bluuebunny/crossref_metadata_2025 huggingface dataset:
Spectral properties of the tubuloglomerular feedback system
Authors: Layton H. E. , Pitman E. Bruce , Moore Leon C.
Published: December 2017

A simple mathematical model was used to investigate the spectral properties of the tubuloglomerular feedback (TGF) system. A perturbation, consisting of small-amplitude broad-band forcing, was applied to simulated thick ascending limb (TAL) flow, and the resulting spectral response of the TGF pathway was assessed by computing a power spectrum from resulting TGF-regulated TAL flow. Power spectra were computed for both open- and closed-feedback-loop cases. Open-feedback-loop power spectra are consistent with a mathematical analysis that predicts a nodal pattern in TAL frequency response, with nodes corresponding to frequencies where oscillatory flow has a TAL transit time that equals the steady-state fluid transit time. Closed-feedback-loop spectra are dominated by the open-loop spectral response, provided that γ, the magnitude of feedback gain, is less than the critical value γc required for emergence of a sustained TGF-mediated oscillation. For γ exceeding γc, closed-loop spectra have peaks corresponding to the fundamental frequency of the TGF-mediated oscillation and its harmonics. The harmonics, expressed in a nonsinusoidal waveform for tubular flow, are introduced by nonlinear elements of the TGF pathway, notably TAL transit time and the TGF response curve. The effect of transit time on the flow waveform leads to crests that are broader than troughs and to an asymmetry in the magnitudes of increasing and decreasing slopes. For feedback gain magnitude that is sufficiently large, the TGF response curve tends to give a square waveshape to the waveform. Published waveforms and power spectra of in vivo TGF oscillations have features consistent with the predictions of this analysis.
Cloning and localization of a double-pore K channel, KCNK1: exclusive expression in distal nephron segments
Authors: Orias Marcelo , Velázquez Heino , Tung Freeman , Lee George , Desir Gary V.
Published: December 2017

The K-selective channel, TOK1, recently identified in yeast, displays the unusual structural feature of having two putative pore regions, in contrast to all previously cloned K channels. Using the TOK1 pore regions as probes, we identified a human kidney cDNA encoding a 337-amino acid protein (hKCNK1) with four transmembrane segments and two pore regions containing the signature sequence of K channels. Amino acid identity to TOK1 is only 15% overall but 40% at the pores. Northern analysis indicates high expression of a 1.9-kb message in brain > kidney >> heart. Nephron segment localization, carried out in rabbit by reverse transcription-polymerase chain reaction, reveals that KCNK1 is expressed in cortical thick ascending limb, connecting tubule, and cortical collecting duct. It was not detected in the proximal tubule, medullary thick ascending limb, distal convoluted tubule, and glomerulus. We conclude that KCNK1 is a unique, double-pore, mammalian K channel, distantly related to the yeast channel TOK1, that is expressed in distal tubule and is a candidate to participate in renal K homeostasis.
An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole
Authors: Yip Kay-Pong , Marsh Donald J.
Published: December 2017

The potential role of integrins in the myogenic mechanism was studied in the rat afferent arteriole (AA) by fluorescence immunolocalization and microperfusion of isolated AA. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for various integrin β- and α-subunits. The β1-, β3-, α3-, α5-, and αV-integrins were found on the plasma membrane in smooth muscle of AA, providing the morphological basis for participation of integrins in mechanotransduction. With 1 mM nitro-l-arginine methyl ester (l-NAME) in the luminal perfusate to inhibit endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP (10−7–10−3M) induced immediate vasoconstriction. The constriction was dose dependent and specific for peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins. In controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM, induced a 21.6 ± 2.6% decrease ( P < 0.05, n = 6) in lumen diameter for 30 s and an 18.3 ± 4.1% increase ( P < 0.05, n = 6) in smooth muscle intracellular calcium concentration for 18 s, as measured by the emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with exposed integrins on AA smooth muscle therefore triggers calcium-dependent vasoconstriction. However, the dose response to RGD was not sensitive to the myogenic tone of the vessel, which suggests that the integrin-mediated vasoconstriction is different from myogenic constriction.
Determination of NH 4 + /NH3fluxes across apical membrane of macula densa cells: a quantitative analysis
Authors: Laamarti M. Anuar , Lapointe Jean-Yves
Published: December 2017

Luminal addition of 20 mM [Formula: see text]produced a rapid acidification of rabbit macula densa (MD) cells from 7.50 ± 0.06 to 6.91 ± 0.05 at an initial rate of 0.071 ± 0.008 pH unit/s. In the luminal presence of 5 μM bumetanide, 5 mM Ba2+or both, the acidification rate was reduced by 57%, 35% and 93% of control levels. In contrast, intracellular pH (pHi) recovery after removing luminal [Formula: see text] was unaffected by bumetanide and Ba2+but was sensitive to 1 mM luminal amiloride (71% inhibition). The bumetanide-sensitive acidification rate represents most certainly the[Formula: see text] flux mediated by the apical Na+:K+([Formula: see text]):2Cl−cotransporter, but the Ba2+-sensitive portion does not seem to be associated with the apical K+channels previously characterized by us. The effects of [Formula: see text]entry across the apical membrane were simulated using a simple model involving five adjustable parameters: apical and basolateral permeabilities for [Formula: see text] and NH3and a parameter describing a pH-regulating mechanism. The model shows that the apical membrane of MD cells is much more permeable to NH3than it is to[Formula: see text] and, under control conditions, the apical [Formula: see text] flux appears surprisingly high (11–20 mM/s) and challenges the notion that MD cells present a low intensity of ionic transport.
Cl− channels in basolateral renal medullary membranes XII. Anti-rbClC-Ka antibody blocks MTAL Cl−channels
Authors: Winters Christopher J. , Zimniak Ludwika , Reeves W. Brian , Andreoli Thomas E.
Published: December 2017

