bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrating multiple paracrine regulators of renal microvascular dynamics
Authors: Navar L. Gabriel
Published: December 2017

There has been tremendous growth in our knowledge about the multiple interacting mechanisms that regulate renal microvascular function. Paracrine signals originating from endothelial and epithelial cells exert profound influences on the basal tone and reactivity of the pre- and postglomerular arterioles. Selective responsiveness of these arterioles to various stimuli is possible because of differential activating mechanisms in vascular smooth muscle cells of afferent and efferent arterioles. Afferent arterioles rely predominantly on voltage-dependent calcium channels, while efferent arterioles utilize other mechanisms for calcium entry as well as intracellular calcium mobilization. The autoregulatory responses of preglomerular arterioles exemplify the selectivity of these complex control mechanisms. The myogenic mechanism responds to increases in renal perfusion pressure through “stretch-activated” cation channels that lead to depolarization, calcium entry, and vascular contraction. Autoregulatory efficiency is enhanced by the tubuloglomerular feedback (TGF) mechanism which responds to flow-dependent changes in tubular fluid composition at the level of the macula densa and transmits signals to the afferent arterioles to alter the activation state of voltage-dependent calcium channels. Recent studies have implicated extracellular ATP as one paracrine factor mediating TGF and autoregulatory related signals to the afferent arterioles. Other paracrine agents including nitric oxide, angiotensin II, adenosine, and arachidonic acid metabolites modulate vascular responsiveness in order to maintain an optimal balance between the metabolically determined reabsorptive capabilities of the tubules and the hemodynamically dependent filtered load.
ANG II and vasopressin stimulate calcium entry in dispersed smooth muscle cells of preglomerular arterioles
Authors: Iversen Bjarne M. , Arendshorst William J.
Published: December 2017

Calcium signaling mechanisms were examined in vessel segments and dispersed single smooth muscle cells (SMC) of interlobular arteries and afferent arterioles (<50 μm diameter) from the rat kidney. These resistance vessels were isolated from rat kidneys, using an iron oxide-sieving technique with subsequent collagenase digestion. Individual cells were identified by their characteristic oval appearance and positive staining for smooth muscle-specific α-actin and heavy chain myosin SM-1 and SM-2. Cytosolic calcium concentration ([Ca2+]i) was measured using fura 2 ratiometric fluorescence at 340 and 380 nm wavelength with a microscope-based photometer. Angiotensin II (ANG II) and arginine vasopressin (AVP), at concentrations of 10−10–10−6M, produced dose-dependent increases in [Ca2+]i; maximum increases were 221 ± 49 nM for ANG II and 237 ± 49 nM for AVP. The temporal response patterns for both agonists were characterized by a square-shaped, immediate step increase in [Ca2+]ito a near maximum level that was maintained through the recording period of 150–200 s. Responses of individual dispersed SMC and short vessel segments were similar. Losartan antagonized the action of ANG II, indicating mediation by AT1receptors on preglomerular arteriolar SMC. The V1-selective antagonist [d(CH2)5Tyr(Me)2Tyr(NH2)9]AVP completely inhibited AVP-induced [Ca2+]ichanges. The importance of calcium entry in hormone-induced changes in [Ca2+]iwas demonstrated by the finding that neither ANG II nor AVP elicited a [Ca2+]iresponse in media rendered nominally calcium free by addition of ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. Calcium entry occurred primarily through L-type, voltage-gated calcium channels as the dihydropyridine, nifedipine, completely prevented or reversed [Ca2+]ichanges normally elicited by either hormone. Our results provide new information about the similarity of calcium signaling in single SMC and short segments freshly isolated from renal interlobular arteries and afferent arterioles. The observations indicate that AT1and V1receptors are coupled to signal transduction pathways leading to rapid changes in [Ca2+]i. Calcium mobilization appears to play a minor to nonexistent role under the experimental conditions. The predominant mechanism involves calcium entry through dihydropyridine-sensitive, voltage-gated calcium channels in single SMC from these resistance vessels.
Soy protein modification of rat polycystic kidney disease
Authors: Ogborn Malcolm R. , Bankovic-Calic Neda , Shoesmith Christen , Buist Richard , Peeling James
Published: December 2017

We undertook a study to determine whether soy protein feeding would ameliorate renal injury in the Han:SPRD- cy rat model of polycystic kidney disease (PKD). Male offspring of Han:SPRD- cy heterozygotes received isocaloric diets based on 20% casein or 20% heat-treated soy protein at weaning ad libitum for 8 wk. Soy-fed animals demonstrated lower serum creatinine (66 vs. 125 μmol/l; P = 0.002), lower urinary ammonium excretion (0.080 vs. 0.173 mmol/kg; P= 0.01), reduced renal cysts (0.98 vs. 4.92 ml/kg body wt, P < 0.0001), renal fibrosis (0.79 vs. 1.4 ml/kg; P = 0.016), macrophage infiltration, renal tubular cell proliferation, and apoptosis. Proton nuclear magnetic resonance (1H-NMR) studies of urine demonstrated that soy diet was associated with increased losses of citric acid cycle organic anions.1H-NMR of perchloric acid-extracted tissue found that levels of succinate were not depleted in soy-fed animals, despite increased urinary losses. Soy-fed animals had marked elevation of tissue betaine ( P < 0.001), with reduced taurine and cholines, compared with casein-fed animals ( P < 0.001). Soy feeding dramatically reduces both tubular and interstitial pathology in the Han:SPRD- cy rat model of PKD, through mechanisms that remain to be determined.
IGF-I and insulin amplify IL-1β-induced nitric oxide and prostaglandin biosynthesis
Authors: Guan Zhonghong , Buckman Shaavhree Y. , Baier Lisa D. , Morrison Aubrey R.
Published: December 2017

