bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of rat laminin gamma 1-chain gene promoter reveals motifs for recognition of multiple transcription factors
Authors: O'Neill B. C. , Suzuki H. , Loomis W. P. , Denisenko O. , Bomsztyk K.
Published: December 2017

We have previously shown that laminin gamma 1 (laminin B2)-chain mRNA levels increase in response to treatment of rat glomerular epithelial cells (GEC) with the cytokine interleukin-1 beta (IL-1 beta) [C. A. Richardson, K. L. Gordon, W. G. Couser, and K. Bomsztyk. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F273-F278, 1995]. IL-1 beta-induced increase in laminin gamma 1-chain gene expression is likely to be transcriptionally regulated. As the laminin gamma 1-chain gene promoter had not previously been cloned in the rat, we cloned the 5'-flanking region of this gene from a rat genomic library. Like the human and murine laminin gamma 1-chain gene promoters, the rat laminin gamma 1-chain gene fragment spanning from nucleotides -1104 to +109, relative to the start codon, is "GC" rich and lacks TATA or CAAT boxes. This rat laminin gamma 1-chain gene promoter region appears to contain at least two transcription initiation sites, i.e., position -169 and -234. In transient transfections in GEC, the -1104/+35 and -1104/-15 fragments cloned upstream of a luciferase reporter gene had very little constitutive activity and were not IL-1 beta responsive. In sharp contrast, a -1104/-234 fragment exhibited constitutive activity and was IL-1 beta responsive. The -1104/-234 fragment contains motifs that recognize Sp1, BCN-1, and ApoE-B1-AP1 DNA-binding activities in GEC nuclear extracts. Collectively, the results of this study suggest that multiple inducible transcription factors may regulate laminin gamma 1-chain gene promoter activity in GEC.
Importance of the mercury-sensitive cysteine on function and routing of AQP1 and AQP2 in oocytes
Authors: Mulders S. M. , Rijss J. P. , Hartog A. , Bindels R. J. , van Os C. H. , Deen P. M.
Published: December 2017

To discriminate between water transport of of aquaporin-2 (AQP2) mutants in nephrogenic diabetes insipidus and that of an AQP2 molecule used to drag them to the oolemma, we investigated the mercury sensitivity of wild-type and AQP2 C181S proteins in oocytes. Incubation with HgCl2 inhibited the osmotic water permeability (Pf) of human (h) AQP2 by 40%, whereas inhibition of hAQP1 was 75%. Oocytes expressing hAQP1 C189S revealed a Pf comparable to wild-type hAQP1, but mercury sensitivity was lost. In contrast, no increase in Pf was obtained when hAQP2 C181S was expressed. Also, expression of rat AQP2 C181A and C181S mutants did not increase the Pf, which contrasts with published observations. Immunocytochemistry and immunoblotting revealed that only AQP1, AQP1 C189S, and AQP2 were targeted to the plasma membrane and that AQP2 mutant proteins are retarded in the endoplasmic reticulum. In conclusion, water transport through AQP2 is less sensitive to mercury inhibition than through AQP1. Furthermore, substitution of the mercury-sensitive cysteine for a serine results in an impaired routing of human and rat AQP2. Similar mutations have no effect on AQP1 function, which is indicative of structural differences between AQP1 and AQP2.
Atrial natriuretic peptide in preeclampsia: metabolic clearance, sodium excretion and renal hemodynamics
Authors: Irons D. W. , Baylis P. H. , Butler T. J. , Davison J. M.
Published: December 2017

To further elucidate the role of atrial natriuretic peptide (ANP) in preeclampsia, its metabolic clearance (MCRANP) was determined concomitantly with its effects on sodium excretion (UNa), glomerular filtration rate (GFR), and effective renal plasma flow (ERPF). Ten untreated preeclamptic primigravidae (PET) were studied at 29-37 wk gestation and again 4 mo postpartum (PP). Basal plasma concentration of ANP was significantly increased in PET compared with PP (14.8 +/- 1.9 vs. 4.1 +/- 0.5 pmol/l, respectively; P < 0.0001). MCRANP in PET and PP was 5.0 +/- 0.8 and 4.9 +/- 0.5 l/min [not significant (NS)], respectively. In PET, infusion of ANP produced (basal vs. ANP) a natriuresis (UNa 0.14 +/- 0.02 vs. 0.28 +/- 0.04 mmol/min, P < 0.001) and an increase in GFR (97 +/- 7 vs. 106 +/- 8 ml/min, P < 0.05), with ERPF unchanged (609 +/- 24 vs. 634 +/- 29 ml/min, NS). In PP, ANP infusion also produced a natriuresis (UNa 0.20 +/- 0.02 vs. 0.25 +/- 0.02 mmol/min, P = 0.01), no significant change in GFR (109 +/- 7 vs. 102 +/- 4 ml/min), and a significant reduction in ERPF (514 +/- 22 vs. 409 +/- 18 ml/min, P < 0.0001). Analysis of variance demonstrated a greater natriuretic effect of ANP in PET compared with PP (P < 0.05), similarly a significant difference in the effect of ANP on ERPF (P < 0.01) and GFR (P < 0.05) was seen but not on filtration fraction (P = 0.35).
Heat shock-induced protection and enhancement of Na+-glucose cotransport by LLC-PK1monolayers
Authors: Sussman Caroline R. , Renfro J. Larry
Published: December 2017