Cl− channels in the medullary thick ascending limb (MTAL) studied by either patch-clamp technique or reconstitution into lipid bilayers are activated by increases in intracellular Cl−concentrations. rbClC-Ka, a ClC Cl− channel, may represent this channel. We therefore evaluated the role of rbClC-Ka in transcellular MTAL Cl− transport in two separate ways. First, an antibody was raised against a fusion protein containing a 153-amino acid fragment of rbClC-Ka. Immunostaining of rabbit kidney sections with the antibody was localized to basolateral regions of MTAL and cortical thick ascending limb (CTAL) segments and also to the cytoplasm of intercalated cells in the cortical collecting duct. Second, Cl− uptake and efflux were measured in suspensions of mouse MTAL segments. Cl− uptake was bumetanide sensitive and was stimulated by treatment with a combination of vasopressin + forskolin + dibutyryl adenosine 3′,5-cyclic monophosphate (DBcAMP). Cl− efflux was also increased significantly by vasopressin + forskolin + DBcAMP from 114 ± 20 to 196 ± 36 nmol ⋅ mg protein−1 ⋅ 45 s−1( P = 0.003). Cl− efflux was inhibited by the Cl− channel blocker diphenylamine-2-carboxylate (154 ± 26 vs. 70 ± 21 nmol ⋅ mg protein−1 ⋅ 45 s−1, P = 0.003). An anti-rbClC-Ka antibody, which inhibits the activity of MTAL Cl− channels in lipid bilayers, reduced Cl− efflux from intact MTAL segments (154 ± 28 vs. 53 ± 14 nmol ⋅ mg protein−1 ⋅ 45 s−1, P = 0.02). These results support the view that rbClC-Ka is the basolateral membrane Cl− channel that mediates vasopressin-stimulated net Cl− transport in the MTAL segment.
Na+-dependent purine nucleoside transporter from human kidney: cloning and functional characterization
Authors: Wang Juan , Su Sheng-Fang , Dresser Mark J. , Schaner Marci E. , Washington Carla B. , Giacomini Kathleen M.
Published: December 2017

Many purine nucleosides and their analogs are actively transported in the kidney. Using homology cloning strategies and reverse transcriptase-polymerase chain reactions, we isolated a cDNA encoding a Na+-dependent nucleoside transporter, hSPNT1, from human kidney. Functional expression in Xenopus laevis oocytes identified hSPNT1 as a Na+-dependent nucleoside transporter that selectively transports purine nucleosides but also transports uridine. The Michaelis constant ( Km) of uridine (80 μM) in interacting with hSPNT1 was substantially higher than that of inosine (4.5 μM). hSPNT1 (658 amino acids) is 81% identical to the previously cloned rat Na+-nucleoside transporter, SPNT, but differs markedly from SPNT in terms of its primary structure in the NH2terminus. In addition, an Alu repetitive element (∼282 bp) is present in the 3′-untranslated region of the hSPNT1 cDNA. Northern analysis revealed that multiple transcripts of hSPNT1 are widely distributed in human tissues including human kidney. In contrast, rat SPNT transcripts are absent in kidney and highly localized to liver and intestine. The hSPNT1 gene was localized to chromosome 15. This is the first demonstration of a purine nucleoside transporter in human kidney.
Effect of reversible and irreversible ischemia on marker enzymes of BBM from renal cortical PT subpopulations
Authors: Khundmiri Syed Jalal , Asghar Mohammed , Khan Farah , Salim Samina , Yusufi Ahad Noor Khan
Published: December 2017

The effect of the reversible and relatively irreversible ischemia induced acute renal failure (ARF) in the activities of alkaline phosphatase (AlkPase) and γ-glutamyltransferase (GGTase) after early (15–30 min) and prolonged (45–60 min) ischemia in the homogenates, and the brush-border membranes (BBM) from rat renal whole, superficial (SC), and juxtamedullary (JMC) cortices were studied. The enzyme activities declined progressively in proportion to the duration of ischemia. Early blood reflow of 15 min to the ischemic rats caused a further decrease in the enzyme activities. However, prolonged reflow (up to 120 min) resulted in partial reversal of the ischemic effect in the early but not in the prolonged ischemic rats. The decrease in the enzyme activities was due to the loss of membrane-bound enzyme components from the damaged BBM into the supernatant fraction as membrane-free enzymes. The activities of AlkPase and GGTase were significantly more decreased by the ischemia in the brush-border membrane vesicles (BBMV)-JMC than in BBMV-SC. The rate of recovery due to reflow for AlkPase was greater in BBMV-SC than apparently for GGTase in BBMV-JMC in early ischemic (15–30 min) rats.
Ouabain reduces net acid secretion and increases pHiby inhibiting NH 4 + uptake on rat tIMCD Na+-K+-ATPase
Authors: Wall Susan M.
Published: December 2017

