). RBF measured with an electromagnetic flow probe was reduced byl-NAME (−50%) and by BQ-788 (−24%). The pressure limits of the autoregulatory plateau (PA∼100 mmHg) and of no RBF autoregulation (Po∼80 mmHg) were significantly lowered by 15 mmHg after l-NAME but were unchanged after bosentan, BQ-610, or BQ-788. During NO inhibition, autoregulatory resetting was completely hindered by bosentan (PA∼100 mmHg) and by ETBreceptor blockade with BQ-788 (PA∼106 mmHg), but not by ETAreceptor blockade with BQ-610 (PA∼85 mmHg). These results suggest that the involvement of ET in the RBF autoregulatory resetting occurs during NO inhibition, possibly by preferential activation of the ETBreceptor. However, the relative contribution of ET receptor subtypes remains to be further specified.
Adenosine A2Aand A2Breceptor activation of erythropoietin production
Authors: Fisher James W. , Brookins Jesse
Published: March 2015

First published July 12, 2001; 10.1152/ajprenal.0083.2001.—We have examined the effects of adenosine receptors and protein kinases A and C in the regulation of erythropoietin (Epo) production using hepatocellular carcinoma (Hep3B) cells in culture and in vivo in normal mice under normoxic and hypoxic conditions. CGS-21680, a selective adenosine A2Aagonist, significantly increased levels of Epo in normoxic Hep3B cell cultures and in serum of normal mice under both normoxic and hypoxic conditions. CGS-21680 also produced a significant increase in Epo mRNA levels in Hep3B cell cultures. SCH-58261, a selective adenosine A2Areceptor antagonist, significantly inhibited the increase in medium levels of Epo in Hep3B cell cultures exposed to hypoxia (1% O2). Enprofylline, a selective adenosine A2Breceptor antagonist, significantly inhibited the increase in plasma levels of Epo in normal mice exposed to hypoxia. Chelerythrine chloride, an antagonist of protein kinase C activation, significantly inhibited hypoxia-induced increases in serum levels of Epo in normal mice. A model is presented for adenosine in hypoxic regulation of Epo production that involves kinases A and C and phospholipase A2pathways.
Renal baroreceptor-stimulated renin in the eNOS knockout mouse
Authors: Beierwaltes William H. , Potter D'Anna L. , Shesely Edward G.
Published: March 2015

The role of endothelium-derived nitric oxide (NO) in renal baroreceptor stimulation of renin was tested comparing endothelial nitric oxide synthase (eNOS)-deficient mice with C57BL/6J (C57) controls. We measured blood pressure, renal blood flow (RBF), and plasma renin concentration (PRC) in Inactin-anesthetized mice. Blood pressure in eNOS knockout mice was higher than in controls (100 ± 3 vs. 86 ± 1 mmHg, respectively; P < 0.001), but RBF was similar (1.71 ± 0.06 vs. 1.66 ± 0.09 ml · min−1· 100 mg kidney wt−1, respectively), so that renal vascular resistance was also higher in the knockouts (59.81 ± 2.07 vs. 51.81 ± 2.66 resistance units, respectively; P < 0.025). PRC was similar (8.24 ± 1.57 in eNOS knockouts vs. 7.10 ± 1.19 ng ANG I · ml−1· h−1in C57). NOS inhibition with nitro-l-arginine methyl ester (l-NAME) in C57 controls increased blood pressure (from 85 ± 2 to 106 ± 1 mmHg, P < 0.001) and decreased RBF (from 1.66 ± 0.09 to 1.08 ± 0.02; P< 0.005), but l-NAME had no effect in eNOS knockout mice. When renal perfusion pressure was reduced in C57 controls to 55 mmHg, PRC increased from 6.6 ± 0.9 to 14.5 ± 1.9 μg · ml−1· h−1( P< 0.025), but this response was blocked by l-NAME. However, in eNOS knockouts, reduced renal perfusion pressure increased PRC from 7.6 ± 1.4 to 15.0 ± 2.8 μg · ml−1· h−1( P< 0.001). Thus in the chronic absence of eNOS, blood pressure was elevated, but RBF was normal. Additionally, the absence of eNOS did not modify baroreceptor-stimulated renin, suggesting that eNOS-derived NO does not directly mediate this renin-regulating pathway.
Localization of thiazide-sensitive Na+-Cl−cotransport and associated gene products in mouse DCT
Authors: Câmpean Valentina , Kricke Jörn , Ellison David , Luft Friedrich C. , Bachmann Sebastian
Published: March 2015

