bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of epoxyeicosatrienoic acids in renal functional response to inhibition of NO production in the rat
Authors: Ogungbade G. O. , Akinsanmi L. A. , Jiang H. , Oyekan A. O.
Published: March 2015

Nitric oxide (NO) inhibits hemoproteins, including cytochrome (CYP) 2C, the gene responsible for the production of epoxyeicosatrienoic acids (EETs). EETs and NO are produced in the kidney, and both regulate renal vascular tone and Na+transport. However, the role of EETs in NO-mediated renal function is not known. This study tested the hypothesis that NO tonically regulates the renal production of EETs, thereby impacting renal vasomotor tone and electrolyte balance. LPS (10 mg/kg iv) inhibited microsomal conversion of14C-labeled arachidonic acid to EETs and reduced mean arterial blood pressure (MABP; Δ = 63 ± 5 mmHg). Nitro-l-arginine methyl ester (l-NAME, 10 mg/kg), an inhibitor of NO synthase, increased MABP (Δ = 26 ± 6 mmHg), reduced cortical (CBF) and medullary (MBF) blood flow (Δ = -0.86 ± 0.15 and -0.34 ± 0.09 V, respectively) and glomerular filtration rate (GFR; from 0.82 ± 0.16 to 0.32 ± 0.10 ml·g kidney-1·min-1), and increased Na+excretion (UNaV, from 0.16 ± 0.04 to 0.30 ± 0.06 μmol·g kidney-1·min-1). 2-(2-Propynyloxy)-benzenehexanoic acid (PPOH), a suicide substrate inhibitor of EET production, did not affect the l-NAME-induced increase in MABP but attenuated the effects of l-NAME on CBF (31 ± 7%, P < 0.05%), GFR (44 ± 6%, P < 0.05), and UNaV (78 ± 7%, P < 0.05). Miconazole (1.3 mg·kg-1·h-1), a heme inhibitor of epoxygenase enzymes, produced effects similar to those of PPOH. Renal intraarterial infusion of 5,6-, 8,9-, 11,12-, and 14,15-EET (1-10 ng/min) elicited dose-dependent reductions in CBF and GFR accompanied by regioisomeric changes in MBF, UNaV, and urine flow rate. In addition, 11,12-EET dose dependently restored the PPOH blunting the effects of l-NAME on CBF, MBF, and GFR. We conclude that NO tonically regulates epoxygenase activity and that EETs are renal vaosoconstrictors in vivo and contribute, at least in part, to the renal functional responses following inhibition of NO production.
p38 Mitogen-activated protein kinase protects glomerular epithelial cells from complement-mediated cell injury
Authors: Aoudjit Lamine , Stanciu Monica , Li Hongping , Lemay Serge , Takano Tomoko
Published: March 2015

In the passive Heymann nephritis (PHN) model of rat membranous nephropathy, complement C5b-9 causes sublytic injury of glomerular epithelial cells (GEC). We previously showed that sublytic concentration of C5b-9 triggers a variety of biological events in GEC. In the current study, we demonstrate that complement activates p38 MAPK in GEC and address the role of p38 in complement-mediated cell injury. When cultured rat GEC were stimulated with complement, p38 kinase activity and phosphorylation were increased by ∼2.4-fold, compared with control. Treatment with p38 inhibitors significantly augmented complement-mediated cytotoxicity. In contrast, when the constitutively active mutant of transforming growth factor-β-activated kinase 1 (TAK1), a kinase upstream of p38, was expressed in GEC in an inducible manner, cytotoxicity was significantly reduced, compared with uninduced cells. p38 inhibitors abolished the protective effect of TAK1 expression. By analogy to cultured cells, p38 activity was also increased in glomeruli from rats with PHN and treatment with the p38 inhibitor FR-167653 increased proteinuria. Complement induced phosphorylation of MAPK-associated protein kinase-2 (MAPKAPK-2), a kinase downstream of p38 in GEC. Heat shock protein (HSP27) is a cytoskeleton-interacting substrate of MAPKAPK-2. Overexpression of the wild-type HSP27, but not a non-phosphorylatable mutant, markedly reduced complement-mediated GEC injury. In summary, complement activates p38 MAPK in GEC in vitro and in glomeruli from rats with PHN. The activation of p38 MAPK appears to be cytoprotective for GEC against complement-mediated GEC injury. Phosphorylation of HSP27 may mediate this cytoprotection.
ATP-sensitive K+channels in renal mitochondria
Authors: Cancherini Douglas V. , Trabuco Leonardo G. , Rebouças Nancy A. , Kowaltowski Alicia J.
Published: March 2015

Isolated kidney mitochondria swell when incubated in hyposmotic solutions containing K+salts in a manner inhibited by ATP, ADP, 5-hydroxydecanoate, and glibenclamide and stimulated by GTP and diazoxide. These results suggest the existence of ATP-sensitive K+channels in these mitochondria, similar to those previously described in heart, liver, and brain. Renal mitochondrial ATP-sensitive K+uptake rates are ∼140 nmol·min–1·mg protein–1. This K+transport results in a slight increase in respiration and decrease in the inner membrane potential. In addition, the activation of ATP-inhibited K+uptake using diazoxide leads to a decrease of ATP hydrolysis through the reverse activity of the F0F1ATP synthase when respiration is inhibited. In conclusion, we characterize an ATP-sensitive K+transport pathway in kidney mitochondria that affects volume, respiration, and membrane potential and may have a role in the prevention of mitochondrial ATP hydrolysis.
Calcium-sensing receptor-mediated TNF production in medullary thick ascending limb cells
Authors: Wang Dairong , Pedraza Paulina L. , Abdullah Huda Ismail , McGiff John C. , Ferreri Nicholas R.
Published: March 2015