In the rat terminal inner medullary collecting duct (tIMCD), Na+pump inhibition reduces transepithelial net acid secretion ( JtAMM) [ JH= total CO2absorption ( JtCO2) + total ammonia secretion] and increases resting intracellular pH (pHi). The increase in pHiand reduction in JHthat follow ouabain addition do not occur in the absence of[Formula: see text] nor when [Formula: see text]is substituted with another weak base. The purpose of this study was to explore the mechanism of the [Formula: see text]-dependent reduction in JtCO2and increase in pHithat follow ouabain addition. We hypothesized that [Formula: see text]enters the tIMCD cell through the Na+-K+-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH4Cl. Since K+and[Formula: see text] compete for a common binding site on the Na+pump, increasing extracellular K+should limit[Formula: see text] (and hence net H+) uptake by the Na+pump. Upon increasing extracellular K+concentration from 3 to 12 mM, the [Formula: see text]-dependent, ouabain-induced increase in pHiand reduction in JtCO2were attenuated. In the presence but not in the absence of[Formula: see text], reducing Na+pump activity by limiting Na+entry reduced JtCO2and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of[Formula: see text]/CO2and was not reproduced with BaCl2or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na+-K+-2Cl−cotransporter or a [Formula: see text] transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell,[Formula: see text] uptake is mediated by the Na+-K+-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K+loading.
Chronic hypoxia induces proliferation of cultured mesangial cells: role of calcium and protein kinase C
Authors: Sahai Atul , Mei Changlin , Pattison Timothy A. , Tannen Richard L.
Published: December 2017

The effect of hypoxia on the proliferation of cultured rat mesangial cells was examined. To evaluate the underlying signaling mechanisms, the roles of intracellular calcium ([Ca2+]i) and protein kinase C (PKC) were determined. Quiescent cultures were exposed to hypoxia (3% O2) or normoxia (18% O2), and [3H]thymidine incorporation, cell number, [Ca2+]i, and PKC were assessed. Mesangial cells exposed to 28 h of hypoxia exhibited a significant increase in [3H]thymidine incorporation followed by a significant increase in cell number at 72 h in comparison with respective normoxic controls. Hypoxia induced a biphasic activation of PKC, reflected by translocation of the enzyme activity from cytosol to membrane at 1 h, a return to baseline at 4 and 8 h, with subsequent reactivation from 16 to 48 h. In addition, hypoxia-induced proliferation was prevented by a PKC inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Cells exposed to hypoxia produced progressive increases in resting [Ca2+]ifrom 15 to 60 min which remain sustained up to 24 h of examination. Verapamil significantly prevented the hypoxia-induced proliferation, and both verapamil treatment and incubations in a calcium-free medium for 1 h blocked the hypoxia-induced stimulation of [Ca2+]ias well as PKC. These results provide the first in vitro evidence that chronic hypoxia induces proliferation of cultured glomerular mesangial cells, which is mediated by the stimulation of [Ca2+]iand the subsequent activation of PKC.
Salt loading enhances rat renal TxA2/PGH2receptor expression and TGF response to U-46,619
Authors: Welch William J. , Peng Bo , Takeuchi Kazuhisa , Abe Keishi , Wilcox Christopher S.
Published: December 2017

The tubuloglomerular feedback (TGF) response is potentiated by thromboxane A2(TxA2) and/or prostaglandin endoperoxide (PGH2) acting on specific receptors. Infusion of the TxA2/PGH2mimetic, U-46,619, into conscious rats leads to hypertension that is potentiated by a high-salt intake. Therefore, we tested the hypothesis that a high-salt intake enhances the expression of transcripts for TxA2/PGH2receptors in the kidney and glomeruli and enhances the response of TGF to TxA2/PGH2receptor stimulation. Groups of rats were accommodated to a low-salt (LS), normal salt (NS), or high-salt (HS) diet for 8–10 days. TxA2/PGH2receptor mRNA was detected by reverse transcription-polymerase chain reaction in kidney cortex, isolated glomeruli, and abdominal aorta. TxA2/PGH2mRNA abundance was significantly ( P< 0.001) increased during intake of high-salt compared with low-salt diets in the kidney cortex (1.34 ± 0.10 vs. 0.84 ± 0.04 arbitrary units) and isolated outer cortical glomeruli (0.68 ± 0.04 vs. 0.32 ± 0.03 arbitrary units), but there was no effect of salt on TxA2/PGH2receptor mRNA expression in the aorta. Maximal TGF responses were assessed from the increase in proximal stop flow pressure (an index of glomerular capillary pressure) during increases in loop of Henle perfusion with artificial tubular fluid from 0 to 40 nl/min. Compared with vehicle, the enhancement of maximal TGF with U-46,619 (10−6M) added to the perfusate was greater in rats adapted to high-salt than normal salt (HS: +9.6 ± 1.1 vs. NS: +5.1 ± 0.4 mmHg; P < 0.001) or low-salt (LS: +3.8 ± 1.3 mmHg; P < 0.001) intakes. Responses to U-46,619 at each level of salt intake were blocked by >70% by the TxA2/PGH2receptor antagonist ifetroban. In contrast, enhancement of TGF by peritubular capillary perfusion of arginine vasopressin (AVP; 10−7M) was similar in high-salt and low-salt rats (HS: +1.5 ± 0.6 vs. LS: +1.6 ± 0.5 mmHg; not significant). We conclude that salt loading increases selectively the abundance of TxA2/PGH2receptor transcripts in the kidney cortex and glomerulus, relative to the aorta, and enhances selectively TGF responses to TxA2/PGH2receptor activation but not to AVP.
Young SHR express increased type 1 angiotensin II receptors in renal proximal tubule
Authors: Cheng Hui-Fang , Wang Jun-Ling , Vinson Gavin P. , Harris Raymond C.
Published: December 2017