Monolayers of the porcine-derived renal epithelial cell line, LLC-PK1, were used to characterize the effects of heat stress on Na+-glucose cotransport. Transepithelial current dependent on 5 mM glucose ( IGlc), phloridzin-sensitive current ( Iphz), and total transepithelial current ( Itotal) were measured as indicators of Na+-glucose cotransport. Severe heat shock (SHS; 45°C for 1 h, then 37°C for measurements) decreased transepithelial electrical resistance (TER), IGlc, Iphz, and Itotal50–70%. Mild heat shock (MHS; 42°C for 3 h, then 37°C for 12 h) induced accumulation of 72-kDa heat shock protein (HSP-72), decreased damage to TER from SHS, and prevented damage to IGlc, Iphz, and Itotal. Kinetic analysis showed that SHS damaged and MHS protected total Na+-glucose transport capacity ( Vmaxof IGlc). MHS alone increased TER (50%), IGlc(20%), Itotal(20%), and Vmaxof IGlc(25%). On enhancement of the Na+gradient by depletion of intracellular Na+, MHS increased IGlc50% and had no effect on transepithelial Na+-dependent sulfate reabsorptive flux measured concurrently or in Na+-replete tissues. These effects of MHS were not reflected in effects on cell survival or luminal membrane surface area as indicated by lactate dehydrogenase or alkaline phosphatase release. In conclusion, HSP-72-inducing heat treatment both protected and enhanced Na+-glucose cotransport independently of the luminal membrane Na+gradient and selectively with respect to effects on TER, reabsorptive sulfate transport, cell survival, and luminal membrane surface area.
Perfusion pressure dependency of in vivo renal tubular dynamics
Authors: Rodriguez-Porcel Martin , Lerman Lilach O. , Sheedy Patrick F. , Romero J. Carlos
Published: December 2017

To examine whether changes in renal perfusion pressure (RPP) within the range of autoregulation induce detectable changes in tubular dynamics in an entire nephron population of the intact kidney, we measured, using electron beam computed tomography (EBCT), transit times (TT, s) and intratubular concentration (%) of filterable contrast media in various nephron segments simultaneously with renal regional perfusion. In seven dogs ( group A) this was performed at the upper and lower limits of autoregulation (RPP = 130 and 95 mmHg, respectively) while group B( n = 5) served as control. In group A alone, a decrease in RPP led to an increase in TT by 40%, 68%, and 32% in the proximal tubules, ascending limb of Henle’s loop, and distal tubules, respectively, in association with an increase in intratubular concentration (+50%, 80%, and 42%, respectively). Papillary perfusion decreased, whereas perfusion of the adjacent, outlying inner medulla increased. The decrease in papillary perfusion correlated positively with the concurrent change in sodium excretion ( R = 0.81). This study demonstrates that changes in RPP within the autoregulatory range elicit changes of tubular sodium reabsorption mainly in proximal, distal, and ascending tubules, in which most of the nephrons participate. These tubular changes are associated with an alteration of perfusion circumscribed to two areas of the inner renal medulla.
The effects of respiratory alkalosis and acidosis on net bicarbonate flux along the rat loop of Henle in vivo
Authors: Unwin R. , Stidwell R. , Taylor S. , Capasso G.
Published: December 2017

We have studied the effects of acute respiratory alkalosis (ARALK, hyperventilation) and acidosis (ARA, 8% CO2), chronic respiratory acidosis (CRA; 10% CO2 for 7–10 days), and subsequent recovery from CRA breathing air on loop of Henle (LOH) net bicarbonate flux ([Formula: see text]) by in vivo tubule microperfusion in anesthetized rats. In ARALK blood, pH increased to 7.6, and blood bicarbonate concentration ([[Formula: see text]]) decreased from 29 to 22 mM. Fractional urinary bicarbonate excretion ([Formula: see text]) increased threefold, but LOH[Formula: see text]was unchanged. In ARA, blood pH fell to 7.2, and blood [[Formula: see text]] rose from 28 to 34 mM; [Formula: see text] was reduced to <0.1%, but LOH[Formula: see text]was unaltered. In CRA, blood pH fell to 7.2, and blood [[Formula: see text]] increased to >50 mM, whereas[Formula: see text]decreased to <0.1%.[Formula: see text]was reduced by ∼30%. Bicarbonaturia occurred when CRA rats breathed air, yet LOH[Formula: see text]increased (by 30%) to normal. These results suggest that LOH[Formula: see text]is affected by the blood-to-tubule lumen [[Formula: see text]] gradient and[Formula: see text] backflux. When the usual perfusing solution at 20 nl/min was made[Formula: see text] free, mean[Formula: see text]was −34.5 ± 4.4 pmol/min compared with 210 ± 28.1 pmol/min plus [Formula: see text]. When a low-NaCl perfusate (to minimize net fluid absorption) containing mannitol and acetazolamide (2 × 10−4 M, to abolish H+-dependent[Formula: see text]) was used,[Formula: see text]was −112.8 ± 5.6 pmol/min. Comparable values for[Formula: see text]at 10 nl/min were −35.9 ± 5.8 and −72.5 ± 8.8 pmol/min, respectively. These data indicate significant backflux of[Formula: see text] along the LOH, which depends on the blood-to-lumen [[Formula: see text]] gradient; in addition to any underlying changes in active acid-base transport mechanisms, [Formula: see text]permeability and backflux are important determinants of LOH[Formula: see text]in vivo.
Recognition of β-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1cells
Authors: Terada Tomohiro , Saito Hideyuki , Mukai Mayumi , Inui Ken-Ichi
Published: December 2017