The inflammatory cytokine interleukin-1β (IL-1β) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric oxide synthase (iNOS) with concomitant release of PGs and nitric oxide (NO) by glomerular mesangial cells. In our current studies, we determine whether insulin and IGF-I are involved in the signal transduction mechanisms resulting in IL-1β-induced NO and PGE2biosynthesis in renal mesangial cells. We demonstrate that both insulin and IGF-I increase IL-1β-induced Cox-2 and iNOS protein expression, which in turn enhance PGE2and NO production. Our data also indicate that both insulin and IGF-I enhance IL-1β-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and SAPK activation. These findings implicate the possible role of the MAPK pathway in mediating the effects of insulin and IGF-I on the upregulation of cytokine-stimulated NO and PG biosynthesis. Together, our results indicate that IGF-I and insulin may function to modulate the renal inflammatory process.
Cis- andtrans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity
Authors: Miyakawa Hiroshi , Woo Seung Kyoon , Chen Ching-Pu , Dahl Stephen C. , Handler Joseph S. , Kwon H. Moo
Published: December 2017

We have previously identified a tonicity-responsive enhancer (TonE) in the promoter region of the canine BGT1 gene. TonE mediates hypertonicity-induced stimulation of transcription. Here, we characterize TonE and TonE binding proteins (TonEBPs) to provide a biochemical basis for cloning of the TonEBPs. Mutational analysis applied to both hypertonicity-induced stimulation of transcription and TonEBP binding reveals that TonE is 11 base pairs in length, with the consensus sequence of (C/T)GGAAnnn(C/T)n(C/T). Activity of the TonEBPs increases in response to hypertonicity with a time course similar to that of transcription of the BGT1 gene. Studies with inhibitors indicate that translation, but not transcription, is required for activation of the TonEBPs. Phosphorylation is required for the stimulation of transcription but not for activation of DNA binding by the TonEBPs. In vivo methylation by dimethyl sulfate reveals that the TonE site of the BGT1 gene is protected with a time course like that of activity of the TonEBPs and activation of transcription. Ultraviolet cross-linking indicates that the TonEBPs share a DNA binding subunit of 200 kDa.
Effects of glycine betaine and glycerophosphocholine on thermal stability of ribonuclease
Authors: Burg Maurice B. , Peters Eugenia M.
Published: December 2017

Urea in renal medullas is sufficiently high to perturb macromolecules, yet the cells survive and function. The counteracting osmolytes hypothesis holds that methylamines, such as glycine betaine (betaine) and glycerophosphocholine (GPC) in renal medullas, stabilize macromolecules and oppose the effects of urea. Although betaine counteracts effects of urea on macromolecules in vitro and protects renal cells from urea in tissue culture, renal cells accumulate GPC rather than betaine in response to high urea both in vivo and in tissue culture. A proposed explanation is that GPC counteracts urea more effectively than betaine. However, we previously found GPC slightly less effective than betaine in counteracting inhibition of pyruvate kinase activity by urea. To test another macromolecule, we now compare GPC and betaine in counteracting reduction of the thermal stability of RNase A by urea. We find that urea decreases the thermal transition temperature and that betaine and GPC increase it, counteracting urea approximately equally. Therefore, the preference for GPC in response to high urea presumably has some other basis, such as a lower metabolic cost of GPC accumulation.
Regional time-dependent changes in vasopressin V2receptor expression in the rat kidney during water restriction
Authors: Park Frank , Koike George , Cowley Allen W.
Published: December 2017

Elevations of arginine vasopressin (AVP) binding to renal vasopressin V2receptors (V2R) enhance water and urea reabsorption in the collecting duct epithelium. This study was designed to quantify the levels of V2R mRNA and protein within the distinct regions of the Sprague-Dawley rat kidney (i.e., the cortex and outer and inner medulla) during 24 and 48 h of water restriction. A competitive reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to quantify changes in the V2R mRNA, in which a deletion mutant RNA transcript was used to control for the efficiency of RT-PCR. Western blot analysis was utilized for the quantification of the V2R protein. The results showed that the steady-state levels of the V2R mRNA decreased in a time-dependent manner in the cortex and outer and inner medulla throughout 48 h of water restriction. Western blot analysis revealed that the V2R protein in the renal cortex decreased after the initial 24 h of water restriction and remained decreased at 48 h. In contrast, outer medullary V2R protein decreased significantly only after 48 h of water restriction, whereas no significant change in the inner medullary V2R protein was observed throughout the 48 h of water restriction. These results suggest that water restriction leads to a regional time-dependent downregulation of the V2R mRNA and protein within the rat kidney. The stability of the plasma membrane V2R protein within the inner medulla may allow for the optimization of urine concentration and minimize water loss during periods of water restriction.
Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip
Authors: Huber Stephan , Schröppel Bernd , Kretzler Matthias , Schlöndorff Detlef , Horster Michael
Published: December 2017

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17( day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 ± 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.
Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats
Authors: Sands Jeff M. , Flores Francisco X. , Kato Akihiko , Baum Michelle A. , Brown Edward M. , Ward Donald T. , Hebert Steve C. , Harris H. William
Published: December 2017