A potential role for the renin-angiotensin system (RAS) in the development and/or maintenance of hypertension in the genetic model of rat hypertension, spontaneously hypertensive rats (SHR), has been suggested by studies indicating that treatment of immature animals with angiotensin-converting enzyme (ACE) inhibitors prevents subsequent development of hypertension. Because young SHR also demonstrate RAS-dependent increased sodium retention, we examined proximal tubule type 1 angiotensin II receptor (AT1R) mRNA expression in young (4 wk) or adult (14 wk) SHR compared with age-matched Wistar-Kyoto (WKY) rats. Proximal tubules were isolated by Percoll gradient centrifugation, and AT1R mRNA expression was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). At 14 wk, when SHR had established hypertension [mean arterial blood pressure (MAP) of SHR vs. WKY: 145 ± 6 vs. 85 ± 5 mmHg, n = 14–15], there were no differences in proximal tubule AT1R mRNA levels [SHR vs. WKY: 79 ± 14 vs. 72 ± 14 counts/min (cpm) per cpm mutant AT1R per cpm β-actin × 10−6, n = 6; not significant (NS)]. In contrast, in 4 wk SHR, at a time of minimal elevations in blood pressure (MAP: 70 ± 8 vs. 63 ± 3), SHR proximal tubule AT1R mRNA levels were 263 ± 30% that of WKY (143 ± 18 vs. 60 ± 11 cpm per cpm of mutant AT1R per cpm β-actin × 10−6, n = 8; P < 0.005). We have recently shown that chronic ACE inhibition decreases proximal tubule AT1R expression and have also shown that chronicl-3,4-dihydroxyphenylalamine (l-DOPA) administration inhibits AT1R expression in adult Sprague-Dawley proximal tubule and cultured proximal tubule, and this inhibition is mediated via Gs-coupled DA1receptors. When 3-wk-old animals were given l-DOPA or captopril for 1 wk, MAP was not altered (70 ± 8 vs. 60 ± 4 or 61 ± 5 mmHg), but proximal tubule AT1R mRNA was no longer significantly different between SHR and WKY (68 ± 9 vs. 38 ± 7 or 20 ± 3 vs. 47 ± 15 cpm per cpm of mutant AT1R per cpm β-actin × 10−6), due to a significant decrease in proximal tubule AT1R expression in SHR ( P < 0.005, compared with untreated SHR). Immunoreactive proximal tubule AT1R expression also was increased in 4 wk SHR and was reversed with captopril orl-DOPA treatment. Therefore, these results indicate that young, but not adult, SHR have increased expression of proximal tubule AT1R and that chronic l-DOPA or captopril treatment decreased the elevated AT1R expression to control levels. These results provide further support for an important role of the RAS in the development of hypertension in SHR.
Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules
Authors: Doctor R. Brian , Chen Jing , Peters Luanne L. , Lux Samuel E. , Mandel Lazaro J.
Published: December 2017

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3200/215. To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3200/215but no Ank3170or Ank3120. Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3200/215. These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3200/215and Na-K-ATPase abundance measured a lower Ank3200/215-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3200/215suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.
Effect of α-ketoglutarate on organic anion transport in single rabbit renal proximal tubules
Authors: Welborn John R. , Shpun Shlomo , Dantzler William H. , Wright Stephen H.
Published: December 2017

The effect of exogenous α-ketoglutarate (αKG) and the peritubular Na+-dicarboxylate (Na-DC) cotransporter on organic anion/dicarboxylate (OA/DC) exchange in S2 segments of single, nonperfused rabbit proximal tubules was measured using 1 μM fluorescein (FL), a model OA, and epifluorescence microscopy. The effect of different transmembrane distributions of 10 μM αKG on peritubular FL uptake was measured at 37°C using bicarbonate-buffered, nutrient-containing buffers, which are conditions similar to those found in vivo. Compared with FL uptake in the absence of exogenous αKG, preloading tubules with αKG ( trans-configuration) or acute exposure to αKG ( cis-configuration) increased FL uptake 62% and 54%, respectively, whereas a cis-trans-configuration of αKG increased FL uptake by 76%. The cis-stimulation of FL uptake by αKG was rapid, within 5–7 s. This stimulation was blocked 96% by simultaneous exposure to 2 mM Li+, indicating that stimulation of transport was secondary to the uptake of exogenous αKG. In the absence of exogenous αKG, selective inhibition of Na-DC cotransport using 2 mM Li+or 1 mM methylsuccinate decreased FL uptake by 25% (effects that were reversible but not additive), suggesting that the Na-DC cotransporter recycles endogenous αKG that has left the cell in exchange for FL and that this activity supports ∼25% of baseline activity of the OA/DC exchanger. With recycling of αKG accounting for ∼25% of FL uptake and with accumulation of exogenous αKG accounting for another ∼75% increase in FL uptake, Na-DC cotransport appears to directly support (25% + 75%)/175%, or ∼57%, of total FL transport.
Role of the NH2terminus of the cloned renal K+channel, ROMK1, in arachidonic acid-mediated inhibition
Authors: Macica Carolyn M. , Yang Yinhai , Lerea Kenneth , Hebert Steven C. , Wang Wenhui
Published: December 2017