Medullary thick ascending limb (mTAL) cells in primary culture express the Ca2+-sensing receptor (CaR), a G protein-coupled receptor that senses changes in extracellular Ca2+(Ca[Formula: see text]) concentration, resulting in increases of intracellular Ca2+concentration and PKC activity. Exposure of mTAL cells to either Ca[Formula: see text] or the CaR-selective agonist poly-l-arginine increased TNF-α synthesis. Moreover, the response to Ca[Formula: see text] was enhanced in mTAL cells transfected with a CaR overexpression vector. Transfection of mTAL cells with a TNF promoter construct revealed an increase in reporter gene activity after exposure of the cells to Ca[Formula: see text], suggesting that intracellular signaling pathways initiated by means of activation of a CaR contribute to TNF synthesis by a mechanism that involves transcription of the TNF gene. Neutralization of TNF activity with an anti-TNF antibody attenuated Ca2+-mediated increases in cyclooxygenase-2 (COX-2) protein expression and PGE2synthesis, suggesting that TNF exerts an autocrine effect in the mTAL, which contributes to COX-2-mediated PGE2production. Preincubation with the PKC inhibitor bisindolylmaleimide I inhibited Ca2+-mediated TNF production. Significant inhibition of COX-2 protein expression and PGE2synthesis also was observed when cells were challenged with Ca[Formula: see text] in the presence of bisindolylmaleimide I. The data suggest that increases in TNF production subsequent to activation of the CaR may be the basis of an important renal mechanism that regulates salt and water excretion.
Substrate-dependent regulation of MAO-A in rat mesangial cells: involvement of dopamine D2-like receptors
Authors: Pizzinat Nathalie , Marchal-Victorion Sophie , Maurel Agnes , Ordener Catherine , Bompart Guy , Parini Angelo
Published: March 2015

In the present study, we investigated the existence of a back-regulation of the catecholamine-degrading enzyme monoamine oxidase (MAO)-A by dopamine in rat renal cells. In proximal tubule cells, MAO-A expression was not modified after dopamine receptor stimulation. In contrast, in mesangial cells, enzyme assay and Western blots showed that MAO activity and protein increased by ∼80% after 48-h incubation with the D2-like receptor agonist bromocriptine and quinpirole but not with the D1-like receptor agonist SKF-38393. This effect was prevented by the D2-receptor antagonist sulpiride and domperidone. The increase in MAO-A protein was preceded by an augmentation of MAO-A mRNA that was prevented by the transcriptional inhibitor actinomycin D. Bromocriptine effect was mimicked by the PKA inhibitor H89 and inhibited by the PKA activator 8-bromo-cAMP. These results show for the first time the existence of a dopamine-dependent MAO-A regulation involving D2-like receptors, inhibition of the cAMP-PKA pathway, and an ex novo enzyme synthesis.
Bone buffering of acid and base in humans
Authors: Lemann Jacob , Bushinsky David A. , Hamm L. Lee
Published: March 2015

The sources and rates of metabolic acid production in relation to renal net acid excretion and thus acid balance in humans have remained controversial. The techniques and possible errors in these measurements are reviewed, as is the relationship of charge balance to acid balance. The results demonstrate that when acid production is experimentally increased among healthy subjects, renal net acid excretion does not increase as much as acid production so that acid balances become positive. These positive imbalances are accompanied by equivalently negative charge balances that are the result of bone buffering of retained H+and loss of bone Ca2+into the urine. The data also demonstrate that when acid production is experimentally reduced during the administration of KHCO3, renal net acid excretion does not decrease as much as the decrease in acid production so that acid balances become negative, or, in opposite terms, there are equivalently positive [Formula: see text] balances. Equivalently positive K+and Ca2+balances, and thus positive charge balances, accompany these negative acid imbalances. Similarly, positive Na+balances, and thus positive charge balances, accompany these negative acid balances during the administration of NaHCO3. These charge balances are likely the result of the adsorption of [Formula: see text] onto the crystal surfaces of bone mineral. There do not appear to be significant errors in the measurements.
Segment-specific ENaC downregulation in kidney of rats with lithium-induced NDI
Authors: Nielsen Jakob , Kwon Tae-Hwan , Praetorius Jeppe , Kim Young-Hee , Frøkiær Jørgen , Knepper Mark A. , Nielsen Søren
Published: March 2015

Lithium-induced nephrogenic diabetes insipidus is associated with increased renal sodium excretion in addition to severe urinary concentrating defects. However, the molecular basis for this altered renal sodium excretion remains undefined. The amiloride-sensitive sodium channel (ENaC) is expressed in the renal connecting tubule and collecting duct and is essential in renal regulation of body sodium balance and blood pressure. We hypothesized that dysregulation of ENaC subunits may be responsible for the increased sodium excretion associated with lithium treatment. Lithium treatment for 28 days resulted in severe polyuria, increased fractional excretion of sodium, and increased plasma aldosterone concentration. Immunoblotting revealed that lithium treatment induced a marked decrease in the protein abundance of β-ENaC and γ-ENaC in the cortex and outer medulla. Moreover, immunohistochemistry and laser confocal microscopy demonstrated an almost complete absence of β-ENaC and γ-ENaC labeling in cortical and outer medullary collecting duct, which was not affected by dietary sodium intake. In contrast, immunohistochemistry showed increased apical labeling of all ENaC subunits in the connecting tubule and inner medullary collecting duct in rats on a fixed sodium intake but not in rats with free access to sodium. Except for a modest downregulation of the thiazide-sensitive Na-Cl cotransporter, the key renal sodium transporters upstream from the connecting tubule (including the α1-subunit of Na-K-ATPase, type 3 Na/H exchanger, and Na-K-2Cl cotransporter) were unchanged. These results identify a marked and highly segment-specific downregulation of β-ENaC and γ-ENaC in the cortical and outer medullary collecting duct, chief sites for collecting duct sodium reabsorption, in rats with a lithium-induced increase in fractional excretion of sodium.
Effect of losartan on renal microvasculature during chronic inhibition of nitric oxide visualized by micro-CT
Authors: Fortepiani Loudes A. , Ruiz M. Clara Ortiz , Passardi Federico , Bentley Michael D. , Garcia-Estan Joaquin , Ritman Erik L. , Romero J. Carlos
Published: March 2015