To investigate how hypercalcemia blunts renal concentrating ability, alterations in basal and arginine vasopressin (AVP)-elicited osmotic water ( Pf) and urea ( Purea) permeabilities were measured in isolated perfused terminal inner medullary collecting ducts (IMCD) from control and chronically hypercalcemic rats after dihydrotachysterol (DHT) (M. Levi, L. Peterson, and T. Berl. Kidney Int. 23: 489–497, 1983) treatment. The IMCD Pfof DHT-treated rats did not increase significantly after AVP and was accompanied by a significant 87 ± 4% reduction in aquaporin-2 (AQP-2) protein but not mRNA. In contrast, both basal and AVP-elicited IMCD Pureafrom DHT rats were significantly increased and accompanied by a significant 41 ± 11% increase in AVP-regulated urea transporter protein content. Immunoblotting with anti-calcium/polyvalent cation-sensing receptor protein (CaR) antiserum revealed specific alterations in CaR bands in endosomes purified from the apical membranes of inner medulla of DHT rats. These data are the first detailed analyses of hypercalcemia-induced alterations in AVP-regulated permeabilities and membrane transporters in IMCD. We conclude that selective alterations in IMCD transport occur in hypercalcemia, permitting the body to dispose of excess calcium without forming calcium-containing renal stones.
Renal microvascular actions of calcitonin gene-related peptide
Authors: Reslerova Martina , Loutzenhiser Rodger
Published: December 2017

Calcitonin gene-related peptide (CGRP) is a potent vasodilator that is suggested to act via ATP-sensitive K channels (KATP). In the present study, we examined the actions of CGRP on pressure- and angiotensin II-induced vasoconstriction, using the in vitro perfused hydronephrotic rat kidney. Elevated pressure (from 80 to 180 mmHg) and 0.1 nM angiotensin II elicited similar decreases in afferent diameter in this model. CGRP inhibited myogenic reactivity in a concentration-dependent manner, completely preventing pressure-induced constriction at 10 nM (95 ± 10% inhibition). These effects were partially attenuated by 10 μM glibenclamide (62 ± 16% inhibition, P = 0.025), indicating both KATP-dependent and -independent actions of CGRP. In contrast, 10 nM CGRP inhibited angiotensin II-induced vasoconstriction by only 54 ± 11%, and this action was not affected by glibenclamide (41 ± 11%, P = 0.31). CGRP also inhibited the efferent arteriolar response to angiotensin II in the absence and presence of glibenclamide. Pinacidil (1.0 μM), a KATP opener also preferentially inhibited pressure- vs. angiotensin II-induced vasoconstriction (97 ± 5 and 59 ± 13% inhibition, respectively; P = 0.034). We conclude that the renal vasodilatory mechanisms of CGRP are pleiotropic and involve both KATP-dependent and -independent pathways. The effectiveness of CGRP in opposing renal vasoconstriction and the role of KATP in this action appear to depend on the nature the underlying vasoconstriction. We suggest that this phenomenon reflects an inhibition of KATP activation by angiotensin II.
Decreased endothelial nitric oxide synthase in gastric mucosa of rats with chronic renal failure
Authors: Tomikawa M. , Ohta M. , Vaziri N. D. , Kaunitz J. D. , Itani R. , Ni Z. , Tarnawski A. S.
Published: December 2017

According to recent reports, chronic renal failure (CRF) increases the susceptibility of gastric mucosa to injury. Since nitric oxide plays a major role in gastric mucosal defense and injury, we investigated, in rats with CRF produced by five-sixths nephrectomy and in control rats, the expression of nitric oxide synthase (NOS) in the stomach and measured mucosal and submucosal gastric blood flow. In CRF rats, gastric mucosal blood flow was significantly reduced compared with control rats, whereas submucosal and serosal blood flow was significantly increased. CRF significantly decreased endothelial NOS (eNOS) mRNA abundance by 53% ( P < 0.01) and reduced expression of eNOS protein by 42% ( P < 0.01) compared with the controls. Enzyme activity of eNOS was significantly reduced in gastric mucosa of CRF rats ( P < 0.05). These data are consistent with reduced gastric mucosal blood flow in CRF rats and can explain altered susceptibility of gastric mucosa to injury in CRF rats.
Cloning, renal distribution, and regulation of the rat Na+- HCO 3 − cotransporter
Authors: Burnham Charles E. , Flagella Michael , Wang Zhaohui , Amlal Hassane , Shull Gary E. , Soleimani Manoocher
Published: December 2017

We recently reported the cloning and expression of a human kidney Na+-[Formula: see text]cotransporter (NBC-1) (C. E. Burnham, H. Amlal, Z. Wang, G. E. Shull, and M. Soleimani. J. Biol. Chem. 272: 19111–19114, 1997). To expedite in vivo experimentation, we now report the cDNA sequence of rat kidney NBC-1. In addition, we describe both the organ and nephron segment distributions and the regulation of NBC-1 mRNA under three models of pH stress: chloride-depletion alkalosis (CDA), metabolic acidosis, and bicarbonate loading. Rat NBC-1 cDNA encodes an open reading frame of 1,035 amino acids, with 96 and 87% identity to human and salamander NBC-1, respectively. Rat NBC-1 mRNA is expressed at high levels in kidney and brain, with lower levels in colon, stomach, and heart. None appears in liver. In the kidney, NBC-1 is expressed mainly in the proximal tubule, with traces found in medullary thick ascending limb and papilla. [Formula: see text] loading decreased NBC-1 mRNA levels, which were unchanged either by metabolic acidosis or by CDA.
Expression of multiple α-adrenoceptor isoforms in rat CCD
Authors: Wilborn Teresa W. , Sun Duo , Schafer James A. ,   (With the Technical Assistance of Li Li and Mary Lou Watkins)
Published: December 2017