We have previously demonstrated that the ROMK channel maintains the property of arachidonic acid (AA) sensitivity observed originally in the native ATP-sensitive K+channel of the rat cortical collecting duct (16). We used the patch-clamp technique to extend these studies to other NH2-terminal splice variants of the ROMK channel family, ROMK2 and ROMK3, expressed in Xenopus oocytes to determine the mechanism by which AA inhibits channel activity. Although the conductance, channel open probability, and open/closed times of the three homologs were determined to be similar, addition of 5–10 μM AA caused only a moderate inhibition of ROMK2 (15 ± 8%) and ROMK3 (13 ± 9%) activity, indicating that differences in the NH2termini of ROMK channels strongly influence the AA action. We consequently examined the effect of AA on a ROMK1 variant, R1ND37, in which the NH2terminal amino acids 2–37 were deleted, and on a mutant ROMK1, R1S4A, in which the serine-4 residue was mutated to alanine. Like ROMK2 and ROMK3, AA had a diminished effect on these variants. Addition of 1 nM exogenous protein kinase C (PKC) inhibited ROMK1 but not the mutant, R1S4A. However, the effect of AA is not a result of stimulation of a membrane bound PKC, since PKC inhibitors, calphostin C and chelerythrine, failed to abolish the AA-induced inhibition. In contrast, application of 5 μM staurosporine, a nonspecific protein kinase inhibitor at high concentration, abolished the effect of AA. We conclude that phosphorylation of serine-4 residue in the NH2terminus plays a key role in determination of AA effect on ROMK channels.
Numerical analysis of viscous flow through fibrous media: a model for glomerular basement membrane permeability
Authors: Palassini Matteo , Remuzzi Andrea
Published: December 2017

Viscous flow through fibrous media is characterized macroscopically by the Darcy permeability ( KD). The relationship between KDand the microscopic structure of the medium has been the subject of experimental and theoretical investigations. Calculations of KDbased on the solution of the hydrodynamic flow at fiber scale exist in literature only for two-dimensional arrays of parallel fibers. We considered a fiber matrix consisting of a three-dimensional periodic array of cylindrical fibers with uniform radius ( r) and length connected in a tetrahedral structure. According to recent ultrastructural studies, this array of fibers can represent a model for the glomerular basement membrane (GBM). The Stokes flow through the periodic array was simulated using a Galerkin finite element method. The dimensionless ratio K* = KD/ r2was determined for values of the fractional solid volume (φ) in the range 0.005 ≤ φ ≤ 0.7. We compared our numerical results, summarized by an interpolating formula relating K* to φ, with previous theoretical determinations of hydraulic permeability in fibrous media. We found a good agreement with the Carman-Kozeny equation only for φ > 0.4. Among the other theoretical analysis considered, only that of Spielman and Goren ( Environ. Sci. Technol. 2: 279–287, 1968) gives satisfactory agreement in the whole range of φ considered. These results can be useful to model combined transport of water and macromolecules through the GBM for the estimation of the radius and length of extracellular protein fibrils.
Serum-stimulated α1type IV collagen gene transcription is mediated by TGF-β and inhibited by estradiol
Authors: Lei Jun , Silbiger Sharon , Ziyadeh Fuad N. , Neugarten Joel
Published: December 2017

We examined the hypothesis that fetal calf serum (FCS) stimulates murine mesangial cell α1type IV collagen ( COL4A1) gene transcription by increasing autocrine production of transforming growth factor-β (TGF-β) through a platelet-derived growth factor (PDGF)-dependent mechanism. PDGF-stimulated COL4A1 gene transcription was inhibited by neutralizing antibody to TGF-β (119.3 ± 3.6 vs. 106.0 ± 6.2 relative luciferase units, expressed as a percentage of control untreated cells, P < 0.003). FCS-stimulated gene transcription was inhibited by neutralizing antibody to PDGF (148.3 ± 4.1 vs. 136.7 ± 0.3 relative luciferase units, P < 0.002) and by neutralizing antibody to TGF-β (148.3 ± 4.1 vs. 127.1 ± 3.4 relative luciferase units, P < 0.036). The inhibitory effect of combined treatment with anti-PDGF and anti-TGF-β antibody on gene transcription was no greater than that of anti-TGF-β antibody alone [129.5 ± 0.53 vs. 127.1 ± 3.4 relative luciferase units, P = not significant (NS)]. FCS-stimulated gene transcription was also inhibited by estradiol (10−7M) (148.4 ± 3.1 vs. 119.4 ± 8.1 relative luciferase units, P < 0.019). In the presence of estradiol, anti-TGF-β antibody failed to further reduce serum-stimulated gene transcription (119.4 ± 8.1 vs. 115.6 ± 9.8, P = NS), suggesting that estradiol reverses FCS-stimulated COL4A1 gene transcription by antagonizing the actions of TGF-β. Measurement of type IV collagen synthesis by Western blotting confirmed that the intact gene responded in a manner analogous to the promoter construct.
Microtubule disruption inhibits AVT-stimulated Cl−secretion but not Na+reabsorption in A6 cells
Authors: Morris Ryan G. , Tousson Albert , Benos Dale J. , Schafer James A.
Published: December 2017

