bluuebunny/crossref_metadata_2025 huggingface dataset:
Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP
Authors: Chen Ruihua , Greene Eddie L. , Collinsworth Georgiann , Grewal Jasjit S. , Houghton Odette , Zeng Haiqun , Garnovskaya Maria , Paul Richard V. , Raymond John R.
Published: December 2017

Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a β-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+cells yielded greater than fivefold enrichment of cells with detectable β-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.
Urea-associated oxidative stress and Gadd153/CHOP induction
Authors: Zhang Zheng , Yang Xiao-Yan , Cohen David M.
Published: December 2017

Urea treatment (100–300 mM) increased expression of the oxidative stress-responsive transcription factor, Gadd153/CHOP, at the mRNA and protein levels (at ≥4 h) in renal medullary mIMCD3 cells in culture, whereas other solutes did not. Expression of the related protein, CCAAT/enhancer-binding protein (C/EBP-β), was not affected, nor was expression of the sensor of endoplasmic reticulum stress, grp78. Urea modestly increased Gadd153 transcription by reporter gene analysis but failed to influence Gadd153 mRNA stability. Importantly, upregulation of Gadd153 mRNA and protein expression by urea was antioxidant sensitive. Accordingly, urea treatment was associated with oxidative stress, as quantitated by intracellular reduced glutathione content in mIMCD3 cells. In addition, antioxidant treatment partially inhibited the ability of urea to activate transcription of an Egr-1 luciferase reporter gene. Therefore oxidative stress represents a novel solute-signaling pathway in the kidney medulla and, potentially, in other tissues.
Pathways for HCO 3 − exit across the basolateral membrane in rat thick limbs
Authors: Leviel Françoise , Eladari Dominique , Blanchard Anne , Poumarat Jean-Stéphane , Paillard Michel , Podevin René-Alexandre
Published: December 2017

We studied the pathways for [Formula: see text] transport in basolateral membrane vesicles (BLMV) purified from rat medullary thick ascending limbs (MTAL). An inward [Formula: see text]gradient in the presence of an inside-positive potential stimulated the rate of22Na uptake minimally and did not induce a22Na overshoot, arguing against the presence of electrogenic Na+-[Formula: see text]cotransport in these membranes. An inside-acid pH gradient stimulated to the same degree uptake of86Rb+(a K+analog) with or without[Formula: see text]. Conversely, applying an outward K+gradient caused a modest intracellular pH (pHi) decrease of ∼0.38 pH units/min, as monitored by quenching of carboxyfluorescein; its rate was unaffected by[Formula: see text], indicating the absence of appreciable K+-[Formula: see text]cotransport. On the other hand, imposing an inward Cl−gradient in the presence of [Formula: see text] caused a marked pHidecrease of ∼1.68 pH units/min; its rate was inhibited by a stilbene derivative. Finally, we could not demonstrate the presence of a[Formula: see text]/lactate exchanger in BLMV. In conclusion, the presence of significant Na+-, K+-, or lactate-linked[Formula: see text] transport could not be demonstrated. These and other data suggest that basolateral Cl−/[Formula: see text]exchange could be the major pathway for[Formula: see text] transport in the MTAL.
Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides
Authors: London Roslyn M. , Eber Sammy L. , Visweswariah Sandhya S. , Krause William J. , Forte Leonard R.
Published: December 2017

Uroguanylin, guanylin, and lymphoguanylin are small peptides that activate renal and intestinal receptor guanylate cyclases (GC). They are structurally similar to bacterial heat-stable enterotoxins (ST) that cause secretory diarrhea. Uroguanylin, guanylin, and ST elicit natriuresis, kaliuresis, and diuresis by direct actions on kidney GC receptors. A 3,762-bp cDNA characterizing a uroguanylin/guanylin/ST receptor was isolated from opossum kidney (OK) cell RNA/cDNA. This kidney cDNA (OK-GC) encodes a mature protein containing 1,049 residues sharing 72.4–75.8% identity with rat, human, and porcine forms of intestinal GC-C receptors. COS or HEK-293 cells expressing OK-GC receptor protein were activated by uroguanylin, guanylin, or ST13peptides. The 3.8-kb OK-GC mRNA transcript is most abundant in the kidney cortex and intestinal mucosa, with lower mRNA levels observed in urinary bladder, adrenal gland, and myocardium and with no detectable transcripts in skin or stomach mucosa. We propose that OK-GC receptor GC participates in a renal mechanism of action for uroguanylin and/or guanylin in the physiological regulation of urinary sodium, potassium, and water excretion. This renal tubular receptor GC may be a target for circulating uroguanylin in an endocrine link between the intestine and kidney and/or participate in an intrarenal paracrine mechanism for regulation of kidney function via the intracellular second messenger, cGMP.
Ultrastructural model for size selectivity in glomerular filtration
Authors: Edwards Aurélie , Daniels Barbara S. , Deen William M.
Published: December 2017