In the rat cortical collecting duct (CCD), epinephrine inhibits vasopressin (AVP)-dependent water permeability and Na+reabsorption. Although inhibition is reversed by the α2-adrenoceptor (AR) antagonist yohimbine, suggesting the epinephrine effect is primarily mediated by an α2-AR [C. T. Hawk, L. H. Kudo, A. J. Rouch, and J. A. Schafer. Am. J. Physiol. 265 ( Renal Fluid Electrolyte Physiol. 34): F449–F460, 1993], there are also suggestions of an effect at an additional receptor, perhaps an α1-AR. For the present experiments, we used RT-PCR of total RNA extracted from 1 to 5 mm of microdissected CCDs from rat kidney to identify the α-AR isoforms expressed. Specific primers for the α2-ARs amplifying from the 6th transmembrane (TM) to the 3′-untranslated regions, revealed the presence of α2Aand α2B. Western blot analysis also indicated the presence of α2B-AR at the protein level. Degenerate α1-AR primers that amplify from conserved regions of TM-1 to TM-5, as well as specific primers that amplify either the same region (α1B), the carboxy terminus (α1A), or within the third cytoplasmic loop (α1D), indicated the presence of all three α1-ARs. Measurement of transepithelial voltage in isolated perfused renal tubules indicated a small inhibitory effect mediated by α1-ARs. Although the functional effects of epinephrine on AVP-dependent transport processes appear to be mediated predominantly by an α2-AR, a small contribution to the overall α-AR effect may be due to simultaneous activation of an α1-AR.
Chronic renal failure in a mouse model of human adenine phosphoribosyltransferase deficiency
Authors: Stockelman Michael G. , Lorenz John N. , Smith Frost N. , Boivin Gregory P. , Sahota Amrik , Tischfield Jay A. , Stambrook Peter J.
Published: December 2017

In humans, adenine phosphoribosyltransferase (APRT, EC 2.4.2.7 ) deficiency can manifest as nephrolithiasis, interstitial nephritis, and chronic renal failure. APRT catalyzes synthesis of AMP from adenine and 5-phosphoribosyl-1-pyrophosphate. In the absence of APRT, 2,8-dihydroxyadenine (DHA) is produced from adenine by xanthine dehydrogenase (XDH) and can precipitate in the renal interstitium, resulting in kidney disease. Treatment with allopurinol controls formation of DHA stones by inhibiting XDH activity. Kidney disease in APRT-deficient mice resembles that seen in humans. By age 12 wk, APRT-deficient male mice are, on average, mildly anemic and smaller than normal males. They have extensive renal interstitial damage (assessed by image analysis) and elevated blood urea nitrogen (BUN), and their creatinine clearance rates, which measure excretion of infused creatinine as an estimate of glomerular filtration rate (GFR), are about half that of wild-type males. APRT-deficient males treated with allopurinol in the drinking water had normal BUN and less extensive visible renal damage, but creatinine clearance remained low. Throughout their lifespans, homozygous null female mice manifested significantly less renal damage than homozygous null males of the same age. APRT-deficient females showed no significant impairment of GFR at age 12 wk. Consequences of APRT deficiency in male mice are more pronounced than in females, possibly due to differences in rates of adenine or DHA synthesis or to sex-determined responses of the kidneys.
Extracellular Ca2+ decreases chloride reabsorption in rat CTAL by inhibiting cAMP pathway
Authors: Ferreira Marie Céleste De Jesus , Bailly Claire
Published: December 2017

The effect of activation of the Ca2+-sensing receptor on net Cl flux ( J Cl) has been investigated on microperfused cortical (C) thick ascending limb (TAL) from rat kidney. Increasing bath Ca2+ from 0.5 to 3 mM or adding 200 μM of the specific Ca2+-sensing receptor agonist neomycin reduced basal as well as antidiuretic hormone (ADH)-stimulated J Cl by 27.7 ± 5.0% and 25.9 ± 4.1%, respectively. J Cl remained unchanged in time control tubules. The effect of neomycin/Ca2+ on J Cl was blocked by two protein kinase A inhibitors, H-9 or H-89, but not by a protein kinase C inhibitor, GF-109203X, regardless of whether ADH was present or not. Moreover, H-89 decreased basal J Cl and prevented a further effect of 3 mM Ca2+. When J Cl was increased by 8-bromo-cAMP plus IBMX, no effect of 3 mM Ca2+ was observed. Inhibitors of phospholipase A2 and cytochrome P-450 monooxygenase failed to modify the effect of 3 mM Ca2+, although these agents dampened significantly the inhibitory effect of bradykinin on medullary TAL. We conclude that extracellular Ca2+ decreases basal and ADH-stimulated Cl reabsorption in CTAL by inhibiting the cAMP pathway, independently of protein kinase C or phospholipase A2 stimulation.
Cerebrospinal fluid formation and absorption in dehydrated sheep
Authors: Chodobski Adam , Szmydynger-Chodobska Joanna , McKinley Michael J.
Published: December 2017