The effects of microtubule disruption on arginine vasotocin (AVT)-stimulated Na+and Cl−transport were studied in A6 cells by measuring short-circuit currents ( Isc) across cell layers grown in tissue culture on permeable supports. Microtubule disruption inhibited an AVT-stimulated secretory Cl−current but did not prevent activation of amiloride-sensitive Na+transport. This AVT-stimulated secretory Cl−current was significantly inhibited by glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). Reverse transcription of RNA isolated from A6 cells followed by polymerase chain reaction (PCR) using primers designed to amplify a portion of the R-domain of CFTR cloned from Xenopus laevis skin and immunocytochemistry demonstrated the presence of CFTR in A6 cells and an apparent recruitment of cytoplasmic CFTR to the apical cell surface after AVT stimulation. In contrast, indirect immunofluorescent labeling of Na+channels using a polyclonal antibody raised against a biochemically isolated Na+channel complex from bovine renal medulla labeled the apical plasma membrane but failed to demonstrate intracellular labeling of Na+channels (except in subconfluent cells) or recruitment of Na+channels to the apical membrane region after AVT stimulation.
Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells
Authors: Dai Long-Jun , Bapty Brian , Ritchie Gordon , Quamme Gary A.
Published: December 2017

Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg2+transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg2+-depleted cells. Intracellular free Mg2+concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+depleted (0.22 ± 0.01 mM) by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg2+]iwas determined. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 164 ± 5 nM/s. Both glucagon and AVP stimulated Mg2+ uptake into MDCT cells, 196 ± 11 and 189 ± 6 nM/s, respectively, at concentrations of 3 × 10−7 M and 10−7 M, respectively. Enhanced Mg2+ uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg2+ entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10−4 M, enhanced Mg2+ uptake (225 ± 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg2+ uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg2+ uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.
Regulation of sorbitol content in cultured porcine urinary bladder epithelial cells
Authors: Mähler Solveigh , Kinne-Saffran Evamaria , Fujisue Hiroshi , Kinne Rolf K. H. , Föllmann Wolfram
Published: December 2017

Sorbitol content was determined in porcine urinary bladder epithelial cells immediately after death of the animals and after primary culture of the cells at different osmolalities. In both instances, sorbitol content increased with urine and medium osmolality, respectively. For example, at 300 mosmol/kg the cultured cells contained 0.84 ± 0.02 nmol/mg protein, at 600 mosmol/kg contained 21.7 ± 0.95 nmol/mg protein, and at 900 mosmol/kg contained 59.5 ± 2.8 nmol/mg protein. Similarly, aldose reductase activity rose from 0.27 ± 0.04 μmol ⋅ h−1⋅ mg protein−1at 300 mosmol/kg to 1.81 ± 0.16 at 600 mosmol/kg and to 3.02 ± 0.33 at 900 mosmol/kg. These changes were, however, only observed when NaCl but not when urea was used to augment the medium osmolality, since urea equilibrated across the cell membrane. In contrast, sorbitol release from cells cultured at 900 mosmol/kg was slowest into a 900 mosmol/kg medium and fastest into a 300 mosmol/kg medium (63 ± 16 nmol/10 min compared with 389 ± 52 nmol/10 min). These studies demonstrate that the sorbitol content of porcine urinary bladder epithelium is regulated by changes both in sorbitol synthesis and sorbitol release. Thus the regulatory mechanisms in the urinary bladder seem to be similar to those present in the embryological related collecting duct.
Potential autocrine and paracrine mechanisms of recovery from mechanical injury of renal tubular epithelial cells
Authors: Anderson Robert J. , Ray Carla J.
Published: December 2017

The present studies were done to clarify potential pathways of the nephrogenic repair process. Media removed from mechanically injured vascular smooth muscle cells and LLC-PK1renal tubular epithelial cells significantly stimulated[Formula: see text]thymidine uptake and cell number in quiescent LLC-PK1cells, demonstrating the existence of potential autocrine and paracrine pathways of nephrogenic repair. The effect of mechanical injury resulting in release of one or more growth factors into culture media was also found in the opossum kidney OK renal tubular cell line. The nonspecific peptide growth factor antagonist suramin inhibited the effect of media from injured LLC-PK1cells to stimulate[Formula: see text]thymidine uptake in quiescent LLC-PK1cells. Exposure of quiescent LLC-PK1cells to six growth factors, including acidic and basic fibroblastic growth factors (aFGF and bFGF), platelet-derived growth factors AA and BB (PDGF-AA and PDGF-BB), endothelin-2, and hepatocyte growth factor, reproduced the biological responses seen when quiescent LLC-PK1cells were exposed to media from injured cells. Immunoblotting and enzyme-linked immunosorbent assay experiments demonstrated the presence of aFGF, bFGF, and PDGF-BB but not other candidate growth factors in the media from injured LLC-PK1cells. A neutralizing antibody directed against bFGF attenuated the effect of media from injured cells to stimulate[Formula: see text]thymidine uptake in serum-starved LLC-PK1cells. These results demonstrate that mechanical injury to renal tubular epithelial cells results in release of aFGF, bFGF, and PDGF-BB into the media and suggests that bFGF may be involved in an autocrine fashion to promote recovery from injury.
Regulation of cyclooxygenase expression in the kidney by dietary salt intake
Authors: Yang Tianxin , Singh Inderjit , Pham Hang , Sun Daqing , Smart Ann , Schnermann Jurgen B. , Briggs Josephine P.
Published: December 2017