PEPT1 and PEPT2 are H+-coupled peptide transporters expressed preferentially in the intestine and kidney, respectively, which mediate uphill transport of oligopeptides and peptide-like drugs such as β-lactam antibiotics. In the present study, we have compared the recognition of β-lactam antibiotics by LLC-PK1cells stably transfected with PEPT1 or PEPT2 cDNA. Cyclacillin (aminopenicillin) and ceftibuten (anionic cephalosporin without an α-amino group) showed potent inhibitory effects on the glycylsarcosine uptake in the PEPT1-expressing cells. Other β-lactams, such as cephalexin, cefadroxil, and cephradine (aminocephalosporins), inhibited modestly the PEPT1-mediated glycylsarcosine uptake. Except for ceftibuten, these β-lactams showed much more potent inhibitions on the glycylsarcosine uptake via PEPT2 than via PEPT1. Comparison of the inhibition constant ( Ki) values between cefadroxil and cephalexin suggested that the hydroxyl group at the NH2-terminal phenyl ring increased affinity for both PEPT1 and PEPT2. It is concluded that PEPT2 has a much higher affinity for β-lactam antibiotics having an α-amino group than PEPT1 and that substituents at the NH2-terminal side chain of these drugs are involved in the recognition by both peptide transporters.
Attenuated response of renal mechanoreceptors to volume expansion in chronically hypoxic rats
Authors: Chien Chiang-Ting , Fu Tsu-Ching , Wu Ming-Shiou , Chen Chau-Fong
Published: December 2017

Multifiber renal afferent nerve activity responds to volume expansion in sea level rats but not in chronically hypoxic (high altitude) rats. We performed single-unit recordings of renal afferent nerve activity to characterize renal sensory receptors and their responses to volume expansion in these animals. Hypoxia was induced by placing Wistar rats in an altitude chamber (380 Torr, 5,500 m) for 4 wk. Spontaneously firing renal R2 chemoreceptor and arterial, ureteropelvic, and venous mechanoreceptors were identified. The basal activity of each receptor was similar among these rats. In response to specific stimulus, the increasing impulse of R2 chemoreceptor was similar between two groups of rats, but the increasing activity of each mechanoreceptor was less in hypoxic rats. When challenged with saline load, R2 chemoreceptor activity decreased, but all mechanoreceptors activated in all rats. Despite similar increases of arterial, renal ureteropelvic, and venous pressure during saline load, the increasing activity of each mechanoreceptor was significantly less in hypoxic rats. These results indicated chronic hypoxia attenuates the sensitivity of renal mechanoreceptors in response to the stimulation of saline load.
Expression of syntaxins in rat kidney
Authors: Mandon Béatrice , Nielsen Søren , Kishore Bellamkonda K. , Knepper Mark A.
Published: December 2017

Previously, we demonstrated that a putative vesicle-targeting protein, syntaxin-4, is expressed in renal collecting duct principal cells and is localized to the apical plasma membrane, suggesting a role in targeting aquaporin-2-containing vesicles to the apical plasma membrane. To investigate whether other syntaxin isoforms are present in the renal collecting duct, we determined the intrarenal localization of syntaxin-2 and -3. Reverse transcription-polymerase chain reaction (RT-PCR) experiments using total RNA extracted from kidney and various organs revealed that both syntaxin-2 and -3 are expressed in kidney cortex and medulla. RT-PCR experiments using microdissected tubules and vascular structures from the kidney revealed that syntaxin-3 mRNA, but not syntaxin-2, is expressed in collecting duct cells. Syntaxin-3 mRNA was also relatively abundant in the thick ascending limb of Henle’s loop and in vasa recta. Syntaxin-2 mRNA was found chiefly in glomeruli. To investigate the localization of syntaxin-3 protein, a peptide-derived polyclonal antibody was raised in rabbits. In immunoblotting experiments, this antibody labeled a 37-kDa protein in inner medulla that was most abundant in plasma membrane-enriched subcellular fractions. Immunoperoxidase labeling of thin cryosections combined with immunogold electron microscopy showed that, in contrast to the labeling seen for syntaxin-4, syntaxin-3 labeling in medullary collecting duct was predominantly in the basolateral plasma membrane of intercalated cells. These results suggest the possibility that syntaxin-3 may be involved in selective targeting of acid-base transporters and/or in basolateral membrane remodeling in response to systemic acid-base perturbations.
Expression of peroxisome proliferator-activated receptors in urinary tract of rabbits and humans
Authors: Guan Youfei , Zhang Yahua , Davis Linda , Breyer Matthew D.
Published: December 2017

Peroxisome proliferator-activated receptors (PPARs, α, β/δ, and γ) are members of the nuclear receptor superfamily of ligand-activated transcription factors. PPARs regulate the expression of genes involved in lipid metabolism. 8( S)-hydroxyeicosatetraenoic acid (8- S-HETE), leukotriene B4(LTB4), and hypolipidemic fibrates activate PPARα, whereas PPARγ is activated by prostaglandin metabolites. The present studies examined the intrarenal and tissue distribution of rabbit and human PPARα, -β/δ, and -γ mRNAs. Nuclease protection showed PPARα predominated in liver, heart, and kidney, whereas PPARγ, a putative adipose-specific transcription factor, was in white adipose tissue, bladder, and ileum, followed by kidney and spleen. Lower expression levels of PPARβ/δ were observed in several tissues. In situ hybridization of kidney showed PPARα mRNA predominated in proximal tubules and medullary thick ascending limbs of both rabbit and human. PPARγ was exclusively expressed in medullary collecting duct and papillary urothelium. Immunoblot confirmed the expression of PPARγ protein in freshly isolated inner medullary collecting ducts. mRNAs for all the PPARs were expressed in the ureter and bladder in both rabbit and human, but PPARγ expression was greatest. This distinct distribution of PPAR isoforms has important implications for lipid-activated gene transcription in urinary epithelia.
AE anion exchanger mRNA and protein expression in vascular smooth muscle cells, aorta, and renal microvessels
Authors: Brosius Frank C. , Pisoni Ronald L. , Cao Xinan , Deshmukh Gayatri , Yannoukakos Drakoulis , Stuart-Tilley Alan. K. , Haller Christlieb , Alper Seth L.
Published: December 2017