A theoretical model was developed to relate the size selectivity of the glomerular barrier to the structural characteristics of the individual layers of the capillary wall. Thicknesses and other linear dimensions were evaluated, where possible, from previous electron microscopic studies. The glomerular basement membrane (GBM) was represented as a homogeneous material characterized by a Darcy permeability and by size-dependent hindrance coefficients for diffusion and convection, respectively; those coefficients were estimated from recent data obtained with isolated rat GBM. The filtration slit diaphragm was modeled as a single row of cylindrical fibers of equal radius but nonuniform spacing. The resistances of the remainder of the slit channel, and of the endothelial fenestrae, to macromolecule movement were calculated to be negligible. The slit diaphragm was found to be the most restrictive part of the barrier. Because of that, macromolecule concentrations in the GBM increased, rather than decreased, in the direction of flow. Thus the overall sieving coefficient (ratio of Bowman’s space concentration to that in plasma) was predicted to be larger for the intact capillary wall than for a hypothetical structure with no GBM. In other words, because the slit diaphragm and GBM do not act independently, the overall sieving coefficient is not simply the product of those for GBM alone and the slit diaphragm alone. Whereas the calculated sieving coefficients were sensitive to the structural features of the slit diaphragm and to the GBM hindrance coefficients, variations in GBM thickness or filtration slit frequency were predicted to have little effect. The ability of the ultrastructural model to represent fractional clearance data in vivo was at least equal to that of conventional pore models with the same number of adjustable parameters. The main strength of the present approach, however, is that it provides a framework for relating structural findings to the size selectivity of the glomerular barrier.
Differential regulation of COX-2 expression in the kidney by lipopolysaccharide: role of CD14
Authors: Yang Tianxin , Sun Daqing , Huang Yuning G. , Smart Ann , Briggs Josephine P. , Schnermann Jurgen B.
Published: December 2017

Induction of the inducible cyclooxygenase isoform COX-2 is likely to be an important mechanism for increased prostaglandin production in renal inflammation. We examined the effect of lipopolysaccharide (LPS) on regional renal COX-2 expression in the rat. In the inner medulla, LPS injection (4 mg/kg ip) induced a twofold and 2.5-fold increase in the levels of COX-2 mRNA and COX-2 protein, respectively. In contrast, COX-2 expression in the renal cortex was not significantly altered. COX-2 promoter transgenic mice were created using the 2.7-kb flanking region of the rat COX-2 gene. In these animals, LPS injection induced reporter gene expression predominately in the inner medulla. The LPS receptor CD14, usually regarded as a monocyte/macrophage-specific marker, was found to be abundantly expressed in the inner medulla and in dissected inner medullary collecting duct (IMCD) cells, suggesting that it may mediate medullary COX-2 induction. CD14 was present only at low levels in cortex and cortical segments, including glomeruli. In cultured cells, it was abundant in mouse IMCD (mIMCD-K2) cells and renal medullary interstitial cells, but largely undetectable in mesangial cells and M1 cells, a cell line derived from mouse cortical collecting ducts. In the mIMCD-K2 cell line, LPS significantly induced COX-2 mRNA expression, with concomitant induction of CD14. LPS-stimulated COX-2 expression was reduced by the addition of an anti-CD14 monoclonal antibody to the culture medium. These results demonstrate that LPS selectively stimulates COX-2 expression in the renal inner medulla through a CD14-dependent mechanism.
Plasma and 24-h NOxand cGMP during normal pregnancy and preeclampsia in women on a reduced NOxdiet
Authors: Conrad Kirk P. , Kerchner Laurie J. , Mosher Monique D.
Published: December 2017

We tested the hypothesis that nitric oxide (NO) biosynthesis increases during normal human pregnancy and decreases in preeclampsia. The major metabolites of NO, nitrate and nitrite (NOx), were measured in both the plasma and 24-h urine of women subjected to a reduced NOxdiet. In this way, the plasma and urinary levels mainly reflected endogenous production rather than dietary intake. Moreover, we assessed cGMP, a second messenger of NO, in the same samples. Both NOxand cGMP assays were validated in our laboratory. We first conducted a cross-sectional study of nonpregnant women ( n = 15), normal pregnant women in the first ( n = 9), second ( n = 17) and third ( n = 22) trimesters, as well as women with preeclampsia ( n = 15) and transient hypertension of pregnancy ( n= 7). We also performed a serial study in the same women ( n = 9) before, during, and after pregnancy. Taken together, the results of the two investigations suggested marked increases in cGMP production especially during the first trimester when the maternal circulation is rapidly vasodilating. In contrast, whole body NO production as estimated by the plasma level and urinary excretion of NOxwas not elevated during the first trimester. Finally, unequivocal demonstration of reduced NO biosynthesis in preeclampsia was not forthcoming.
Cyclooxygenase inhibitors increase Na-K-2Cl cotransporter abundance in thick ascending limb of Henle’s loop
Authors: Fernández-Llama Patricia , Ecelbarger Carolyn A. , Ware Joseph A. , Andrews Peter , Lee Alanna J. , Turner Rachel , Nielsen Søren , Knepper Mark A.
Published: December 2017