Cerebrospinal fluid (CSF) plays an important role in the brain’s adaptive response to acute osmotic disturbances. In the present experiments, the effect of 48-h dehydration on CSF formation and absorption rates was studied in conscious adult sheep. Animals had cannulas chronically implanted into the lateral cerebral ventricles and cisterna magna to enable the ventriculocisternal perfusion. A 48-h water deprivation altered neither CSF production nor resistance to CSF absorption. However, in the water-depleted sheep, intraventricular pressure tended to be lower than that found under control conditions. This likely resulted from decreased extracellular fluid volume and a subsequent drop in central venous pressure occurring in dehydrated animals. In conclusion, our findings provide evidence for the maintenance of CSF production during mild dehydration, which may play a role in the regulation of fluid balance in the brain during chronic hyperosmotic stress.
Myeloma light chains are ligands for cubilin (gp280)
Authors: Batuman Vecihi , Verroust Pierre J. , Navar Gabriel L. , Kaysen James H. , Goda Fatime O. , Campbell Wendy C. , Simon Eric , Pontillon Francoise , Lyles Michelle , Bruno Joanne , Hammond Timothy G.
Published: December 2017

Although myeloma light chains are known to undergo receptor-mediated endocytosis in the kidney, the molecular identity of the receptor has not been characterized. We examined the interaction between cubilin (gp280) and four species of light chains isolated from the urine of patients with multiple myeloma. Four lines of evidence identify cubilin, a giant glycoprotein receptor, which is restricted in distribution to endocytic scavenger pathways and which has potent effects on endosomal trafficking, as a potentially physiologically relevant binding site for light chains: 1) light chains coeluted during immunoaffinity purification of cubilin; 2) polyclonal antisera to cubilin but not control sera, displaced human light chain binding from rat renal brush-border membranes; 3) cubilin bound to multiple species of light chains during surface plasmon resonance; 4) anti-cubilin antiserum interfered with light chain endocytosis by visceral yolk sac epithelial cells. However, both binding of light chains to brush-border membranes and endocytosis of light chains by yolk sac epithelial cells were only partially inhibited by anticubilin antibodies, suggesting presence of additional or alternate binding sites for light chains. Excess light chain had a potent inhibitory effect on endosomal fusion in vitro. Binding showed dose and time-dependent saturability with low-affinity, high-capacity equilibrium binding parameters. These data demonstrate that cubilin plays a role in the endocytosis and trafficking of light chains in renal proximal tubule cells.
Expression of HKα2protein is increased selectively in renal medulla by chronic hypokalemia
Authors: Codina Juan , Delmas-Mata Juan T. , DuBose Thomas D.
Published: December 2017

Our laboratory has demonstrated by Northern analysis that chronic hypokalemia increases HKα2(i.e., α-subunit of the colonic H+-K+-ATPase) mRNA abundance in the rat. To determine whether the increase in mRNA correlated with an increase in HKα2protein, an antibody was raised against a synthetic peptide derived from amino acids 686–698 of the HKα2sequence. The anti-HKα2antibody hybridized to rat distal colon membranes which migrated at ∼100 kDa (expected mobility of HKα2). HKα2protein was not detected in plasma membranes from rat whole kidney or stomach (100 μg) derived from control animals. The antibody was then used to investigate changes in expression of HKα2in renal cortex, renal medulla, and distal colon in two pathophysiological conditions: 1) chronic hypokalemia (LK) and 2) chronic metabolic acidosis (CMA). In LK rats there was a marked, but selective, increase in the abundance of HKα2protein in membranes prepared from renal medulla. Nevertheless, a corresponding increase in HKα2protein abundance was not observed in membranes prepared from the distal colon of LK rats. HKα2protein abundance in CMA was indistinguishable from controls. Moreover, chronic hypokalemia had no effect on expression of α1-Na+-K+-ATPase or HKα1in kidney or distal colon under any experimental condition. Therefore, HKα2protein is tissue- and site-specifically upregulated in response to chronic hypokalemia but not by CMA. Furthermore, this regulatory response is localized to the renal medulla.
Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension
Authors: Yip Kay-Pong , Tse Chung-Ming , McDonough Alicia A. , Marsh Donald J.
Published: December 2017

Redistribution of apical Na+/H+exchangers (NHE) in the proximal tubules as a plausible mechanism of pressure natriuresis was investigated with confocal immunofluorescence microscopy in Sprague-Dawley rats (SD), spontaneously hypertensive rats (SHR), and two-kidney, one-clip Goldblatt hypertensive rats (GH). NHE isoform NHE3 was localized in the brush border of proximal tubules in SD. Twenty minutes of induced acute hypertension (20–40 mmHg) resulted in a pronounced redistribution of isoform NHE3 from the brush border into the base of microvilli, where clathrin-coated pits were localized. Prehypertensive young SHR (5 wk old, mean blood pressure 105 ± 3 mmHg, n = 11) produced similar findings. However, NHE3 was found to concentrate in the base of microvilli in adult SHR (12 wk old, mean blood pressure 134 ± 6 mmHg, n = 12) and nonclipped kidneys of GH (mean blood pressure 131 ± 6 mmHg, n = 6). In clipped kidneys of GH, which were not exposed to the hypertension because of the arterial clips, NHE3 was localized on the brush border as in normal SD. No further redistribution of NHE3 was detected in adult SHR or GH when acute hypertension was induced. Since both acute and chronic increase of arterial pressure can provoke the redistribution of apical NHE in proximal tubules, the pressure-induced NHE redistribution could be a physiological response and an integral part of pressure natriuresis.
Inactivation of kinase cascades in mesangial cells grown on collagen type I
Authors: Miralem Tiho , Templeton Douglas M.
Published: December 2017