First published August 8, 2001; 10.1152/ajprenal.00148.2001.—The mammalian distal nephron develops a complex assembly of specialized cell types to accomplish the fine adjustment of urinary electrolyte composition. The epithelia of the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD) show an axial structural heterogeneity that has been functionally elucidated by the localization of proteins involved in transepithelial ion transport. We compared the distribution of the thiazide-sensitive Na+-Cl−cotransporter (TSC), basolateral Na+/Ca2+exchanger (Na/Ca), cytosolic calcium-binding proteins calbindin D28Kand parvalbumin, and the key enzyme for selective aldosterone actions, 11β-hydroxysteroid-dehydrogenase 2 (11HSD2), in the distal convolutions of the mouse. In the mouse, as opposed to the rat, we found no clear subsegmentation of the DCT into a proximal (DCT1) and a distal (DCT2) portion. The TSC was expressed along the entire DCT. Na/Ca and calbindin D28Kwere similarly expressed along most of the DCT, with minor exceptions in the initial portion of the DCT. Both were also present in the CNT. Parvalbumin was found in the entire DCT, with an occasional absence from short end portions of the DCT, and was not present in CNT. 11HSD2 was predominantly located in the CNT and CCD. Short end portions of DCT only occasionally showed the 11HSD2 signal. We also observed an overlap of 11HSD2 immunoreactivity and mRNA staining. Our observations will have implications in understanding the physiological effects of gene disruption and targeting experiments in the mouse.
Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways
Authors: Elkjær Marie-Louise , Nejsum Lene N. , Gresz Veronika , Kwon Tae-Hwan , Jensen Uffe B. , Frøkiær Jørgen , Nielsen Søren
Published: March 2015

First published August 8, 2001; 10.1152/ajprenal.00158.2001.—The purpose of this study was to determine the cellular and subcellular localization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR analyses and by immunoblotting and immunohistochemistry using peptide-derived rabbit antibodies to rat AQP8. RT-PCR and Southern blotting revealed the presence of AQP8 mRNA in all kidney zones. LLC-PK1cells transfected with a rat AQP8 construct exhibited strong labeling with the affinity-purified antibodies, whereas controls using cells transfected with the vector, but without the insert, were negative. The labeling was almost exclusively associated with intracellular vesicles. Immunoblotting of kidney membrane fractions revealed a predominant single band of 26–28 kDa. AQP8 immunoreactivity was mainly present in the cortex and outer stripe of the outer medulla. Sequential ultracentrifugation of rat kidney membrane revealed that AQP8 resides predominantly in intracellular vesicular fractions. Immunocytochemistry revealed modest labeling of proximal tubules and weak labeling of collecting ducts in cortex and medulla of rat kidney. The labeling was confined to cytoplasmic areas with no labeling of the brush border. Immunoblotting and RT-PCR/Southern blotting also revealed the presence of AQP8 protein and mRNA in rat liver, testis, epididymis, duodenum, jejunum, colon, and bronchi/trachea. Consistent with this, immunohistochemistry revealed AQP8 labeling in the hepatocytes and spematogenic cells in testis and in the basal cells in ductus epididymis, trachea, and bronchial epithelia. Moreover, AQP8 labeling was observed in the myoepithelial cells in salivary, bronchial, and tracheal glands with no labeling of acini or ductal epithelial cells. AQP8 is also present in the surface epithelial cells in duodenum, jejunum, and colon. In conclusion, AQP8 is expressed at low levels in rat kidney proximal tubules and collecting ducts, and it is present in distinct cell types in liver, testis, epididymis, duodenum, jejunum, colon, trachea, and principal bronchi as well as in multiple glands, including salivary glands.