Cyclooxygenase inhibitors, such as indomethacin and diclofenac, have well-described effects to enhance renal water reabsorption and urinary concentrating ability. Concentrating ability is regulated in part at the level of the thick ascending limb of Henle’s loop, where active NaCl absorption drives the countercurrent multiplication mechanism. We used semiquantitative immunoblotting to test the effects of indomethacin and diclofenac, given over a 48-h period, on the expression levels of the ion transporters responsible for active NaCl transport in the thick ascending limb. Both agents strongly increased the expression level of the apical Na-K-2Cl cotransporter in both outer medulla and cortex. Neither agent significantly altered outer medullary expression levels of other thick ascending limb proteins, namely, the type 3 Na/H exchanger (NHE-3), Tamm-Horsfall protein, or α1- or β1-subunits of the Na-K-ATPase. Administration of the EP3-selective PGE2analog, misoprostol, to indomethacin-treated rats reversed the stimulatory effect of indomethacin on Na-K-2Cl cotransporter expression. We conclude that cyclooxygenase inhibitors enhance urinary concentrating ability in part through effects to increase Na-K-2Cl cotransporter expression in the thick ascending limb of Henle’s loop. This action is most likely due to elimination of an EP3-receptor-mediated tonic inhibitory effect of PGE2on cAMP production.
Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells
Authors: Moyer Bryan D. , Loffing-Cueni Dominique , Loffing Jan , Reynolds Donna , Stanton Bruce A.
Published: December 2017

Sodium butyrate and its derivatives are useful therapeutic agents for the treatment of genetic diseases including urea cycle disorders, sickle cell disease, thalassemias, and possibly cystic fibrosis (CF). Butyrate partially restores cAMP-activated Cl−secretion in CF epithelial cells by stimulating ΔF508 cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) gene expression and increasing the amount of ΔF508-CFTR in the plasma membrane. Because the effect of butyrate on Cl−secretion by renal epithelial cells has not been reported, we examined the effects of chronic butyrate treatment (15–18 h) on the function, expression, and localization of CFTR fused to the green fluorescent protein (GFP-CFTR) in stably transfected MDCK cells. We report that sodium butyrate reduced Cl−secretion across MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold and increased apical membrane Cl−currents 30-fold. Although butyrate also increased Na-K-ATPase protein expression twofold, the drug reduced the activity of the Na-K-ATPase by 55%. Our findings suggest that butyrate inhibits cAMP-stimulated Cl−secretion across MDCK cells in part by reducing the activity of the Na-K-ATPase.
Lysophosphatidylcholine activates mesangial cell PKC and MAP kinase by PLCγ-1 and tyrosine kinase-Ras pathways
Authors: Bassa Babu V. , Roh Daeyoung D. , Vaziri Nosratola D. , Kirschenbaum Michael A. , Kamanna Vaijinath S.
Published: December 2017

Although lysophosphatidylcholine (LPC)-mediated cellular responses are attributed to the activation of protein kinase C (PKC), relatively little is known about the upstream signaling mechanisms that regulate the activation of PKC and downstream mitogen-activated protein (MAP) kinase. LPC activated p42 MAP kinase and PKC in mesangial cells. LPC-mediated MAP kinase activation was inhibited (but not completely) by PKC inhibition, suggesting additional signaling events. LPC stimulated protein tyrosine kinase (PTK) activity and induced Ras-GTP binding. LPC-induced MAP kinase activity was blocked by the PTK inhibitor genistein. Because LPC increased PTK activity, we examined the involvement of phospholipase Cγ-1 (PLCγ-1) as a key participant in LPC-induced PKC activation. LPC stimulated the phosphorylation of PLCγ-1. PTK inhibitors suppressed LPC-induced PKC activity, whereas the same had no effect on phorbol 12-myristate 13-acetate-mediated PKC activity. Other lysophospholipids [e.g., lysophosphatidylinositol and lysophosphatidic acid (LPA)] also induced MAP kinase activity, and only LPA-induced MAP kinase activation was sensitive to pertussis toxin. These results indicate that LPC-mediated PKC activation may be regulated by PTK-dependent activation of PLCγ-1, and both PKC and PTK-Ras pathways are involved in LPC-mediated downstream MAP kinase activation.
Cortical and medullary betaine-GPC modulated by osmolality independently of oxygen in the intact kidney
Authors: Cowin Gary J. , Crozier Stuart , Endre Zoltan H. , Leditschke I. Anne , Brereton Ian M.
Published: December 2017

Renal osmolyte concentrations are reduced during reflow following ischemia. Osmolyte decreases may follow oxygen depletion or loss of extracellular osmolality in the medulla. Image-guided volume-localized magnetic resonance (MR) microspectroscopy was used to monitor regional osmolytes during hyposmotic shock and hypoxia in the intact rat kidney. Alternate spectra were acquired from 24-μl voxels in cortex and medulla of the isolated perfused kidney. There was a progressive decrease in the combined betaine-glycerophosphorylcholine (GPC) peak intensity of 21% in cortex and 35% in medulla of normoxic kidneys between 60 and 160 min after commencing perfusion. Hypoxia had no significant effect on the betaine-GPC peak intensity in cortex or medulla, despite a dramatic reduction in tubular sodium, potassium, and water reabsorption. The results suggest that cortical and medullary intracellular osmolyte concentrations depend on osmotically regulated channels that are insensitive to oxygen and dissociated from the oxygen-dependent parameters of renal function, the fractional excretion of sodium, the fractional excretion of potassium, and urine-to-plasma inulin concentration ratio.
Nitric oxide inhibits sodium/hydrogen exchange activity in the thick ascending limb
Authors: Garvin Jeffrey L. , Hong Nancy J.
Published: December 2017