Chronic inhibition of nitric oxide (NO) synthase with the competitive l-arginine analog NG-nitro-l-arginine methyl ester (l-NAME) leads to an elevated systemic blood pressure and reduction in renal blood flow without significant changes in urinary sodium and water excretion. Simultaneous administration of ANG II AT1 receptor antagonist losartan and l-NAME prevents the alterations in blood pressure and renal hemodynamics. Microcomputed tomography (micro-CT) was used to investigate the role of ANG II in the changes of renal microvasculature during chronic NO inhibition. Sprague-Dawley rats were given l-NAME with or without AT1 receptor antagonist losartan (40 mg · kg-1 · day-1 each) in their drinking water for 19 days. Kidneys from each group (control, l-NAME-, and l-NAME + losartan-treated rats) were perfusion-fixed in situ, infused with a silicon-based polymer containing lead chromate, and scanned by micro-CT. The microvasculature in the reconstructed three-dimensional renal images was studied using computerized analytic techniques. Kidneys of l-NAME-treated rats had significantly fewer normal glomeruli (28,824 ± 838) than those of control rats (36,266 ± 3,572). Losartan normalized the number to control values (34,094 ± 1,536). The amount of vasculature in the cortex, outer medulla, and inner medulla of l-NAME-treated rats was about two-thirds that of control rats; losartan normalized the values to control levels. These data indicate that chronic treatment with the NO synthase inhibitor l-NAME produces a generalized rarefaction of renal capillaries. Because simultaneous AT1 receptor blockade abolished those changes, the data suggest that the reduction in vasculature is mediated by ANG II through AT1 receptors.
Determinants of renal afferent arteriolar actions of bradykinin: evidence that multiple pathways mediate responses attributed to EDHF
Authors: Wang Xuemei , Trottier Greg , Loutzenhiser Rodger
Published: March 2015

The determinants of bradykinin (BK)-induced afferent arteriolar vasodilation were investigated in the in vitro perfused hydronephrotic rat kidney. BK elicited a concentration-dependent vasodilation of afferent arterioles that had been preconstricted with ANG II (0.1 nmol/l), but this dilation was transient in character. Pretreatment with the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (100 μmol/l) and the cyclooxygenase inhibitor ibuprofen (10 μmol/l) did not prevent this dilation when tone was established by ANG II but fully blocked the response when tone was established by elevated extracellular KCl, which suggests roles for both NO and endothelium-derived hyperpolarizing factor (EDHF). We had previously shown that the EDHF-like response of the afferent arteriole evoked by ACh was fully abolished by a combination of charybdotoxin (ChTX;10 nmol/l) and apamin (AP; 1 μmol/l). However, in the current study, treatment with ChTX plus AP only reduced the EDHF-like component of the BK response from 98 ± 5 to 53 ± 6% dilation. Tetraethylammonium (TEA; 1 mmol/l), which had no effect on the EDHF-induced vasodilation associated with ACh, reduced the EDHF-like response to BK to 88 ± 3% dilation. However, the combination of TEA plus ChTX plus AP abolished the response (0.3 ± 1% dilation). Similarly, 17-octadecynoic acid (17-ODYA) did not prevent the dilation when it was administered alone (77 ± 9% dilation) but fully abolished the EDHF-like response when added in combination with ChTX plus AP (-0.5 ± 4% dilation). These findings suggest that BK acts via multiple EDHFs: one that is similar to that evoked by ACh in that it is blocked by ChTX plus AP, and a second that is blocked by either TEA or 17-ODYA. Our finding that a component of the BK response is sensitive to TEA and 17-ODYA is consistent with previous suggestions that the EDHF released by BK is an epoxyeicosatrienoic acid.
Chronic acidosis-induced alteration in bone bicarbonate and phosphate
Authors: Bushinsky David A. , Smith Susan B. , Gavrilov Konstantin L. , Gavrilov Leonid F. , Li Jianwei , Levi-Setti Riccardo
Published: March 2015

Chronic metabolic acidosis increases urinary calcium excretion without altering intestinal calcium absorption, suggesting that bone mineral is the source of the additional urinary calcium. In vivo and in vitro studies have shown that metabolic acidosis causes a loss of mineral calcium while buffering the additional hydrogen ions. Previously, we studied changes in femoral, midcortical ion concentrations after 7 days of in vivo metabolic acidosis induced by oral ammonium chloride. We found that, compared with mice drinking only distilled water, ammonium chloride induced a loss of bone sodium and potassium and a depletion of mineral HCO3− and phosphate. There is more phosphate than carbonate in neonatal mouse bone. In the present in vitro study, we utilized a high-resolution scanning ion microprobe with secondary ion mass spectroscopy to test the hypothesis that chronic acidosis would decrease bulk (cross-sectional) bone phosphate to a greater extent than HCO3− by localizing and comparing changes in bone HCO3− and phosphate after chronic incubation of neonatal mouse calvariae in acidic medium. Calvariae were cultured for a total of 51 h in medium acidified by a reduction in HCO3− concentration ([HCO3−]; pH ∼7.14, [HCO3−] ∼13) or in control medium (pH ∼7.45, HCO3− ∼26). Compared with incubation in control medium, incubation in acidic medium caused no change in surface total phosphate but a significant fall in cross-sectional phosphate, with respect to the carbon-carbon bond (C2) and the carbon-nitrogen bond (CN). Compared with incubation in control medium, incubation in acidic medium caused no change in surface HCO3− but a significant fall in cross-sectional HCO3− with respect to C2 and CN. The fall in cross-sectional phosphate was significantly greater than the fall in cross-sectional HCO3−. The fall in phosphate indicates release of mineral phosphates, and the fall in HCO3− indicates release of mineral HCO3−, both of which would be expected to buffer the additional protons and help restore the pH toward normal. Thus a model of chronic acidosis depletes bulk bone proton buffers, with phosphate depletion exceeding that of HCO3−.
PDZ domain-mediated interaction of rabbit podocalyxin and Na+/H+exchange regulatory factor-2
Authors: Li Yong , Li Jian , Straight Samuel W. , Kershaw David B.
Published: March 2015