Growth on collagen type I gels is known to suppress the mitogenic responsiveness of mesangial cells. Because these cells proliferate in some renal diseases and themselves synthesize collagen type I, we examined the influence of growth on collagen upon several kinase signaling cascades involved in mesangial cell proliferation. Quiescent mesangial cells grown on collagen type I and then stimulated with serum showed a markedly diminished induction of the protooncogene c- fos, compared with their counterparts on plastic or fibronectin. This effect was accompanied by decreased activation of mitogen-activated (Erk family) and Ca2+/calmodulin-dependent protein kinases. Cells on collagen showed lower basal protein kinase C (PKC) activity and diminished levels of PKC-α and -ζ isoforms. Global phosphorylation of tyrosine residues was diminished on collagen, and tyrosine phosphorylation of Erk and focal adhesion kinase in response to serum was not detected, in contrast to cells on plastic. We conclude that attachment of mesangial cells to collagen type I results in a broad suppression of protein phosphorylation that is reflected in diminished induction of the c- fos gene and probably underlies the conversion of cultured mesangial cells to a nonproliferative phenotype.
Cyclooxygenase-2 participates in tubular flow-dependent afferent arteriolar tone: interaction with neuronal NOS
Authors: Ichihara Atsuhiro , Imig John D. , Inscho Edward W. , Navar L. Gabriel
Published: December 2017

To delineate the microvascular role of cyclooxygenase-2 (Cox-2) in modulating tubuloglomerular feedback (TGF) signals and to determine its relationship to neuronal nitric oxide synthase (nNOS), afferent (AA) and efferent (EA) arteriolar diameters of rat kidneys were assessed using the blood-perfused juxtamedullary nephron technique. The Cox-2 inhibitor NS-398 (10 μM) did not alter AA diameters in untreated kidneys but significantly constricted AAs by 17.0 ± 2.2% in kidneys treated with 10 mM acetazolamide, which enhances TGF-mediated AA constriction by increasing distal volume delivery. The NS-398-induced AA constriction was prevented after interruption of distal delivery by transection of the loops of Henle. The effect was selective for AAs since NS-398 did not influence EAs of untreated or acetazolamide-treated kidneys. Pretreatment with the nNOS inhibitor S-methyl-l-thiocitrulline (10 μM) prevented the NS-398-induced AA constriction observed during acetazolamide treatment. Although we previously demonstrated that acetazolamide treatment enhanced AA constrictor response to S-methyl-l-thiocitrulline, the enhancement by acetazolamide was inhibited by pretreatment with 10 μM NS-398 (16.4 ± 1.9 and 15.0 ± 0.5% with and without acetazolamide, respectively, P > 0.05). These results indicate that, during increased activation of TGF-dependent vasoconstrictor signals, Cox-2 generates vasodilatory metabolites in response to increased nNOS activity and thus participates in the counteracting modulation of TGF-mediated AA constriction.
Transport and regulatory properties of the apical Na-K-2Cl cotransporter of macula densa cells
Authors: Laamarti M. Anuar , Bell P. Darwin , Lapointe Jean-Yves
Published: December 2017

[Formula: see text]/NH3fluxes were used to probe apical Na-K-2Cl transport activity of macula densa (MD) cells from rabbit kidney. In the presence of 25 mM NaCl and 5 mM Ba2+, addition of 20 mM[Formula: see text] to the lumen produced a profound intracellular acidification, and ∼80% of the initial acidification rate was bumetanide sensitive. The[Formula: see text]-induced acidification rate was dependent on luminal Cl−and Na+with apparent affinities of 17 ± 4 mM (Hill number 1.45) and 1.0 ± 0.3 mM, respectively. In the presence of saturating luminal NaCl concentration ([NaCl]L), blockade of basolateral Cl−efflux with 10 μM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) reduced the [Formula: see text]-induced acidification rate by 51 ± 6% ( P> 0.01, n = 5). Under similar conditions, dibutyryl-cAMP (DBcAMP) + forskolin increased the[Formula: see text]-induced acidification rate by 27%, whereas it produced no detectable effect at low luminal NaCl concentration. Most of the observed DBcAMP + forskolin effect was probably due to the stimulation of the basolateral Cl−conductance, since, in the presence of basolateral NPPB, this activation was changed to a 17.1% and 16.6% inhibition of the[Formula: see text]-induced acidification rate observed at high or low [NaCl]L, respectively. We conclude that the cotransporter found in MD cells displays, with respect to other Na-K-2Cl cotransporters, a relatively high affinity for luminal Na+and luminal Cl−and can be specifically inhibited by increases in intracellular Cl−and cAMP concentrations.
Intrarenal and subcellular localization of rat CLC5
Authors: Luyckx Valerie A. , Goda Fatime O. , Mount David B. , Nishio Toshiyuki , Hall Amy , Hebert Steven C. , Hammond Timothy G. , Yu Alan S. L.
Published: December 2017

Dent’s disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi’s syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unknown. We have investigated the intrarenal and subcellular localization of CLC5 in rat kidney by in situ hybridization and immunohistochemistry. By in situ hybridization, CLC5 mRNA was detected predominantly in cortical medullary ray and outer medullary tubule epithelial cells. Polyclonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 isoform-specific antiserum. By immunohistochemistry, CLC5 protein was localized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending limb. By subcellular membrane fractionation and flow cytometry, CLC5 expression was found in outer medullary endosomes. These findings are consistent with a model in which CLC5 encodes an endosomal chloride channel that facilitates acidification and trafficking of renal epithelial endosomes.
Dietary lipids regulate β-oxidation enzyme gene expression in the developing rat kidney
Authors: Ouali F. , Djouadi F. , Merlet-Bénichou C. , Bastin J.
Published: December 2017