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb (TAL) expresses nitric oxide synthase (NOS). However, the effects of NO on TAL transport have not been extensively studied. We tested the hypothesis that NO inhibits apical and basolateral Na+/H+exchange by the TAL by measuring intracellular pH (pHi) of isolated, perfused rat TALs using the fluorescent dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NO donor spermine NONOate (SPM, 10 μM) decreased steady-state pHiin medullary TALs from 7.18 ± 0.13 to 7.13 ± 0.14 ( P < 0.02), whereas controls did not decrease significantly. We next measured the buffering capacity of medullary TALs and the rate at which they recovered from acid loads to investigate the mechanism whereby NO reduces steady-state pHi. SPM decreased H+flux ( JH) from 2.41 ± 0.66 to 0.97 ± 0.19 pmol ⋅ min−1⋅ mm−1, 55%. To assure that the decrease in JHwas due to NO, another donor, nitroglycerin (NTG; 10 μM), was used. NTG decreased JHfrom 1.65 ± 0.11 to 1.07 ± 0.24 pmol ⋅ min−1⋅ mm−1, 37%. To determine the relative contributions of the apical and basolateral Na+/H+exchangers, 5-( N, N-dimethyl)amiloride (DMA; 100 μM) was added to either bath or lumen. With DMA added to the bath, SPM decreased JHfrom 4.78 ± 1.08 to 2.74 ± 0.54 pmol ⋅ min−1⋅ mm−1, an inhibition of 41%; and with DMA added to the lumen, SPM decreased JHfrom 2.31 ± 0.29 to 1.74 ± 0.27 pmol ⋅ min−1⋅ mm−1, a reduction of 26%. Addition of DMA alone to both bath and lumen resulted in an 87% inhibition of JH. We conclude that NO inhibits both apical and basolateral Na+/H+exchangers and consequently may play an important role in regulating pHiand may alter acid/base balance by directly affecting bicarbonate absorption in the TAL.
Selective A2Aadenosine receptor activation reduces ischemia-reperfusion injury in rat kidney
Authors: Okusa Mark D. , Linden Joel , Macdonald Timothy , Huang Liping
Published: December 2017

A2Aadenosine receptors (A2A-ARs) are known modulators of renal hemodynamics and potent inhibitors of inflammation. We sought to determine whether selective activation of A2A-ARs protects kidneys from ischemia-reperfusion injury. The ester derivative of DWH-146 (DWH-146e), a selective A2Aagonist, was found to be more potent and selective for A2A-ARs than the prototype compound CGS-21680. Osmotic minipumps were implanted subcutaneously to infuse into rats either vehicle or DWH-146e (0.004 μg ⋅ kg−1⋅ min−1), during and after ischemia-reperfusion injury. Following 24 and 48 h of reperfusion, the rise in serum creatinine and blood urea nitrogen for vehicle-treated rats was substantially elevated compared with DWH-146e-treated rats. Histological examination revealed widespread tubular epithelial necrosis and vascular congestion in the outer medulla of vehicle-treated compared with DWH-146e-treated animals. ZM-241385, a selective A2Aantagonist, blocked the protective effect of DWH-146e. Delaying administration of DWH-146e until the initiation of reperfusion also decreased serum creatinine. We conclude that 1) selective A2A-AR activation by DWH-146e reduces ischemia-reperfusion injury in rat kidneys, 2) the effect of DWH-146e is A2Areceptor mediated, and 3) the protective effects are mediated by preventing injury during the reperfusion period.
Inhibition of PARP prevents oxidant-induced necrosis but not apoptosis in LLC-PK1cells
Authors: Filipovic Dragana M. , Meng Xianmin , Reeves W. Brian
Published: December 2017

Oxidant-induced cell injury has been implicated in the pathogenesis of several forms of acute renal failure. The present studies examined whether activation of poly(ADP-ribose)polymerase (PARP) by oxidant-induced DNA damage contributes to oxidant injury of renal epithelial cells. H2O2exposure resulted in an increase in PARP activity and decreases in cell ATP and NAD content. These changes were significantly inhibited by 10 mM 3-aminobenzamide (3-ABA), a PARP inhibitor. In contrast, H2O2-induced DNA damage was not prevented by 3-ABA. Exposure of LLC-PK1 cells to 1 mM H2O2for 2 h induced necrotic cell death as measured by increased lactate dehydrogenase (LDH) release. 3-ABA completely prevented the H2O2-induced LDH release. Live/dead fluorescent staining confirmed the protection by 3-ABA. These results are consistent with the view that oxidant-induced DNA damage activates PARP and that the subsequent ATP and NAD depletion contribute to necrotic cell death. Of note, although protected from necrosis, cells treated with H2O2and 3-ABA underwent apoptosis as evidenced by DNA fragmentation and bis-benzimide staining. In conclusion, activation of PARP contributes to oxidant-induced ATP depletion and necrosis in LLC-PK1 cells. However, PARP inhibition may target cells toward an apoptotic form of cell death.
Dexamethasone increases eNOS gene expression and prevents renal vasoconstriction induced by cyclosporin
Authors: Bobadilla Norma A. , Tapia Edilia , Jiménez Fabiola , Sánchez-Lozada Laura G. , Santamaría José , Monjardín Alberto , Bolio Alexis , Gamba Gerardo , Herrera-Acosta Jaime
Published: December 2017