The present studies were undertaken to determine the effect of dietary salt intake on the renal expression of cyclooxygenase-1 (COX-1) and -2 (COX-2). Protein levels were assessed by Western blotting, and mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) on cDNA prepared from kidney regions, dissected nephron segments, and cultured renal cells. Both isoforms were expressed at high levels in inner medulla (IM), with low levels detected in outer medulla and cortex. COX-1 mRNA was present in the glomerulus and all along the collecting duct, whereas COX-2 mRNA was restricted to the macula densa-containing segment (MD), cortical thick ascending limb (CTAL), and, at significantly lower levels, in the inner medullary collecting duct. Both isoforms were highly expressed at high levels in cultured medullary interstitial cells and at lower levels in primary mesangial cells and collecting duct cell lines. Maintaining rats on a low- or high-NaCl diet for 1 wk did not affect expression of COX-1. In IM of rats treated with a high-salt diet, COX-2 mRNA increased 4.5-fold, and protein levels increased 9.5-fold. In contrast, cortical COX-2 mRNA levels decreased 2.9-fold in rats on a high-salt diet and increased 3.3-fold in rats on a low-salt diet. A low-salt diet increased COX-2 mRNA 7.7-fold in MD and 3.3-fold in CTAL. Divergent regulation of COX-2 in cortex and medulla by dietary salt suggests that prostaglandins in different kidney regions serve different functions, with medullary production playing a role in promoting the excretion of salt and water in volume overload, whereas cortical prostaglandins may protect glomerular circulation in volume depletion.
The endogenous insulin-like growth factor system in radiocontrast nephropathy
Authors: Symon Z. , Fuchs S. , Agmon Y. , Weiss O. , Nephesh I. , Moshe R. , Brezis M. , Flyvbjerg A. , Raz I.
Published: December 2017

The response of insulin-like growth factor (IGF) I in acute renal failure was evaluated in a model of radiocontrast nephropathy associated with selective necrosis of medullary thick ascending limbs. In brief, rats were administered radiocontrast medium or vehicle injections for controls after combined inhibition of prostanoids and nitric oxide. Twenty-four hours after the insult, tissue mRNAs for IGF-I, the IGF-I receptor, and IGF-binding proteins (IGFBP) 1 and 3 were assayed in cortex, medulla, and liver by solution hybridization-RNase protection assay, and IGFBPs were measured in serum and tissue by Western ligand blotting. Cortical IGF-I increased, whereas medullary IGF-I mRNA decreased. Renal IGFBPs decreased, whereas IGFBP-1 mRNA increased. The IGF system in the liver was unchanged. We conclude that general changes in renal IGFBPs in this experimental model of acute renal failure might increase the level of cortical IGF-I in a way that could modulate medullary recovery.
Neuronal nitric oxide synthase modulates rat renal microvascular function
Authors: Ichihara Atsuhiro , Inscho Edward W. , Imig John D. , Navar L. Gabriel
Published: December 2017

This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 ± 0.2 μm) and efferent (18.5 ± 0.2 μm) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-l-thiocitrulline (l-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses tol-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, Nω-nitro-l-arginine (l-NNA) were only attenuated by papillectomy.l-SMTC (10 μM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, l-NNA (100 μM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.
Phosphoinositide signaling in rat inner medullary collecting duct
Authors: Chou Chung-Lin , Rapko Sonia I. , Knepper Mark A.
Published: December 2017

Previous studies in microdissected rat inner medullary collecting duct (IMCD) segments have demonstrated that carbachol, arginine vasopressin (AVP), and the V2vasopressin receptor agonist 1-desamino-8-d-arginine vasopressin (DDAVP) induce a similar increase in intracellular Ca2+. The present study tested whether these agents activate the phosphoinositide hydrolysis pathway. In intracellular inositol 1,4,5-trisphosphate (IP3) measurements, we found that IMCD suspensions incubated with AVP or DDAVP (10−8M) displayed no measurable increase in IP3, whereas IMCD suspensions incubated with the muscarinic cholinergic agent carbachol (100 μM) induced a significant increase in IP3production. Similarly, carbachol, but not AVP or DDAVP, induced a significant increase in membrane-associated protein kinase C (PKC) enzyme activity. To test what specific PKC isoforms are activated by carbachol in IMCD, we first characterized the PKC isoforms in IMCD suspensions by immunoblotting using affinity-purified antibodies against different PKC isoforms. We identified one classic PKC isoform (α), three novel PKC isoforms (δ, ε, η), and one atypical PKC isoform (ζ) in the IMCD. Carbachol induced a cytosol-to-membrane translocation of the PKC-η isoform but did not alter the distribution of any other isoform. In contrast, AVP had no effect on the distribution of any PKC isoform tested. These data, taken together, demonstrate that carbachol is an activator of the phosphoinositide hydrolysis pathway in IMCD but do not demonstrate signaling via this pathway in response to AVP or DDAVP. These results suggest that the previously observed AVP-stimulated Ca2+mobilization in IMCD may be due to a mechanism other than activation of the phosphoinositide hydrolysis pathway.
Differential effects of ACE and AT1receptor inhibition on chemoattractant and adhesion molecule synthesis
Authors: Morrissey Jeremiah J. , Klahr Saulo
Published: December 2017