Intracellular pH (pHi) is an important regulator of vascular smooth muscle cell (VSMC) tone, contractility, and intracellular Ca2+concentration. Among the multiple transport processes that regulate VSMC pHi, Na+-independent Cl−/[Formula: see text]exchange is the major process that acidifies VSMCs in response to an alkaline load. Here, we characterize, in native and cultured VSMCs, the expression of the AE family of band 3-related anion exchangers, the best studied of these Cl−/[Formula: see text]exchangers. A 4.2-kb AE2 mRNA was present in aorta and in all cultured VSMCs tested. Cultured VSMCs and aorta both expressed a ∼165-kDa AE2 polypeptide, but a ∼115-kDa polypeptide was the major AE2-related protein in aorta. AE3 mRNA levels in VSMCs and in arterial tissue were significantly lower than those for AE2, but AE3 or related polypeptides were readily detected by immunoblot and immunolocalization experiments. The ∼125-kDa AE3 polypeptide was present in an immortalized aortic VSMC line, but the predominant AE3 epitope in aorta and most cultured cells was associated with a polypeptide of Mr∼80 kDa. These data demonstrate the expression in native arteries and in VSMCs of products of the AE2 and AE3 genes, which may contribute to Na+-independent Cl−/[Formula: see text]exchange activity in these tissues and cells.
Urea and methylamines have similar effects on aldose reductase activity
Authors: Burg Maurice B. , Peters Eugenia M.
Published: December 2017

The concentration of urea in renal medullary cells is sufficiently high to inhibit activity of many enzymes, yet the cells survive and function. The generally accepted explanation is the counteracting osmolytes hypothesis, which holds that methylamines, such as glycerophosphorylcholine (GPC) and glycine betaine (betaine), found in the renal medulla stabilize biological macromolecules and oppose the effects of urea. The present study tests this hypothesis by determining the effects of urea and methylamines, singly and in combination, on the activity of aldose reductase, an enzyme that is important in renal medullas for catalyzing production of sorbitol from glucose. In apparent contradiction to the counteracting osmolytes hypothesis, urea (1.0 M) and three different methylamines (trimethylamine N-oxide, betaine, and GPC; 0.5 M) all have similar and partially additive inhibitory effects. They all decrease substantially both the Michaelis constant ( Km) and the maximum velocity ( Vmax). Also, a high concentration (0.5 M) of other organic osmolytes that are abundant in the renal medulla, namely inositol, sorbitol, or taurine, has a similar but lesser effect. KCl (0.3 M) causes a small increase in activity. We discuss the significance of these findings with regard to function of aldose reductase in the renal medulla and the counteracting osmolytes hypothesis.
Neurosteroid inhibition of cell death
Authors: Waters Shayla L. , Miller Gary W. , Aleo Michael D. , Schnellmann Rick G.
Published: December 2017

Diverse γ-aminobutyric acid (GABAA) receptor modulators exhibited novel cytoprotective effects and mechanisms of action in rabbit renal proximal tubules subjected to mitochondrial inhibition (antimycin A) or hypoxia. Cytoprotective potencies (50% effective concentration, EC50) were 0.3 nM allopregnanolone (AP) > 0.4 nM 17α-OH-allopregnanolone (17α-OH-AP) > 30 nM dehydroepiandrosterone sulfate (DHEAS) = 30 nM pregnenolone sulfate (PS) > 500 nM pregnenolone (PREG) > 30 μM muscimol > 10 mM GABA following antimycin A exposure. Maximal protection with AP and 17α-OH-AP was 70%, whereas DHEAS, PS, PREG, and muscimol produced 100% cytoprotection. Experiments with AP, PS, and muscimol revealed the return of mitochondrial function and active Na+transport following hypoxia/reoxygenation. Muscimol inhibited the antimycin A-induced influx of both extracellular Ca2+and Cl−that occurs during the late phase of cell injury, whereas the neurosteroids only inhibited influx of Cl−. Radioligand binding studies with AP and PS did not reveal a specific binding site; however, structural requirements were observed for cytoprotective potency and efficacy. In conclusion, we suggest that the GABAAreceptor modulators muscimol and neurosteroids are cytoprotective at different cellular sites in the late phase of cell injury; muscimol inhibits Ca2+and subsequent Cl−influx, whereas the neurosteroids inhibit Cl−influx.
G protein mRNA expression in renal microvessels from spontaneously hypertensive and Wistar-Kyoto rats
Authors: Mokkapatti Rupa , Vyas Subhash J. , Jackson Edwin K.
Published: December 2017