Cyclosporin A (CsA)-induced renal vasoconstriction (RV) is attributed to an imbalance in vasoactive factors release. Dexamethasone (Dex) exerts a renal vasodilatory effect by a mechanism not yet characterized. This study evaluates whether the effect of Dex is mediated by NO and whether it prevents CsA-induced RV. Micropuncture studies were performed in six groups of uninephrectomized rats treated for 7 days with the following: vehicle (Veh); Veh + 4 mg/kg dexamethasone (Veh+Dex); 30 mg/kg CsA; CsA+Dex; vehicle + 10 mg/kg nitro-l-arginine methyl ester (Veh+l-NAME); and Veh+Dex+l-NAME. NO synthase (NOS) isoform mRNA levels were evaluated in renal cortex and medulla by semiquantitative RT-PCR analysis in the first four groups. Dex produced renal vasodilation, which was blocked by concomitant l-NAME administration, and the effect of Dex was associated with higher cortical and medullary endothelial NOS (eNOS) and cortical inducible NOS (iNOS) mRNA levels. In the CsA group, Dex prevented RV, restoring glomerular hemodynamics to control values. These changes were associated with further enhancement of eNOS and restoration of medullary iNOS and neuronal NOS (nNOS) expression. We conclude that Dex prevents CsA-induced RV, and its vasodilator effect could be mediated by increased intrarenal generation of NO, secondary to enhanced expression of eNOS and iNOS.
Cytosolic [Ca2+] signaling pathway in macula densa cells
Authors: Peti-Peterdi János , Bell P. Darwin
Published: December 2017

Previous micropuncture studies suggested that macula densa (MD) cells might detect variations in luminal sodium chloride concentration ([NaCl]l) through changes in cytosolic calcium ([Ca2+]c). To test this hypothesis, MD [Ca2+]cwas measured with fluorescence microscopy using fura 2 in the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney. Tubules were bathed and perfused with a Ringer solution, [NaCl]lwas varied and isosmotically replaced with N-methyl-d-glucamine cyclamate. Control [Ca2+]c, during perfusion with 25 mM NaCl and 150 mM NaCl in the bath, averaged 101.6 ± 8.2 nM ( n = 21). Increasing [NaCl]lto 150 mM elevated [Ca2+]cby 39.1 ± 5.2 nM ( n = 21, P < 0.01). This effect was concentration dependent between zero and 60 mM [NaCl]l. The presence of either luminal furosemide or basolateral nifedipine or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a potent Cl−channel blocker, significantly reduced resting [Ca2+]cand abolished the increase in [Ca2+]cin response to increased [NaCl]l. Nifedipine failed to produce a similar inhibitory effect when added exclusively to the luminal perfusate. Also, 100 nM BAY K 8644, a voltage-gated Ca2+channel agonist, added to the bathing solution increased [Ca2+]cby 33.2 ± 8.1 nM ( n = 5, P < 0.05). These observations suggest that MD cells may detect variations in [NaCl]lthrough a signaling pathway that includes Na+-2Cl−-K+cotransport, basolateral membrane depolarization via Cl−channels, and Ca2+entry through voltage-gated Ca2+channels.
Chloride dependency of renal brush-border membrane phosphate transport
Authors: Yanagawa Norimoto , Pham Chi , Shih Remi N. J. , Miao Stephen , Jo Oak Don
Published: December 2017

In our present study, we examined the effect of Cl−on rabbit renal brush-border membrane (BBM) phosphate (Pi) uptake. It was found that the Na+-dependent BBM32P uptake was significantly inhibited by Cl−replacement in the uptake solution with other anions, or by Cl−transport inhibitors, including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid (NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Intravesicular formate or Cl−increased BBM36Cl−uptake but did not affect BBM32P uptake. BBM22Na+uptake was lowered by Cl−replacement in the uptake solution but not by Cl−transport inhibitors. Changes in transmembrane electrical potential altered BBM36Cl−and32P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the Cl−-dependent BBM Piuptake was not affected by changes in transmembrane electrical potential. Finally, a similar Cl−dependency of Piuptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of Na+-dependent Piuptake was also Cl−dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this Cl−dependency remains to be identified.
A role for intracellular calcium in tight junction reassembly after ATP depletion-repletion
Authors: Ye Jiuming , Tsukamoto Tatsuo , Sun Adam , Nigam Sanjay K.
Published: December 2017

The integrity of the tight junction (TJ), which is responsible for the permeability barrier of the polarized epithelium, is disrupted during ischemic injury and must be reestablished for recovery. Recently, with the use of an ATP depletion-repletion model for ischemia and reperfusion injury in Madin-Darby canine kidney cells, TJ proteins such as zonula occludens-1 (ZO-1) were shown to reversibly form large complexes and associate with cytoskeletal proteins (T. Tsukamoto and S. K. Nigam, J. Biol. Chem. 272: 16133–16139, 1997). In this study, we examined the role of intracellular calcium in TJ reassembly after ATP depletion-repletion by employing the cell-permeant calcium chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM (BAPTA-AM). Lowering intracellular calcium during ATP depletion is associated with significant inhibition of the reestablishment of the permeability barrier following ATP repletion as measured by transepithelial electrical resistance and mannitol flux, marked alterations in the subcellular localization of occludin by immunofluorescent analysis, and decreased solubility of ZO-1 and other TJ proteins by Triton X-100 extraction assay, suggesting that lowering intracellular calcium potentiates the interaction of TJ proteins with the cytoskeleton. Coimmunoprecipitation studies indicated that decreased solubility may partly result from the stabilization of large TJ protein-containing complexes with fodrin. Although ionic detergents (SDS and deoxycholate) appeared to cause a dissociation of ZO-1-containing complexes from the cytoskeleton, sucrose gradient analyses of the solubilized proteins suggested that calcium chelation leads to self-association of these complexes. Together, these results raise the possibility that intracellular calcium plays an important facilitatory role in the reassembly of the TJ damaged by ischemic insults. Calcium appears to be necessary for the dissociation of TJ-cytoskeletal complexes, thus permitting functional TJ reassembly and paracellular permeability barrier recovery.
Capacitative calcium entry in smooth muscle cells from preglomerular vessels
Authors: Fellner Susan K. , Arendshorst William J.
Published: December 2017