Ureteral obstruction causes infiltration of the kidney by monocytes/macrophages. This infiltrate is significantly reduced by administration of an angiotensin-converting enzyme (ACE) inhibitor but not by a specific angiotensin II type 1 receptor (AT1receptor) antagonist. Chemoattractants and cell surface adhesive molecules mediate monocyte/macrophage infiltration. Rats with unilateral ureteral obstruction (UUO) of 1, 3, or 5 days duration were untreated or given enalapril or SC-51316 in the drinking water. We measured the mRNA levels of monocyte chemoatactic peptide 1 (MCP-1), a chemoattractant, and levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), two cell surface adhesion proteins. MCP-1 mRNA increased significantly after 1 day of UUO and increased further through 5 days of UUO in the obstructed kidney. ICAM-1 mRNA also increased significantly after 1 day but steadily declined through 5 days of UUO in the obstructed kidney. VCAM-1 mRNA did not increase significantly until after 3 days of UUO and increased further through 5 days of obstruction. Enalapril or SC-51316 treatment had no significant effect on ICAM-1 mRNA levels. MCP-1 mRNA levels were reduced but remained significantly elevated. Enalapril significantly blunted the increase in VCAM-1 mRNA levels and VCAM-1 protein determined by immunocytochemistry; SC-51316 had no significant effect. Thus changes in VCAM-1 levels may account for the differential effect of enalapril and SC-51316 on monocyte/macrophage infiltration of the kidney during ureteral obstruction.
Identification of gene family of caspases in rat kidney and altered expression in ischemia-reperfusion injury
Authors: Kaushal Gur P. , Singh Amar B. , Shah Sudhir V.
Published: December 2017

In the present study, we demonstrate that rat kidney contains caspase activity that was markedly inhibited by specific peptide inhibitors of caspases but not by inhibitors of Ser, Cys, Asp, or metalloproteinases. Using primers based on the nucleotide sequence of known members of Ced-3/interleukin-1β-converting enzyme (ICE) family from human origin, we have identified by reverse-transcription (RT) polymerase chain reaction (PCR) analyses that rat kidney transcribes the genes for caspase-1 (ICE), caspase-2 (Nedd2), caspase-3 (CPP32), and caspase-6 (Mch2). RT-PCR products, when subcloned and sequenced, provided full-length cDNAs for ICE (1,209 bp) and CPP32 (786 bp) and partial cDNA products for Mch2 (561 bp) and Nedd2 (811 bp). The sequence analysis of the caspase cDNAs showed conserved catalytic site QACRG as well as Asp cleavage site. Rat kidneys subjected to ischemia-reperfusion injury revealed differential expression of caspases with marked increase in CPP32 and ICE mRNA and proteins during reperfusion, transient increase in Nedd2 mRNA and proteins during ischemia and the early period of reperfusion, and little change in Mch2 expression during the ischemia or reperfusion period. The altered expression suggests that caspases may act in concert in a cascade and may play an important role in ischemic acute renal failure.
Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance
Authors: Fejes-Tóth Géza , Rusvai Erzsébet , Cleaveland Emily S. , Náray-Fejes-Tóth Anikó
Published: December 2017

AE2 mRNA and protein is expressed in several nephron segments, one of which is the cortical collecting duct (CCD). However, the distribution of AE2 among the different cell types of the CCD and the function of AE2 in the kidney are not known. The purpose of this study was to determine the distribution of AE2 mRNA among the three CCD cell types and to examine the effects of changes in acid/base balance on its expression. Following NH4Cl (acid) or NaHCO3(base) loading of rabbits for ∼18 h, CCD cells were isolated by immunodissection. AE2 mRNA levels were determined by RT-PCR and were normalized for β-actin levels. We found that CCD cells express high levels of AE2 mRNA (∼500 copies/cell). AE2 mRNA levels were significantly higher in CCD cells originating from base-loaded than acid-loaded rabbits, with an average increase of 3.7 ± 1.07-fold. The effect of pH on AE2 mRNA levels was also tested directly using primary cultures of CCD cells. CCD cells incubated in acidic media expressed significantly lower levels of AE2 mRNA than those in normal or alkaline media. Experiments with isolated principal cells, α-intercalated cells, and β-intercalated cells (separated by fluorescence-activated cell sorting) demonstrated that AE2 mRNA levels are comparable in the three collecting duct cell subtypes and are similarly regulated by changes in acid/base balance. Based on these results, we conclude that adaptation to changes in extracellular H+concentration is accompanied by opposite changes in AE2 mRNA expression. The observations that AE2 mRNA is not expressed in a cell-type-specific manner and that changes in acid/base balance have similar effects on each CCD cell subtype suggest that AE2 might serve a housekeeping function rather than being the apical anion exchanger of β-intercalated cells.