The exaggerated sensitivity of spontaneously hypertensive rat (SHR) renal microvasculature to angiotensin II (ANG II) may be due to an imbalance between the effectiveness of Gαs-utilizing vasodilator pathways and vasoconstrictor pathways activated by ANG II (mediated by Gαi-1, Gαi-2, Gαi-3, and Gαq). Because the alteration appears to be distal to the hormone receptors and proximal to the effector adenylyl cyclase, we hypothesized that SHR have altered amounts of signal-transducing G proteins. This was examined by quantifying the steady-state mRNA levels of specific Gα subunits in renal microvessels of 12- to 14-wk-old SHR and control Wistar-Kyoto (WKY) rats, using a quantitative-competitive polymerase chain reaction technique coupled to reverse transcription. No significant differences were detected in the absolute levels of Gαs(0.96 ± 0.35 vs. 0.74 ± 0.25 amol/50 ng RNA) or in the relative levels of Gαi-1(0.44 ± 0.05 vs. 0.48 ± 0.13), Gαi-2(40.9 ± 7.8 vs. 45.2 ± 8.9), or Gαi-3(0.79 ± 0.05 vs. 0.82 ± 0.15) normalized to the level of Gαsfor WKY vs. SHR, respectively. The ratio of Gαqto Gαstended to be higher in SHR, but this difference did not achieve statistical significance (0.41 ± 0.08 vs. 1.04 ± 0.32, P = 0.08). In conclusion, the steady-state levels of Gαs, Gαi-1, Gαi-2, Gαi-3, and Gαqare similar in SHR and WKY renal microvasculature, suggesting that other components of the ANG II signal transduction mechanism are responsible for the enhanced renal vascular responsiveness in SHR.
Regulation of adenylyl cyclase in polarized renal epithelial cells by G protein-coupled receptors
Authors: Okusa Mark D. , Huang Liping , Momose-Hotokezaka Akemi , Huynh Long P. , Mangrum Amy J.
Published: December 2017

We employed two guanine nucleotide binding protein (G protein)-coupled receptors known to be targeted to opposite domains in renal epithelial cells to test the hypothesis that the polarized receptor expression of receptors regulates the activity of the receptor’s effector molecule, adenylyl cyclase. We used LLC-PK1 cells stably transfected with cDNA encoding the α2B-adrenergic receptor (α2B-AR) or A1-adenosine receptor (A1-AdR). Immunohistochemistry and Western blot analysis confirmed the basolateral and apical expression of α2B-ARs and A1-AdRs, respectively. Adenylyl cyclase activity was assessed by measuring cAMP accumulation following the addition of forskolin (10 μM) in the presence of 3-isobutyl-1-methylxanthine to apical or basolateral chambers of confluent monolayers. A five- to sixfold increase in cAMP accumulation occurred following apical (or basolateral) stimulation of LLC-PK1 cells expressing apical (or basolateral) receptors in comparison to forskolin stimulation of corresponding domains of untransfected cells. We conclude 1) adenylyl cyclase activity is present at or near the apical and basolateral domains of LLC-PK1 cells, and 2) factors that regulate the polarized expression of inhibitory G protein-coupled receptors may also regulate local adenylyl cyclase activity.
PI-3-kinase and MAPK regulate mesangial cell proliferation and migration in response to PDGF
Authors: Choudhury Goutam Ghosh , Karamitsos C. , Hernandez James , Gentilini Alessandra , Bardgette John , Abboud Hanna E.
Published: December 2017

Proliferation and migration are important biological responses of mesangial cells to injury. Platelet-derived growth factor (PDGF) is a prime candidate to mediate these responses in glomerular disease. PDGF and its receptor (PDGFR) are upregulated in the mesangium during glomerular injury. We have recently shown that PDGF activates phosphatidylinositol 3-kinase (PI-3-kinase) in cultured mesangial cells. The role of this enzyme and other more distal signaling pathways in regulating migration and proliferation of mesangial cells has not yet been addressed. In this study, we used two inhibitors of PI-3-kinase, wortmannin (WMN) and LY-294002, to investigate the role of this enzyme in these processes. Pretreatment of mesangial cells with WMN and LY-294002 dose-dependently inhibited PDGF-induced PI-3-kinase activity assayed in antiphosphotyrosine immunoprecipitates. WMN pretreatment also inhibited the PI-3-kinase activity associated with anti-PDGFRβ immunoprecipitates prepared from mesangial cells treated with PDGF. Pretreatment of the cells with different concentrations of WMN resulted in a dose-dependent inhibition of PDGF-induced DNA synthesis. Both WMN and LY-294002 inhibited PDGF-stimulated migration of mesangial cells in a dose-dependent manner. It has recently been shown that PI-3-kinase physically interacts with Ras protein. Because Ras is an upstream regulator of the kinase cascade leading to the activation of mitogen-activated protein kinase (MAPK), we determined whether activation of PI-3-kinase is necessary for activation of MAPK. Pretreatment of mesangial cells with WMN and LY-294002 significantly inhibited PDGF-induced MAPK activity as measured by immune complex kinase assay of MAPK immunoprecipitates. Furthermore, PD-098059, an inhibitor of MAPK-activating kinase inhibited PDGF-induced MAPK activity and resulted in significant reduction of mesangial cell migration in response to PDGF. These data indicate that MAPK is a downstream target of PI-3-kinase and that both these enzymes are involved in regulating proliferation and migration of mesangial cells.
Identification of Mg-transporting renal tubules and cells by ion microscopy imaging of stable isotopes
Authors: Chandra Subhash , Morrison George H. , Beyenbach Klaus W.
Published: December 2017