Calcium entry via voltage-gated L-type channels is responsible for at least half of the increase in cytosolic calcium ([Ca2+]i) in afferent arterioles following agonist stimulation. We sought the presence of capacitative calcium entry in fresh vascular smooth muscle cells (VSMC) derived from rat preglomerular vessels. [Ca2+]iwas measured using fura-2 ratiometric fluorescence. Vasopressin V1 receptor agonist (V1R) (10−7M) increased [Ca2+]iby ∼100 nM. A calcium channel blocker (CCB), nifedipine or verapamil (10−7M), inhibited the response by ∼50%. V1R in the presence of CCB increased [Ca2+]ifrom 106 to 176 nM, confirming that calcium mobilization and/or entry may occur independent of voltage-gated channels. In nominally Ca2+-free buffer, V1R increased [Ca2+]ifrom 94 to 129 nM, denoting mobilization; addition of CaCl2(1 mM) further elevated [Ca2+]ito 176 nM, indicating a secondary phase of Ca2+entry. Similar responses were obtained when CCB was present in calcium-free buffer or when EGTA was present. In nominally Ca2+-free medium, the sarcoplasmic reticulum Ca2+-ATPase inhibitors (SRCAI), thapsigargin and cyclopiazonic acid (CPA), increased [Ca2+]ifrom 97 to 128 and 143 nM, respectively, and to 214 and 220 nM, respectively, when 1 mM extracellular Ca2+was added. In the presence of verapamil, the results with CPA acid were nearly identical. In Ca2+-free buffer, the stimulatory effect of V1R or SRCAI on the Ca2+/fura signal was quenched by the addition of Mn2+(1 mM), demonstrating divalent cation entry. These studies provide evidence for capacitative (store- operated) calcium entry in VSMC freshly isolated from rat preglomerular arterioles.
NaPO4cotransport type III (PiT1) expression in human embryonic kidney cells and regulation by PTH
Authors: Fernandes Isabelle , Béliveau Richard , Friedlander Gérard , Silve Caroline
Published: December 2017

The aim of the present study was to characterize the type(s) of NaPO4cotransporter expressed in the human renal cell line HEK-293 and its regulation by parathyroid hormone (PTH) in wild-type cells and in cells transfected by the PTH/PTH-related protein (PTHrP) receptor. The results showed that human embryonic kidney HEK-293 cells expressed NaPO4cotransporter type III (PiT1) mRNA and protein. In contrast, type I (NPT1) or II (NPT2) cotransporter mRNA were not expressed. Na+-dependent phosphate uptake followed a Michaelis-Menten model (apparent maximal transport rate and affinity constant: 23.32 ± 0.69 nmol PO4⋅ mg protein−1⋅ 10 min−1and 0.147 ± 0.014 mM KH2PO4, respectively), was stimulated by phosphate deprivation (maximal increase 24.5 ± 0.8%, P < 0.001, after 15 h of phosphate deprivation), and was inhibited by increasing pH (3.6 ± 0.2-fold decrease at pH 8.5, P < 0.0001). It was inhibited in a time- and concentration-dependent fashion by PTH in HEK-293 cells stably transfected by PTH/PTHrP receptors but not in parental HEK-293 cells. Maximal inhibition of Na+-dependent phosphate transport was observed at 30 min after the addition of 72 nM PTH-(1—34) (31.5 ± 2.4% inhibition, P < 0.01). PTH inhibition of phosphate transport was maintained in phosphate-deprived cells and reversed by both GF109203X (10−6M) or staurosporine (5.5 nM), two protein kinase C inhibitors. Na+-dependent phosphate uptake was also significantly inhibited by phorbol 12-myristate 13-acetate (20.9 ± 3.9% inhibition, P < 0.001) but not by dibutyril-cAMP (10−4M) or forskolin (50 μM). The physiological role played by type III NaPO4cotransport expression in the overall renal regulation of phosphate homeostasis remains to be established.
Mechanisms of HCO 3 − secretion in the rabbit connecting segment
Authors: Tsuruoka Shuichi , Schwartz George J.
Published: December 2017