This study examines the ability of dietary lipids to regulate gene expression of mitochondrial and peroxisomal fatty acid β-oxidation enzymes in the kidney cortex and medulla of 3-wk-old rats and evaluates the role of glucagon or of the α-isoform of peroxisome proliferator-activated receptor (PPARα) in mediating β-oxidation enzyme gene regulation in the immature kidney. The long-chain (LCAD) and medium-chain acyl-CoA dehydrogenases (MCAD) and acyl-CoA oxidase (ACO) mRNA levels were found coordinately upregulated in renal cortex, but not in medulla, of pups weaned on a high-fat diet from day 16 to 21. Further results establish that switching pups from a low- to a high-fat diet for only 1 day was sufficient to induce large increases in cortical LCAD, MCAD, and ACO mRNA levels, and gavage experiments show that this upregulation of β-oxidation gene expression is initiated within 6 h following lipid ingestion. Treatment of pups with clofibrate, a PPARα agonist, demonstrated that PPARα can mediate regulation of cortical β-oxidation enzyme gene expression, whereas glucagon was found ineffective. Thus dietary lipids physiologically regulate gene expression of mitochondrial and peroxisomal β-oxidation enzymes in the renal cortex of suckling pups, and this might involve PPARα-mediated mechanisms.
Morphological and functional characterization of a conditionally immortalized collecting tubule cell line
Authors: Takacs-Jarrett Marcia , Sweeney William E. , Avner Ellis D. , Cotton Calvin U.
Published: December 2017

A conditionally immortalized collecting tubule cell line, mCT1, was derived from the H-2Kb-ts A58 transgenic mouse (ImmortoMouse), which harbors a temperature-sensitive mutant of the SV40 large T antigen oncogene. Cells maintained under permissive conditions [33°C with interferon-γ (IFN-γ)] form epithelial monolayers, express large T antigen, and proliferate (>50 passages). The cells retain properties characteristic of the renal collecting tubule (CT) including: vasopressin (VP)-stimulated cAMP accumulation, aquaporin-2 expression, high transepithelial electrical resistance, VP-stimulated ion transport, and amiloride-sensitive sodium absorption. When the cells are transferred to nonpermissive conditions (39°C without IFN-γ), the steady-state level of large T antigen protein declines (>95% decrease) and cell proliferation is arrested. This conditionally immortalized, murine renal cell line should prove useful for studies of CT physiology and large T antigen biology.
Chronic metabolic acidosis reversibly inhibits extracellular matrix gene expression in mouse osteoblasts
Authors: Frick Kevin K. , Bushinsky David A.
Published: December 2017

Chronic metabolic acidosis induces net calcium efflux from bone mineral through an increase in osteoclastic resorption and a decrease in osteoblastic matrix deposition and mineralization. To determine the effects of chronic metabolic acidosis on the expression of genes necessary for mineralization, we grew primary bone cells, which are principally osteoblasts, to confluence in neutral pH (7.5) medium and then switched the cells either to a neutral pH or to an acidic pH (7.1) differentiation medium. Cells were harvested for RNA at 4- to 7-day intervals for up to 44 days. By 36 days, there was extensive bone nodule formation and mineralization in cells cultured in neutral medium; however, there was a substantial decrease in nodule formation and mineralization in cells cultured in acidic medium. There was a marked increase in matrix Gla protein RNA and an increase in osteopontin RNA in neutral cultures; however, acidic medium almost completely prevented any increase. In contrast, RNA levels for osteonectin and transforming growth factor-β1 were not altered by chronic acidosis. Additional cells were incubated in acid differentiation medium for 1, 2, or 3 wk and then transferred to neutral medium; in each case, there was recovery of matrix Gla protein RNA and osteopontin RNA expression. Still other cells were incubated in neutral differentiation medium for 1, 2, or 3 wk and then transferred to acid medium; in each case there was inhibition of matrix Gla protein RNA and osteopontin RNA expression. Thus metabolic acidosis appears to specifically inhibit RNA accumulation of certain genes whose products may be essential for formation of mature bone matrix.
Effects of okadaic acid, calyculin A, and PDBu on state of phosphorylation of rat renal Na+-K+-ATPase
Authors: Li Dailin , Cheng Sam Xian Jun , Fisone Gilberto , Caplan Michael J. , Ohtomo Yoshiyuki , Aperia Anita
Published: December 2017

Several indirect lines of evidence suggest that protein kinases and phosphatases modulate the activity of renal Na+-K+-ATPase. The aim of this study was to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were prelabeled with [32P]orthophosphate and incubated with the inhibitors of protein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase α-subunit was evaluated by measuring the amount of [32P]phosphate incorporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [32P]phosphate incorporation into immunoprecipitated Na+-K+-ATPase α-subunit. OA and PDBu had a synergistic effect on the state of phosphorylation of Na+-K+-ATPase α-subunit. FK-506 did not affect Na+-K+-ATPase phosphorylation, neither alone nor in the presence of PDBu. Each of the drugs, OA, CL-A, and PDBu, inhibited the activity of Na+-K+-ATPase in microdissected proximal tubules. PDBu potentiated OA-induced inhibition of Na+-K+-ATPase activity. Inhibition of Na+-K+-ATPase required a lower dose of CL-A than of OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was ∼1.5 nmol Pi⋅ mg protein−1⋅ min−1. Using a monoclonal anti-α antibody that fails to recognize the subunit when Ser23is phosphorylated by PKC, we demonstrated that the dose response of PDBu inhibition of Na+-K+-ATPase correlated with the dose response of phosphorylation of the enzyme. The results suggest that the state of phosphorylation and activity of proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases.
Urinary concentrating function in mice lacking EP3receptors for prostaglandin E2
Authors: Fleming Eric F. , Athirakul Krairek , Oliverio Michael I. , Key Mikelle , Goulet Jennifer , Koller Beverly H. , Coffman Thomas M.
Published: December 2017