Sites of renal Mg transport were identified in seawater killifish ( Fundulus heteroclitus) using a Cameca model IMS-3f ion microscope. Killifish were given an intraperitoneal injection of the stable isotope26Mg (99.5% enrichment) to stimulate and trace renal Mg excretion. We identified two sites of26Mg transport in frozen freeze-dried cryosections of kidney: the proximal tubule, known to secrete Mg, and the collecting duct, heretofore not known to handle Mg. In epithelial cells of the proximal tubule, the punctate distribution of injected26Mg suggests transcytotic excretion of Mg in bound form. In collecting ducts, a subpopulation of Mg/Ca-rich cells was identified with high accumulations of injected26Mg. Here, the punctate distribution of26Mg decreased from the apical to the basal region of the cells, revealing a transcytotic gradient of apparently bound Mg. Since proximal tubules of fish are implicated with Mg secretion, Mg/Ca-rich cells in the collecting duct may reabsorb Mg, thereby providing the usual two-step of renal regulation, now also for Mg.
Glucocorticoids mediate a decrease in AVP-regulated urea transporter in diabetic rat inner medulla
Authors: Klein Janet D. , Price S. Russ , Bailey James L. , Jacobs Joely D. , Sands Jeff M.
Published: December 2017

Providing glucocorticoids to adrenalectomized (Adx) rats results in downregulation of the vasopressin (AVP)-regulated urea transporter (VRUT) in the renal inner medullary (IM) tip. To examine the physiological relevance of this response, we studied rats with uncontrolled diabetes mellitus induced by streptozotocin (STZ), since these rats have increased corticosterone production and urea excretion. We measured VRUT protein in extracts from the IM tip or base of pair-fed control and diabetic rats by Western analysis using an antibody to rat VRUT. In the IM tip, VRUT was significantly reduced by 39% in diabetic compared with control rats. In the IM base, there was no significant difference between diabetic and control rats. To determine whether the decrease in VRUT in the IM tip was mediated by glucocorticoids, the experiment was repeated using the following three groups of rats: 1) Adx alone, 2) Adx + STZ, and 3) Adx + STZ + replacement with a physiological dose of glucocorticoid. There was no significant difference in VRUT between Adx and Adx + STZ rats. However, VRUT was significantly reduced by 32% in the IM tip of glucocorticoid-treated Adx + STZ rats compared with control Adx + STZ rats. We conclude that glucocorticoids regulate the abundance of VRUT protein independently of insulin in diabetic rats.
Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes
Authors: Kawachi Hiroshi , Kurihara Hidetake , Topham Peter S. , Brown Dennis , Shia Michael A. , Orikasa Michiaki , Shimizu Fujio , Salant David J.
Published: December 2017

Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates showed that the decline in staining was due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, while the slit diaphragms remained morphologically intact. These findings suggest that MAb 5-1-6 alters the molecular composition of the slit diaphragm and thereby affects the glomerular permeability barrier.
Contrasting endocrine responses to acute oral compared with intravenous sodium loading in normal humans
Authors: Singer Donald R. J. , Markandu Nirmala D. , Buckley Martin G. , Miller Michelle A. , Sagnella Giuseppe A. , MacGregor Graham A.
Published: December 2017

There is evidence in animals and in humans for accelerated natriuresis after oral compared with intravenous sodium loading. To assess the role of atrial natriuretic peptide (ANP) as a contributory mechanism, we compared the hormonal responses to an intravenous sodium load and to the same sodium load taken orally in three separate groups of healthy subjects in balance on low, normal, or high sodium intake. On each diet, there was a trend for an early delay in sodium excretion, followed by increased natriuresis after the oral compared with intravenous sodium load. On all levels of dietary sodium intake, there was a significant (∼2-fold) increase in plasma ANP levels after intravenous saline infusion. There was a significant suppression of the renin system both after oral and intravenous sodium loading. However, there was no acute increase in plasma ANP levels after the oral sodium load, except on the very low sodium intake. This striking and unexpected observation suggests that changes in plasma ANP levels appear to play little role in the early response to an acute oral sodium load in subjects with sodium intake in the range of 150–350 mmol/day. Endocrine mechanisms for the accelerated increase in sodium excretion after oral compared with intravenous sodium loading remain to be elucidated.
Protein kinase C regulation of organic anion transport in renal proximal tubule
Authors: Miller David S.
Published: December 2017

Fluorescence microscopy and digital image analysis were used to examine the role of protein kinase C (PKC) in the control of organic anion (fluorescein, FL) transport in killifish renal proximal tubules. Phorbol ester (1–100 nM) reduced cellular and luminal accumulation of FL, and protein kinase inhibitors [staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 10–1,000 nM] increased cellular and luminal accumulation. Phorbol ester effects were blocked by staurosporine. The increases in tissue fluorescence caused by staurosporine were blocked by p-aminohippurate, indicating that they represent increased FL transport on the organic anion system. Neither phorbol ester nor staurosporine had any effects on the cell-to-lumen transport of a fluorescent organic anion that was generated intracellularly from a nonfluorescent, uncharged precursor. Finally, studies with a fluorescent PKC inhibitor showed that phorbol ester caused PKC translocation from cytoplasm to the plasma membrane. Together, these findings indicate that renal organic anion transport is negatively correlated with PKC activity and that PKC directly or indirectly controls the basolateral step in transport.
Hypoxia triggers release of an endogenous inhibitor of Na+-K+-ATPase from midbrain and adrenal
Authors: De Angelis C. , Haupert G. T.
Published: December 2017