The transmembrane sialoglycoprotein podocalyxin is thought to be essential in the fine interdigitating foot process structure of the podocyte. The intracellular COOH-terminal amino acids Asp-Thr-His-Leu (DTHL) of podocalyxin comprise a putative ligand for a type I PSD95-Dlg-zona occludens-1 (PDZ) domain. A 20-amino acid synthetic peptide containing this motif was used to screen a cDNA library, and clones of rabbit Na+/H+exchange regulatory factor-2 (NHERF-2) were obtained. In vitro analysis demonstrated that each PDZ domain of NHERF-2 could bind podocalyxin independently. NHERF-2 coprecipitated from glomerular extracts with podocalyxin, and podocalyxin and NHERF-2 colocalized in the glomerular capillary loops, indicating that podocalyxin and NHERF-2 may interact in vivo. Podocalyxin peptide missing the terminal leucine (−DTHL) failed to interact with NHERF-2 in vitro. Podocalyxin localized to the apical membrane of transfected Madin-Darby canine kidney (MDCK) cells. However, mutant podocalyxin (missing a functional DTHL COOH-terminal motif) showed cytoplasmic and apical membrane localization in transfected cells and was also less stable at the apical membrane, as assessed by confocal microscopy and biotinylation studies. Mutant podocalyxin did lower the transepithelial resistance of MDCK cell monolayers, albeit to a lesser extent than full-length podocalyxin. We conclude that podocalyxin can interact with both PDZ domains of NHERF-2 and that this interaction requires the intact COOH terminus of podocalyxin, which is also responsible for the efficient apical localization of podocalyxin in transfected MDCK cells. These results suggest that the interaction of podocalyxin with NHERF-2 may function to efficiently retain podocalyxin at the apical surface of the podocyte and provide a mechanism linking podocalyxin to the actin cytoskeleton.
Role of glycosylation in the renal electrogenic Na+-HCO 3 − cotransporter (NBCe1)
Authors: Choi Inyeong , Hu Lihui , Rojas José D. , Schmitt Bernhard M. , Boron Walter F.
Published: March 2015

The electrogenic Na+-HCO[Formula: see text] cotransporter NBCe1 is important for the regulation of intracellular pH (pHi) and for epithelial HCO[Formula: see text] transport in many tissues, including kidney, pancreas, and brain. In the present study, we investigate glycosylation sites in NBCe1. Treatment of rat kidney membrane extracts with peptide N-glycosidase F (PNGase F) shifted the apparent molecular weight (MW) of NBCe1 from 130 to 116, the MW predicted from the deduced amino acid sequence. Treatment with endoglycosidase F2or H or O-glycosidase did not affect the MW of NBCe1. Lectin-binding studies, together with the enzyme data, suggest that the N-linked carbohydrates are of tri- or tetra-antennary type. To localize glycosylation sites, we individually mutated the seven consensus N-glycosylation sites by replacing asparagine (N) with glutamine (Q) and assessing mutant transporters in Xenopus laevis oocytes. Immunoblotting of oocyte membrane extracts treated with PNGase F indicates that NBCe1 is normally glycosylated at N597 and N617 (both on the third extracellular loop). However, N592 (on the same loop) is glycosylated when the other two sites are mutated. The triple mutant (N592Q/N597Q/N617Q) is completely unglycosylated but, based on microelectrode measurements of membrane potential and pHiin oocytes, preserves the Na+and HCO[Formula: see text] dependence and electrogenicity of wild-type NBCe1.
Distribution of cytochromeP-450 4A and 4F isoforms along the nephron in mice
Authors: Stec David E. , Flasch Averia , Roman Richard J. , White Jared A.
Published: March 2015

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney is thought to be involved in the control of renal vascular tone and tubular sodium and chloride reabsorption. 20-HETE production in the kidney has been extensively studied in rats and humans and occurs primarily via the actions of P-450 enzymes of the CYP4A and -4F families. Recent advancements in molecular genetics of the mouse have made it possible to disrupt genes in a cell-type-specific fashion. These advances could help in the creation of models that could distinguish between the vascular and tubular actions of 20-HETE. However, isoforms of the CYP4A and -4F families that may be responsible for the production of 20-HETE in the vascular and tubular segments in the kidney of the mouse are presently unknown. The goal of this study was to identify the isoforms of the CYP4A and -4F families along the nephron by RT-PCR of RNA isolated from microdissected renal blood vessels and nephron segments from 16- to 24-wk-old male and female C57BL/6J mice. CYP4A and -4F isoforms were detected in every segment analyzed, with sex differences only observed in the proximal tubule and glomeruli. In the proximal tubular segments from male mice, the 4A10 and -12 isoforms were present, whereas the 4A10 and -14 isoforms were detected in segments from female mice. In glomeruli, sex differences in the expression pattern of CYP4F isoforms were also observed, with male mice expressing the 4F13, -14, and -15 isoforms, whereas female mice expressed the 4F13, -16, and -18 isoforms. These results demonstrate that isolated nephron and renal vessel segments express multiple isoforms of the CYP4A and -4F families; therefore, elimination of a single CYP4A or -4F isoform may not decrease 20-HETE production in all nephron segments or the renal vasculature of male and female mice. However, the importance of CYP4A vs. -4F isoforms to the production of 20-HETE in each of these renal tubular and vascular segments of the mouse remains to be determined.
Expression, localization, and regulation of aquaporin-1 to -3 in rat urothelia
Authors: Spector David A. , Wade James B. , Dillow Russell , Steplock Deborah A. , Weinman Edward J.
Published: March 2015