The connecting tubule (CNT) contains α-(H+-secreting) and β-([Formula: see text]-secreting) intercalated cells and is therefore likely to contribute to acid-base homeostasis. To characterize the mechanisms of [Formula: see text]transport in the rabbit CNT, in which there is little definitive data presently available, we microdissected the segments from the superficial cortical labyrinth, perfused them in vitro, measured net[Formula: see text] transport ( J[Formula: see text]) by microcalorimetry, and examined the effects of several experimental maneuvers. Mean ± SE basal J[Formula: see text]was −3.4 ± 0.1 pmol ⋅ min−1⋅ mm−1(net [Formula: see text] secretion), and transepithelial voltage was −13 ± 1 mV ( n = 47). Net[Formula: see text] secretion was markedly inhibited by removal of luminal Cl−or application of basolateral H+-ATPase inhibitors (bafilomycin or concanamycin), maneuvers that inhibit β-intercalated cell function. Net [Formula: see text] secretion was not affected by inhibitors of α-intercalated cell function (basolateral Cl−removal, basolateral DIDS, or luminal H+-ATPase inhibitors). Net [Formula: see text] secretion was stimulated by isoproterenol and inhibited by acetazolamide. These data indicate that 1) CNTs secrete[Formula: see text] via an apical DIDS-insensitive Cl−/[Formula: see text]exchanger, mediated by a basolateral bafilomycin- and concanamycin-sensitive H+-ATPase; 2) inhibition of cytosolic carbonic anhydrase decreases [Formula: see text] secretion; and 3) stimulation of β-adrenergic receptors increases [Formula: see text] secretion. The failure to influence net [Formula: see text]transport by inhibiting α-intercalated cell apical H+-ATPases or basolateral Cl−/[Formula: see text]exchange suggests that the CNT has fewer functioning α-intercalated cells than the cortical collecting duct. These are the first studies to examine the rate and mechanisms of[Formula: see text] secretion by the rabbit CNT; this is clearly an important segment in mediating acid-base homeostasis.
Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice
Authors: Igarashi Peter , Shashikant Cooduvalli S. , Thomson R. Brent , Whyte Dilys A. , Liu-Chen Shuxian , Ruddle Frank H. , Aronson Peter S.
Published: December 2017

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of β-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.
Dissimilar mechanisms of Ca2+response to bradykinin in different types of juxtamedullary glomerular arterioles
Authors: Praddaude F. , Marchetti J. , Alhenc-Gelas F. , Ader J.-L.
Published: December 2017

Bradykinin (BK)-induced changes in intracellular calcium level ([Ca2+]i) were studied on fura 2-loaded afferent (AA) and efferent glomerular arterioles (EA) microdissected from juxtamedullary renal cortex. A distinction was made between thin and muscular EA. In AA and both types of EA, BK increased [Ca2+]ithrough activation of B2receptors located only on the endothelium. The responses were not affected by nifedipine (10−6M) and were smaller in a Ca2+-free medium, providing evidence that BK opens voltage-independent Ca2+channels and mobilizes intracellular Ca2+. Thin EA differed from AA and muscular EA by a lower sensitivity to BK (EC50= 6.95 ± 3.81 vs. 0.21 ± 0.08 and 0.18 ± 0.13 nM, respectively; P < 0.05), a higher maximal response (89 ± 5 vs. 57 ± 5 and 44 ± 7 nM; P < 0.001), and a spontaneous return to basal Ca2+level, even in the presence of BK. Genistein (10−4M) and herbimycin A (25 × 10−6M), specific inhibitors of tyrosine kinases, inhibited the [Ca2+]iresponses exclusively in AA. Genistein reduced the peak and plateau phases of responses by 69 ± 9 and 82 ± 6%, respectively, in a medium with Ca2+and the peak by 48 ± 9% in a Ca2+-free medium. Similar reductions were observed with herbimycin A. These results show that dissimilar signal transduction pathways are involved in BK effects on juxtamedullary arterioles and that a tyrosine kinase activity could participate in the regulation of BK effect on AA but not on EA.
Albumin is a major serum survival factor for renal tubular cells and macrophages through scavenging of ROS
Authors: Iglesias José , Abernethy Vivian Elizabeth , Wang Zhiyong , Lieberthal Wilfred , Koh Jason S. , Levine Jerrold S.
Published: December 2017

We have previously shown that lysophosphatidic acid (LPA), an abundant serum lipid that binds with high affinity to albumin, is a potent survival factor for mouse proximal tubular cells and peritoneal macrophages. We show here that BSA also has potent survival activity independent of bound lipids. Delipidated BSA (dBSA) protected cells from apoptosis induced by FCS withdrawal at concentrations as low as 1% of that in FCS. dBSA did not activate phosphatidylinositol 3-kinase, implying that its survival activity occurs via a mechanism distinct from that for most cytokines. On the basis of the following evidence, we propose that dBSA inhibits apoptosis by scavenging reactive oxygen species (ROS): 1) FCS withdrawal leads to ROS accumulation that is inhibitable by dBSA; 2) during protection from apoptosis, sulfhydryl and hydroxyl groups of dBSA are oxidized; and 3) chemical blockage of free sulfhydryl groups or preoxidation of dBSA with H2O2removes its survival activity. Moreover, dBSA confers almost complete protection from cell death in a well-established model of oxidative injury (xanthine/xanthine oxidase). These results implicate albumin as a major serum survival factor. Inhibition of apoptosis by albumin occurs through at least two distinct mechanisms: carriage of LPA and scavenging of ROS.
In vitro metabolic and respiratory acidosis selectively inhibit osteoblastic matrix gene expression
Authors: Frick Kevin K. , Bushinsky David A.
Published: December 2017