An endogenous inhibitor of Na+-K+-ATPase has been isolated from bovine hypothalamus and human plasma and structurally characterized as an isomer of the plant cardiac glycoside, ouabain (A. A. Tymiak, J. A. Norman, M. Bolgar, G. C. DiDonato, H. Lee, W. L. Parker, L.-C. Lo, N. Berova, K. Nakanishi, E. Haber, and G. T. Haupert, Jr. Proc. Natl. Acad. Sci. USA 90: 8189–8193, 1993; N. Zhao, L.-C. Lo, N. Berova, K. Nakanishi, J. H. Ludens, and G. T. Haupert, Jr. Biochemistry 34: 9893–9896, 1995). This hypothalamic inhibitory factor (HIF) acts on cardiovascular and renal tissues consistent with physiological regulation in vivo. Stimuli for the release of HIF from tissue are unknown. Hypoxia may be a stimulus for the elaboration of digitalis-like activity in humans, and high NaCl concentration in central nervous system stimulates ouabain-like activity in animals. We examined the ability of low O2tension in vivo and in vitro to stimulate HIF release from midbrain and adrenal tissues in Wistar rats. In both tissues, hypoxia stimulated a remarkable release of an inhibitor cochromatographing with HIF, and this release was enhanced by 300 mM NaCl. Plasma from hypoxic rats also showed increased levels of the purified inhibitory activity. We conclude that hypoxia is a potent stimulus for the release of HIF or HIF-like activity and discuss the possibility that an Na+-K+-ATPase inhibitor could be involved in energy-conserving cellular adaptive responses to hypoxic or ischemic insult through ATP conservation.
Disruption of guinea pig urinary bladder permeability barrier in noninfectious cystitis
Authors: Lavelle John P. , Apodaca Gerard , Meyers Susan A. , Ruiz Wily G. , Zeidel Mark L.
Published: December 2017

Although most cell membranes permit rapid flux of water, small nonelectrolytes, and ammonia, the apical membranes of bladder epithelial umbrella cells, which form the bladder permeability barrier, exhibit strikingly low permeabilities to these substances. In cystitis, disruption of the bladder permeability barrier may irritate the bladder wall layers underlying the epithelium, causing or exacerbating inflammation, and increasing urinary frequency, urgency, and bladder pain. To determine the effects of inflammation on the integrity of the permeability barrier, guinea pigs were sensitized with ovalbumin, and the bladders were exposed subsequently to antigen by instillation on the urinary side. Inflammation of the bladder wall markedly reduced transepithelial resistance of dissected epithelium mounted in Ussing chambers and increased water and urea permeabilities modestly at 2 h and more strikingly at 24 h after induction of the inflammation. Transmission and scanning electron microscopy of bladders at 30 min and 24 h after antigen exposure revealed disruption of tight junctions, denuding of patches of epithelium, and occasional loss of apical membrane architecture. These permeability and structural effects did not occur in nonsensitized animals in which the bladders were exposed to antigen and in sensitized animals exposed to saline vehicle rather than antigen. These results demonstrate that inflammation of the underlying muscle and lamina propria can disrupt the bladder permeability barrier by damaging tight junctions and apical membranes and causing sloughing of epithelial cells. Leakage of urinary constituents through the damaged epithelium may then exacerbate the inflammation in the underlying muscle layers.
Distinct localization of renin and GLUT-4 in juxtaglomerular cells of mouse kidney
Authors: Anderson Timothy J. , Martin Sally , Berka Jennifer L. , James David E. , Slot Jan W. , Stow Jennifer L.
Published: December 2017

The insulin-responsive glucose transporter, GLUT-4, is found primarily in adipocytes and skeletal muscle cells, where it is sequestered in a specialized recycling compartment, from which it can be recruited to the cell surface following insulin stimulation. Lower levels of GLUT-4 are also expressed in other tissues, including the kidney, where it is present particularly in cells of the afferent arteriole and juxtaglomerular apparatus (JGA). The exact nature of GLUT-4-containing compartments and their relationship to other regulated trafficking pathways in different cells are not yet well defined. The trafficking of GLUT-4 has been studied in different cells with regulated secretory pathways, and a recent study shows that, in cardiomyocytes, GLUT-4 is sorted and packaged into multiple regulated pathways (J. W. Slot, G. Garruti, S. Martin, V. Oorschot, G. Pshuma, E. W. Kraegen, R. Laybutt, G. Thibault, and D. E. James. J. Cell Biol. 137: 1243–1254, 1997). In the kidney, cells of the JGA synthesize and secrete their major product, renin, via a well-established, regulated, secretory pathway. These cells also express GLUT-4 and thus offer the potential to directly compare the localization and trafficking of GLUT-4 and renin in a unique cell type. The present study was undertaken to investigate the intracellular distribution of GLUT-4 in mouse kidney cortex and to determine whether GLUT-4 and renin are trafficked in the same or in separate regulated pathways. Ultrathin cryosections of mouse kidney were labeled by the immunogold technique and viewed by electron microscopy, demonstrating the distribution of GLUT-4 in cells of the JGA, afferent arteriole, and distal tubule. In granular cells of the JGA, renin was localized in secretory granules of the regulated secretory pathway, whereas GLUT-4 labeling in the same cells was found in a distinct tubulovesicular compartment located adjacent to the trans-Golgi network. We show that granular cells have separate, morphologically distinct compartments for the sequestration of renin and GLUT-4, providing evidence that there may be distinct pathways for the sorting and trafficking of these two proteins.
Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles
Authors: Coury Larry A. , Mathai John C. , Prasad G. V. Ramesh , Brodsky Jeffrey L. , Agre Peter , Zeidel Mark L.
Published: December 2017