Although mammalian urothelia are generally considered impermeable to constituents of urine, in vivo studies in several species indicate urothelial transport of water and solutes under certain conditions. This study investigates the expression, localization, and regulation of aquaporin (AQP)-1, -2, and -3 in ureteral and bladder tissues in 48-h dehydrated and water-loaded female Wistar rats. Immunoblots of homogenates of whole ureter and bladder identified characteristic ∼28- and 35- to 44-kDa bands for AQP-1, -2, and -3. AQP-1 was localized to capillary and arteriole endothelial cells, whereas AQP-2 and -3 circumferentially lined the epithelial cell membranes except for the apical membrane of the epithelial cells adjacent to the lumens of both ureter and bladder. AQP-2 was also present in epithelial cell cytoplasm. Dehydration resulted in 160–200% increases of AQP-3 signal and 24–49% increases of AQP-2 signal but no change in AQP-1 signal on immunoblots of homogenates of ureters and bladders. AQPs in genitourinary tract urothelia likely play a role in the regulation of epithelial cell volume and osmolality and may play a role in bulk water movement across urothelia.
Localization of pendrin in mouse kidney
Authors: Wall Susan M. , Hassell Kathryn A. , Royaux Ines E. , Green Eric D. , Chang Judy Y. , Shipley Gregory L. , Verlander Jill W.
Published: March 2015

Pendrin is an anion exchanger expressed in type B intercalated cells of the cortical collecting duct (CCD). Whether pendrin localizes to other nephron segments with intercalated cells is unknown. Moreover, whether pendrin is expressed in proximal tubule is debated. Thus the distribution of pendrin mRNA and protein expression in mouse kidney was investigated by using light and electron microscopic immunohistochemistry and quantitative real-time PCR. We observed that pendrin mRNA is expressed mainly in cortex. Within cortex, pendrin mRNA is at least fivefold higher in CCD and the connecting tubule (CNT) than in the other segments. Pendrin protein was observed in a subset of cells within the distal convoluted tubule as well as in type B and in non-A-non-B intercalated cells of the CNT and CCD. In type B intercalated cells, pendrin immunoreactivity was highest in apical cytoplasmic vesicles with little immunolabel along the apical plasma membrane. In non-A-non-B intercalated cells, intense pendrin immunoreactivity was detected along the apical plasma membrane. These differences in the subcellular distribution of pendrin immunolabel were confirmed by morphometric analysis. In conclusion, pendrin is expressed in the mouse distal convoluted tubule, CCD, and CNT along the apical plasma membrane of non-A-non-B intercalated cells and in subapical cytoplasmic vesicles of type B intercalated cells.
Structural determinants and significance of regulation of electrogenic Na+-HCO 3 − cotransporter stoichiometry
Authors: Gross Eitan , Kurtz Ira
Published: March 2015

Na+-HCO[Formula: see text]cotransporters play an important role in intracellular pH regulation and transepithelial HCO[Formula: see text] transport in various tissues. Of the characterized members of the HCO[Formula: see text]transporter superfamily, NBC1 and NBC4 proteins are known to be electrogenic. An important functional property of electrogenic Na+-HCO[Formula: see text] cotransporters is their HCO[Formula: see text]:Na+coupling ratio, which sets the transporter reversal potential and determines the direction of Na+-HCO[Formula: see text] flux. Recent studies have shown that the HCO[Formula: see text]:Na+transport stoichiometry of NBC1 proteins is either 2:1 or 3:1 depending on the cell type in which the transporters are expressed, indicating that the HCO[Formula: see text]:Na+coupling ratio can be regulated. Mutational analysis has been very helpful in revealing the molecular mechanisms and signaling pathways that modulate the coupling ratio. These studies have demonstrated that PKA-dependent phosphorylation of the COOH terminus of NBC1 proteins alters the transport stoichiometry. This cAMP-dependent signaling pathway provides HCO[Formula: see text]-transporting epithelia with an efficient mechanism for modulating the direction of Na+-HCO[Formula: see text] flux through the cotransporter.
Expression of PGT in MDCK cell monolayers: polarized apical localization and induction of active PG transport
Authors: Endo Shinichi , Nomura Teruhisa , Chan Brenda S. , Lu Run , Pucci Michael L. , Bao Yi , Schuster Victor L.
Published: March 2015

The PG transporter (PGT) is expressed in subapical vesicles in the kidney collecting duct. To gain insight into the possible function of the PGT in this tubule segment, we tagged rat PGT with green fluorescent protein at the COOH terminus and generated stable PGT-expressing Madin-Darby canine kidney cell lines. When grown on permeable filters, green fluorescent protein-PGT was expressed predominantly at the apical membrane. Although the basal-to-apical transepithelial flux of [3H]PGE2 was little changed by PGT expression, the apical-to-basolateral flux was increased 100-fold compared with wild-type cells. Analysis of driving forces revealed that this flux represents PGT-mediated active transepithelial PGE2 transport. We propose that endogenous PGT is exocytically inserted into the collecting duct apical membrane, where it could control the concentration of luminal PGs.
Effects of amino acids and glucagon on renal hemodynamics in type 1 diabetes
Authors: Tuttle Katherine R. , Puhlman Mark E. , Cooney Sheryl K. , Short Robert A.
Published: March 2015