Clinically, a decrease in blood pH may be due to either a reduction in bicarbonate concentration ([H[Formula: see text]], metabolic acidosis) or an increase in[Formula: see text] (respiratory acidosis). In mammals, metabolic acidosis induces a far greater increase in urine calcium excretion than respiratory acidosis. In cultured bone, metabolic acidosis induces a marked increase in calcium efflux and a decrease in osteoblastic collagen synthesis, whereas isohydric respiratory acidosis has little effect on either parameter. We have shown that metabolic acidosis prevents the normal developmental increase in the expression of RNA for matrix Gla protein and osteopontin in chronic cultures of primary murine calvarial bone cells (predominantly osteoblasts) but does not alter expression of osteonectin. To compare the effects of isohydric metabolic and respiratory acidosis on expression of these genes, bone cell cultures were incubated in medium at pH ∼7.2 to model metabolic ([H[Formula: see text]], ∼13 mM) or respiratory ([Formula: see text], ∼80 mmHg) acidosis or at pH ∼7.4 as a control. Cells were sampled at weeks 4, 5, and 6 to assess specific RNA content. At all time periods studied, both metabolic and respiratory acidosis inhibited the expression of RNA for matrix Gla protein and osteopontin to a similar extent, whereas there was no change in osteonectin expression. In contrast to the significant difference in the effects of metabolic and respiratory acidosis on bone calcium efflux and osteoblastic collagen synthesis, these two forms of acidosis have a similar effect on osteoblastic RNA expression of both matrix Gla protein and osteopontin. Thus, although several aspects of bone cell function are dependent on the type of acidosis, expression of these two matrix genes appears to be regulated by extracellular pH, independently of the type of acidosis.
A comparison of the natriuretic/diuretic effects of rat vs. human leptin in the rat
Authors: Jackson Edwin K. , Herzer William A.
Published: December 2017

Intrarenal artery infusions of low-dose human, but not mouse, leptin cause diuresis/natriuresis in rats [E. K. Jackson and P. Li. Am. J. Physiol. 272 ( Renal Physiol. 41): F333–F338, 1997]. The lack of effect of mouse leptin in the rat could be due to slight differences in the primary structure of mouse vs. rat leptin. To test this hypothesis, we infused single doses of rat (0.1, 0.3, 1, or 3 μg/min) or human (3 μg/min) leptin into the renal artery of rats for 140 min while continuously measuring blood pressure and the renal excretion rate of urine and electrolytes. Intrarenal infusions of rat leptin did not alter any measured parameter. Human leptin caused a delayed diuresis/natriuresis ( P < 0.0006 and P < 0.0049, respectively) that required ∼2 h to achieve a maximum effect and that was not accompanied by changes in blood pressure or potassium excretion. We conclude that low-dose human, but not low-dose rodent, leptin has direct diuretic/natriuretic activity. Our results can be explained from an evolutionary perspective, since obesity-induced hypertension would be a much greater selective force in hominids compared with rodents.
Inhibition of NFκB activation with antioxidants is correlated with reduced cytokine transcription in PTC
Authors: Rangan Gopala K. , Wang Yiping , Tay Yuet-Ching , Harris David C. H.
Published: December 2017

We recently reported that inhibition of the transcription factor nuclear factor-κB (NFκB) with pyrrolidinedithiocarbamate (PDTC) reduced interstitial monocyte infiltration in rats with proteinuric tubulointerstitial disease, whereas N-acetylcysteine (NAC) was not effective. Here we investigate the effects of antioxidants (PDTC, NAC, and quercetin) on NFκB activation and cytokine transcription in primary cultured rat proximal tubular epithelial cells (PTC) stimulated with lipopolysaccharide. Antioxidant-mediated inhibition of NFκB activation (PDTC, 20–100 μM; NAC, 100 mM; and quercetin, 50 μM) diminished the induction of both pro- [interleukin (IL)-1β, tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1α, and MIP-2] and anti-inflammatory (IL-10, transforming growth factor-β1) cytokine transcription in PTC (RT-PCR analysis). PDTC and quercetin did not affect PTC viability, but NAC (100 mM) caused a threefold increase in lactate dehydrogenase leakage ( P < 0.001). We conclude that NAC is unable to suppress NFκB activation in PTC at subtoxic and physiologically relevant concentrations. Furthermore, antioxidant-mediated inhibition of NFκB is correlated with the nonselective reduction of cytokine transcription in activated tubular cells. These data might explain the protective effects of PDTC-mediated NFκB inhibition in tubulointerstitial disease in vivo.
Cisplatin induces renal expression of P-glycoprotein and canalicular multispecific organic anion transporter
Authors: Demeule Michel , Brossard Mathieu , Béliveau Richard
Published: December 2017

The expression of two members of the ATP-binding cassette family of transport proteins, P-glycoprotein (P-gp) and the canalicular multispecific organic anion transporter (cMOAT or Mrp2), was evaluated in renal brush-border membranes (BBM) and various rat tissues after cisplatin treatment. One administration of cisplatin (5 mg/kg) increased P-gp expression by >200–300% in renal BBM and in crude membranes from liver and intestine. The increase in P-gp expression in the kidney was also detected in photolabeling experiments, suggesting the induction of functional P-gp. cMOAT expression was increased by >10-fold in renal BBM after cisplatin administration, although it had no effect on liver cMOAT expression. The increase in the levels of both proteins was maximal at 2 days after cisplatin treatment and lasted for at least 8 days. These results indicate that a single administration of cisplatin induces overexpression of P-gp and cMOAT in specific tissues. This may be of significant relevance to the design of clinical trials using cisplatin as a single chemotherapeutic agent or in combination with other drugs.