Aquaporins 1 (AQP1) and 2 (AQP2) were expressed in the yeast secretory mutant sec6-4. The mutant accumulates post-Golgi, plasma membrane-targeted vesicles and may be used to produce large quantities of membrane proteins. AQP1 or AQP2 were inducibly expressed in yeast and were localized within isolated sec6-4 vesicles by immunoblot analysis. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeabilities and low activation energies for water flow, indicating expression of functional AQP1 and AQP2. AQP1 solubilized from secretory vesicles was successfully reconstituted into proteoliposomes, demonstrating the ability to use the yeast system to express aquaporins for reconstitution studies. The AQP2-containing secretory vesicles showed no increased permeability toward formamide, urea, glycerol, or protons compared with control vesicles, demonstrating that AQP2 is highly selective for water over these other substances. We conclude that the expression of aquaporins in yeast sec6 vesicles is a valid system to further study mammalian water channel function.
Graded ATP depletion can cause necrosis or apoptosis of cultured mouse proximal tubular cells
Authors: Lieberthal Wilfred , Menza Sarah A. , Levine Jerrold S.
Published: December 2017

The mechanisms of cell death induced by ATP depletion were studied in primary cultures of mouse proximal tubular (MPT) cells. Graded ATP depletion, ranging in severity from ∼2 to 70% of control levels, was induced by incubating cells with either antimycin or 2-deoxyglucose, with varying concentrations of dextrose. We found that cells subjected to ATP depletion below ∼15% of control died uniformly of necrosis. In contrast, cells subjected to ATP depletion between ∼25 and 70% of control all died by apoptosis. The rapidity of cell death was proportional to the severity of reduction of cell ATP content and was independent of the mechanism of cell death. Renal growth factors, epidermal growth factor (EGF) and high-dose insulin, did not ameliorate apoptotic cell death induced by ATP depletion. We conclude that ATP depletion can cause either necrosis or apoptosis in MPT cells. Furthermore, we have identified a narrow range of ATP depletion (∼15 to 25% of control) representing a threshold that determines whether cells die by necrosis or apoptosis.
In vitro demonstration of a mitogenic activity in renal tissue extracts during regenerative hyperplasia
Authors: Piron Annie , Leonard Isabelle , Nonclercq Denis , Toubeau Gerard , Falmagne Paul , Heuson-Stiennon Jeanine-Anne , Laurent Guy
Published: December 2017

Normal rat kidney (NRK-52E) cells, an established cell line of renal origin, were used as a bioassay system to reveal a possible mitogenic activity in tissue extracts prepared from kidneys undergoing tubular regeneration. Acute tubular injury was induced in female Wistar rats by a 4-day treatment with gentamicin at daily doses of 50 or 100 mg/kg twice daily. Animals were killed either 1 or 4 days after cessation of gentamicin administration. Proximal tubule regeneration in treated animals was confirmed by morphological examination after proliferating cell nuclear antigen staining. Tissue extracts from regenerating kidneys stimulated DNA synthesis in growth-arrested cells to a higher extent than extracts from intact kidneys. Sera from treated and control animals showed no difference with respect to mitogenic activity. The mitogenic effect of tissue extracts was sensitive to the tyrosine kinase inhibitor tyrphostin A46. The cell proliferative response to regenerating kidney extracts, but not that to intact kidney extracts, was partly suppressed by the addition of anti-insulin-like growth factor I (anti-IGF-I) antiserum. These data indicate that nephrogenic repair entails an elevation of biologically active IGF-I in kidney tissue.
Cloning and functional expression of rNBC, an electrogenic Na+- HCO 3 − cotransporter from rat kidney
Authors: Romero Michael F. , Fong Peying , Berger Urs V. , Hediger Matthias A. , Boron Walter F.
Published: December 2017

We have recently cloned the renal electrogenic Na+-bicarbonate cotransporter of the salamander Ambystoma tigrinum(aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409–413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na+-bicarbonate cotransporter. NBC constitutes the major route for[Formula: see text] reabsorption and assists in Na+reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in λgt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3′-untranslated region of ∼500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (∼30%) to the AE family of Cl−/[Formula: see text]exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na+-[Formula: see text]cotransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO2/10 mM[Formula: see text] elicits a hyperpolarization of >50 mV and a rapid decrease of intracellular pH (pHi), followed by an increase in pHi. Subsequent removal of Na+in the presence of CO2/[Formula: see text]causes a depolarization of >50 mV and a concomitant decrease of pHi. Thus rNBC is in the same newly identified family of Na+-linked[Formula: see text] transporters as is aNBC.
Model explaining the relation between distal nephron Li+ reabsorption and urinary Na+ excretion in rats
Authors: Shalmi Michael , Jonassen Thomas , Thomsen Klaus , Kibble Jonathan D. , Bie Peter , Christensen Sten
Published: December 2017

Li+ may be reabsorbed via an amiloride-sensitive mechanism in the collecting ducts of rats administered a low-Na+ diet. This was investigated by measuring the increase in fractional urinary excretion of Li+(FELi) in response to amiloride in conscious rats at two different levels of plasma Li+ concentration and after administration of bendroflumethiazide (BFTZ), angiotensin III (ANG III), and aldosterone (Aldo). The results confirmed that amiloride increased (FELi) in rats on a low-Na+ diet (20 ± 1 to 35 ± 1%, means ± SE), whereas no increase was observed in rats on a normal Na+ diet (37 ± 1 to 38 ± 1%). The lithiuretic effect of amiloride was 1) abolished by preadministration of BFTZ (32 ± 1 to 33 ± 2%) to Na+-deprived rats and 2) increased by ANG III (27 ± 3 to 33 ± 2%) and Aldo (25 ± 2 to 37 ± 2%) in Na+-replete rats. Amiloride-induced changes in FELiwere independent of plasma Li+concentration but inversely related to the fractional excretion of Na+ and the amiloride-sensitive excretion of K+. These results are compatible with the hypothesis that a low tubular Na+ concentration reduces end-tubular Na+ reabsorption and results in hyperpolarization of the apical membrane, thus favoring Li+ uptake into the cells.