Increased dietary protein and circulating amino acids raise glomerular filtration rate (GFR) and pressure. In diabetes, this glomerular hyperfiltration response is augmented. The purpose of this study was to determine whether glucagon mediates the augmented GFR response to amino acids in diabetes and whether the responses to amino acids and glucagon depend on prostaglandins. Patients with type 1 diabetes mellitus ( n = 12) and normal control subjects ( n= 12) were studied in a series of six experiments, each on different occasions. Baseline GFR was not significantly increased, but filtration fraction was higher in diabetes. In response to amino acid infusion, GFR increased more and filtration fraction was greater among those with diabetes. Their augmented GFR response to amino acids was not inhibited by octreotide or indomethacin. Participants with diabetes also had enhanced GFR and renal plasma flow responses to glucagon infusion, both of which were inhibited by indomethacin. Glomerular hyperfiltration responses induced by amino acids or glucagon occur by divergent pathways in diabetes; only the response to glucagon is prostaglandin dependent.
Relaxin increases ubiquitin-dependent degradation of fibronectin in vitro and ameliorates renal fibrosis in vivo
Authors: McDonald Glenn A. , Sarkar Pradip , Rennke Helmut , Unemori Elaine , Kalluri Raghu , Sukhatme Vikas P.
Published: March 2015

Fibronectin, a large adhesive glycoprotein, is a prominent constituent of the extracellular matrix. Abnormalities in fibronectin homeostasis occur in numerous disease states, ranging from primary fibrosing conditions to neoplastic transformation. We demonstrate that fibronectin is a target protein substrate for ubiquitin-dependent degradation. Coimmunoprecipitation experiments and confocal microscopy demonstrated ubiquitin-fibronectin interaction. In an in vitro model of renal fibrosis, relaxin, an insulin-like growth factor, increased ubiquitin-dependent fibronectin degradation. Relaxin also was evaluated in an anti-glomerular basement membrane model of renal fibrosis. Animals treated with relaxin experienced renoprotection, manifested by decreased serum creatinine and proteinuria. Histological evaluation of kidney sections from animals treated with relaxin showed decreased glomerulosclerosis and interstitial fibrosis. We conclude that relaxin might be developed as a useful agent for the treatment of renal fibrosis.
Calcineurin is activated in diabetes and is required for glomerular hypertrophy and ECM accumulation
Authors: Gooch Jennifer L. , Barnes Jeffrey L. , Garcia Sergio , Abboud Hanna E.
Published: March 2015

Diabetic nephropathy is characterized by the rapid onset of hypertrophy and ECM expansion. Previously, we showed that calcineurin phosphatase is required for hypertrophy and ECM synthesis in cultured mesangial cells. Therefore, we examined the effect of calcineurin inhibition on renal hypertrophy and ECM accumulation in streptozotocin-induced diabetic rats. After 2 wk of diabetes, calcineurin protein was increased in whole cortex and glomeruli in conjunction with increased phosphatase activity. Daily administration of cyclosporin A blocked accumulation of both calcineurin protein and calcineurin activity. Also associated with calcineurin upregulation was nuclear localization of the calcineurin substrate NFATc1. Inhibition of calcineurin reduced whole kidney hypertrophy and abolished glomerular hypertrophy in diabetic rats. Furthermore, calcineurin inhibition substantially reduced ECM accumulation in diabetic glomeruli but not in cortical tissue, suggesting a differential effect of calcineurin inhibition in glomerular vs. extraglomerular tissue. Corresponding increases in fibronectin mRNA and transforming growth factor-β mRNA were observed in tubulointerstitium but not in glomeruli. In summary, calcineurin plays an important role in glomerular hypertrophy and ECM accumulation in diabetic nephropathy.
Angiotensin II mediates LDL-induced superoxide generation in mesangial cells
Authors: Park So Yeon , Song Chi Young , Kim Bong Cho , Hong Hye Kyoung , Lee Hyun Soon
Published: March 2015

Lipid abnormalities and activation of the local renin-angiotensin system (RAS) may be involved in the pathogenesis of chronic glomerular disease. This study investigated whether low-density lipoprotein (LDL) activates local RAS in cultured human mesangial cells (HMC) and, at the same time, whether ANG II mediates LDL-induced mesangial cell proliferation, hypertrophy, and superoxide ([Formula: see text]) generation. Quiescent HMC were exposed to 50 to 200 μg/ml of LDL or 10-7to 10-10M ANG II for 0.5 to 24 h in the presence or absence of 10-6M losartan, an ANG II type I (AT1) receptor antagonist, or 10-5M diphehylendieodonium (DPI) or 10-4M apocynin, inhibitors of nicotinamide adenine dinucleotide phosphate oxidase. LDL induced an up to threefold increase in the ANG II levels in the culture medium of HMC. LDL upregulated AT1receptor and angiotensinogen mRNA expression in HMC. LDL incubated with HMC increased [Formula: see text] production by up to 3.3 times compared with the level of control cells. The LDL-induced, increased [Formula: see text] generation was suppressed by losartan, DPI, or apocynin. LDL significantly increased mesangial [3H]thymidine or [3H]leucine incorporation, whereas these processes were abrogated by losartan. In conclusion, LDL increases ANG II production by mesangial cells, which in turn results in increased [Formula: see text] production, and cell proliferation and hypertrophy, these effects of ANG II being mediated by the AT1receptor.
Chemical anoxia of tubular cells induces activation of c-Src and its translocation to the zonula adherens
Authors: Sinha Diviya , Wang Zhiyong , Price Valerie R. , Schwartz John H. , Lieberthal Wilfred
Published: March 2015