The actions of prostaglandin (PG) E2are mediated by four distinct classes of PGE2E-prostanoid (EP) receptors (EP1through EP4). However, the in vivo functions of the individual EP receptor subtypes have not been delineated. To study the functions of one of these subtypes, the EP3receptor, we generated EP3-deficient (−/−) mice by gene targeting. EP3−/− animals survived in expected numbers, reproduced, and had no obvious abnormalities in their major organ systems. Because the EP3receptor is expressed at high levels in the renal medulla and cortical collecting duct, and because previous studies have suggested that the EP3receptor might antagonize the effects of vasopressin in the distal nephron, we examined urinary concentrating functions in EP3−/− mice. Basal urine osmolality (UOsm) was similar in groups of EP3−/− and wild-type (EP3+/+) mice. However, after inhibition of endogenous PGE2production by indomethacin, UOsmincreased significantly in EP3+/+ but not in EP3−/− mice. Despite this insensitivity to acute inhibition of prostanoid production, EP3−/− mice concentrated and diluted their urine normally in response to a series of physiological stimuli. This suggests that PGE2acts through the EP3receptor to modulate urinary concentrating mechanisms in the kidney, but these effects are not essential for normal regulation of urinary osmolality.
pH-dependent modulation of the cloned renal K+ channel, ROMK
Authors: McNicholas Carmel M. , MacGregor Gordon G. , Islas Leon D. , Yang Yinhai , Hebert Steven C. , Giebisch Gerhard
Published: December 2017

pH is an important modulator of the low-conductance ATP-sensitive K+ channel of the distal nephron. To examine the mechanism of interaction of protons with the channel-forming protein, we expressed the cloned renal K channel, ROMK (Kir1.x), in Xenopus oocytes and examined the response to varied concentrations of protons both in the presence and in the absence of ATP. Initial experiments were performed on inside-out patches in the absence of ATP in Mg2+-free solution, which prevents channel rundown. A steep sigmoidal relationship was shown between bath pH and ROMK1 or ROMK2 channel function with intracellular acidification reducing channel activity. We calculated values for p K = 7.18 and 7.04 and Hill coefficients = 3.1 and 3.3, for ROMK1 and ROMK2, respectively. Intracellular acidification (pH 7.2) also increased the Mg-ATP binding affinity of ROMK2, resulting in a leftward shift of the relationship between ATP concentration and the reduction in channel activity. The K 1/2 for Mg-ATP decreased from 2.4 mM at pH 7.4 to ∼0.5 mM at pH 7.2. Mutation of lysine-61 to methionine in ROMK2, which abolishes pH sensitivity, modulated but did not eliminate the effect of pH on ATP inhibition of channel activity. We previously demonstrated that the putative phosphate loop in the carboxy terminus of ROMK2 is involved in ATP binding and channel inhibition [C. M. McNicholas, Y. Yang, G. Giebisch, and S. C. Hebert. Am. J. Physiol. 271 ( Renal Fluid Electrolyte Physiol. 40): F275–F285, 1996]. Conceivably, therefore, protonation of the histidine residue within this region could alter net charge (i.e., positive shift) and increase affinity for the negatively charged nucleotide.
Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney
Authors: Hosoyamada Makoto , Sekine Takashi , Kanai Yoshikatsu , Endou Hitoshi
Published: December 2017

Recently, we isolated the multispecific organic anion transporter (OAT1) from the rat kidney, which plays important roles in the renal elimination of endogenous and exogenous organic anions including clinically important drugs. In the present study, we cloned and characterized human OAT1. Two cDNA clones, hOAT1–1 cDNA and hOAT1–2 cDNA, were isolated from a human kidney cDNA library, whose amino acid sequences were 86.0% and 87.8% identical to that of rat OAT1, respectively. When expressed in Xenopus laevis oocytes, hOAT1 mediated sodium-independent uptake of p-aminohippurate (PAH) ( K m = 9.3 ± 1.0 μM). hOAT1-mediated PAH uptake was inhibited by bulky inorganic anions, various xenobiotics, and endogenous substances, including benzylpenicillin, furosemide, indomethacin, probenecid, phenol red, urate, and α-ketoglutarate. Northern blot analysis revealed that hOAT1 mRNA is strongly expressed in human kidney; transcripts of different sizes are expressed in skeletal muscle, brain, and placenta. Immunohistochemical analysis using rabbit IgG antibody against the carboxy-terminal 14 peptides of hOAT1 revealed that hOAT1 is expressed at the basolateral membrane of the proximal tubule. hOAT1 gene was located on human chromosome 11q13.1 by fluorescent in situ hybridization analysis. These results indicate that hOAT1 is a multispecific organic anion transporter on the basolateral membrane of the proximal tubule in human kidney.