Cyanide (CN)-induced chemical anoxia of cultured mouse proximal tubular (MPT) cells increased the kinase activity of c-Src by approximately threefold. 4-Amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), a specific inhibitor of c-Src, prevented Src activation. CN also increased the permeability of MPT cell monolayers, an event ameliorated by PP2. During CN treatment, the proteins of the zonula adherens (ZA; E-cadherin and the catenins) disappeared from their normal location at cell-cell borders and appeared within the cytosol. CN also resulted in the appearance of c-Src at cell-cell borders. PP2 prevented these CN-induced alterations in the distribution of ZA proteins and c-Src. CN also increased the association of c-Src with β-catenin and p120 and induced a substantial increase in tyrosine phosphorylation of both catenins. PP2 prevented the CN-induced phosphorylation of these catenins. In summary, we show that CN-induced chemical anoxia activates c-Src and induces its translocation to cell-cell junctions where it binds to and phosphorylates β-catenin and p120. Our findings suggest that these events contribute to the loss of the epithelial barrier function associated with chemical anoxia.
Role of NO in endothelin-regulated drug transport in the renal proximal tubule
Authors: Notenboom Sylvia , Miller David S. , Smits Paul , Russel Frans G. M. , Masereeuw Rosalinde
Published: March 2015

We previously demonstrated in intact killifish renal proximal tubules that endothelin (ET), acting through an ETBreceptor and protein kinase C (PKC), reduced transport mediated by multidrug resistance-associated protein 2 (Mrp2), i.e., luminal accumulation of fluorescein methotrexate (FL-MTX) (Masereeuw R, Terlouw SA, Van Aubel RAMH, Russel FGM, and Miller DS. Mol Pharmacol 57: 59–67, 2000). In the present study, we used confocal microscopy and quantitative image analysis to measure Mrp2-mediated transport of FL-MTX in killifish tubules as an indicator of the status of this ET-fired, intracellular signaling pathway. Exposing tubules to sodium nitroprusside (SNP), a nitric oxide (NO) donor, signaled a reduction in luminal accumulation of FL-MTX, which suggested pathway activation. NG-monomethyl-l-arginine (l-NMMA), an NO synthase inhibitor, blocked the action of ET-1 on transport. Because SNP effects on transport were blocked by bisindoylmaleide, a PKC-selective inhibitor, but not by RES-701–1, an ETB-receptor antagonist, generation of NO occurred after ETBreceptor signaling but before PKC activation. NO generation was implicated in the actions of several nephrotoxicants, i.e., diatrizoate, gentamicin, amikacin, HgCl2, and CdCl2, each of which decreased Mrp2-mediated transport by activating ET signaling. For each nephrotoxicant, decreased FL-MTX transport was prevented when tubules were exposed tol-NMMA. ET-1 and each nephrotoxicant stimulated NO production by the tubules, as determined by a fluorescence-based assay. Together, the data show that NO generation follows ET binding to the basolateral ETBreceptor and that, in activating the ET-signaling pathway, nephrotoxicants produce NO, a molecule that could contribute to subsequent toxic effects.
Hyperosmotic mannitol activates basolateral NHE in proximal tubule from P-glycoprotein null mice
Authors: Miyata Yukio , Asano Yasushi , Muto Shigeaki
Published: March 2015

Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we examined the effects of hyperosmotic mannitol on basolateral Na+/H+exchange (NHE) activity in isolated nonperfused proximal tubule S2 segments from mice lacking both the mdr1a and mdr1b genes (KO) and wild-type mice (WT). All experiments were performed in CO2/HCO[Formula: see text]-free HEPES solutions. Osmolality of the peritubular solution was raised from 300 to 500 mosmol/kgH2O by the addition of mannitol. NHE activity was assessed by Na+-dependent acid extrusion rates ( JH) after an acid load with NH4Cl prepulse. Under isosmotic conditions, JHvalues at a wide intracellular pH (pHi) range of 6.20–6.90 were not different between the two groups. In WT mice, hyperosmotic mannitol had no effect on JHat the wide pHirange. In contrast, in KO mice, hyperosmotic mannitol increased JHat a pHirange of 6.20–6.45 and shifted the JH-pHirelationship by 0.15 pH units in the alkaline direction. In KO mice, hyperosmotic mannitol caused an increase in maximal velocity without changing the Michaelis-Menten constant for peritubular Na+. Exposure of cells from WT mice to the hyperosmotic mannitol solution including the P-gp inhibitor cyclosporin A increased JH(at pHi6.30) to an extent similar to that in cells from KO mice exposed to hyperosmotic mannitol alone. In KO mice, staurosporine and calphostin C inhibited the hyperosmotic mannitol-induced increase in JH. The stimulatory effect of hyperosmotic mannitol on JHwas mimicked by addition to the isosmotic control solution, including phorbol 12-myristate 13-acetate (PMA; the PKC activator). In WT mice, hyperosmotic mannitol with PMA increased JH. We conclude that, in the absence of P-gp activity, hyperosmotic mannitol activates basolateral NHE via protein kinase C, whereas in the presence of P-gp activity, it does not.
Altered regulation of SHP-2 and PTP 1B tyrosine phosphatases in cystic kidneys from bcl-2 −/− mice
Authors: Sorenson Christine M. , Sheibani Nader
Published: March 2015

Protein tyrosine phosphorylation is a dynamic reversible process in which the level of phosphorylation, at any time, is the result of phosphatase and/or kinase activity. This balance is critical for control of growth and differentiation. The role of tyrosine phosphatases during nephrogenesis and in kidney disease requires delineation. Appropriate regulation of focal adhesion proteins such as focal adhesion kinase (FAK) and paxillin are important in cell adhesion, migration, and differentiation. We have previously shown that B cell lymphoma/leukemia-2 (bcl-2) −/− mice develop cystic kidneys and exhibit sustained phosphorylation of FAK and paxillin. We have examined the expression and activity of focal adhesion tyrosine phosphatases [Src homology-2 domain phosphatase (SHP-2), protein tyrosine phosphatase (PTP 1B), and PTP-proline, glutamate, serine, and threonine sequences (PEST)] during normal nephrogenesis and in cystic kidneys from bcl-2 −/− mice. Cystic kidneys from postnatal day 20 bcl-2 −/− mice demonstrate a reduced expression, sixfold decrease in activity, and altered distribution of SHP-2 and PTP 1B. PTP-PEST expression and distribution were similar in both bcl-2 +/+ and bcl-2 −/− mice. The altered regulation of PTP 1B and SHP-2 in kidneys from bcl-2 −/− mice correlates with sustained phosphorylation of FAK and paxillin. Thus renal cyst formation in the bcl-2 −/− mice may be the result of an inability of complete differentiation due to continued activation of growth processes, including activation of FAK and paxillin.
Chloride channels in the kidney: lessons learned from knockout animals
Authors: Devuyst Olivier , Guggino William B.
Published: March 2015

Cl−channels are involved in a range of functions, including regulation of cell volume and/or intracellular pH, acidification of intracellular vesicles, and vectorial transport of NaCl across many epithelia. Numerous Cl−channels have been identified in the kidney, based on single-channel properties such as conductance, anion selectivity, gating, and response to inhibitors. The molecular counterpart of many of these Cl−channels is still not known. This review will focus on gene-targeted mouse models disrupting two structural classes of Cl−channels that are relevant for the kidney: the CLC family of voltage-gated Cl−channels and the CFTR. Disruption of several members of the CLC family in the mouse provided useful models for various inherited diseases of the kidney, including Dent's disease and diabetes insipidus. Mice with disrupted CFTR are valuable models for cystic fibrosis (CF), the most common autosomal recessive, lethal disease in Caucasians. Although CFTR is expressed in various nephron segments, there is no overt renal phenotype in CF. Analysis of CF mice has been useful to identify the role and potential interactions of CFTR in the kidney. Furthermore, observations made in CF mice are potentially relevant to all other models of Cl−channel knockouts because they emphasize the importance of alternative Cl−pathways in such models.
Rat proximal NHE3 adapts to chronic acid-base disorders but not to chronic changes in dietary NaCl intake
Authors: Eladari Dominique , Leviel Françoise , Pezy Françoise , Paillard Michel , Chambrey Régine
Published: March 2015

In the proximal tubule, the apical Na+/H+exchanger identified as NHE3 mediates most NaCl and NaHCO3absorption. The purpose of this study was to analyze the long-term regulation of NHE3 during alkalosis induced by dietary NaHCO3loading and changes in NaCl intake. Sprague-Dawley rats exposed to a low-NaCl, high-NaCl, or NaHCO3diet for 6 days were studied. Renal cortical apical membrane vesicles (AMV) were prepared from treated and normal rats. Na+/H+exchange was assayed as the initial rate of22Na+uptake in the presence of an outward H+gradient.22Na+uptake measured in the presence of high-dose 5-( N-ethyl- N-isopropyl) amiloride was not different among models. Changes in NaCl intake did not affect NHE3 activity, whereas NaHCO3loading inhibited22Na+uptake by 30%. AMV NHE3 protein abundance assessed by Western blot analysis was unaffected during changes in NaCl intake. During NaHCO3loading, NHE3 protein abundance was decreased by 65%. We conclude that proximal NHE3 adapts to chronic metabolic acid-base disorders but not to changes in dietary NaCl intake.
Ontogeny of flow-stimulated potassium secretion in rabbit cortical collecting duct: functional and molecular aspects
Authors: Woda Craig B. , Miyawaki Nobuyuki , Ramalakshmi Santhanam , Ramkumar Mohan , Rojas Raul , Zavilowitz Beth , Kleyman Thomas R. , Satlin Lisa M.
Published: March 2015

High urinary flow rates stimulate K secretion in the fully differentiated but not neonatal or weanling rabbit cortical collecting duct (CCD). Both small-conductance secretory K and high-conductance Ca2+/stretch-activated maxi-K channels have been identified in the apical membrane of the mature CCD by patch-clamp analysis. We reported that flow-stimulated net K secretion in the adult rabbit CCD is 1) blocked by TEA and charybdotoxin, inhibitors of intermediate- and high-conductance (maxi-K) Ca2+-activated K channels, and 2) associated with increases in net Na absorption and intracellular Ca2+concentration ([Ca2+]i). The present study examined whether the absence of flow-stimulated K secretion early in life is due to a 1) limited flow-induced rise in net Na absorption and/or [Ca2+]iand/or 2) paucity of apical maxi-K channels. An approximately sixfold increase in tubular fluid flow rate in CCDs isolated from 4-wk-old rabbits and microperfused in vitro led to an increase in net Na absorption and [Ca2+]i, similar in magnitude to the response observed in 6-wk-old tubules, but it failed to generate an increase in net K secretion. By 5 wk of age, there was a small, but significant, flow-stimulated rise in net K secretion that increased further by 6 wk of life. Luminal perfusion with iberiotoxin blocked the flow stimulation of net K secretion in the adult CCD, confirming the identity of the maxi-K channel in this response. Maxi-K channel α-subunit message was consistently detected in single CCDs from animals ≥4 wk of age by RT-PCR. Indirect immunofluorescence microscopy using antibodies directed against the α-subunit revealed apical labeling of intercalated cells in cryosections from animals ≥5 wk of age; principal cell labeling was generally intracellular and punctate. We speculate that the postnatal appearance of flow-dependent K secretion is determined by the transcriptional/translational regulation of expression of maxi-K channels. Furthermore, our studies suggest a novel function for intercalated cells in mediating flow-stimulated K secretion.