bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional and molecular characterization of the human neutral solute channel aquaporin-9
Authors: Tsukaguchi Hiroyasu , Weremowicz Stanislawa , Morton Cynthia C. , Hediger Matthias A.
Published: December 2017

In metabolically active cells, the coordinated transport of water and solutes is important for maintaining osmotic homeostasis. We recently identified a broad selective-neutral solute channel, AQP9, from rat liver that allows the passage of a wide variety of water and neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273: 24737–24743, 1998). A human homolog (hAQP9) with 76% amino acid sequence identity to rat AQP9 (rAQP9) was described, but its permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Biochem. Biophys. Res. Commun. 244: 268–274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its gene, and its chromosomal localization. When expressed in Xenopus oocytes, hAQP9 allowed passage of a wide variety of noncharged solutes, including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercurial-sensitive manner. These functional characteristics are similar to those of rAQP9. Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene is composed of 6 exons and 5 introns distributed over approximately ∼25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1–22.2. Our data show that hAQP9 serves as a promiscuous solute channel expressed in both liver and peripheral leukocytes, where it is ideally suited to transport of metabolites and/or nutrients into and out of these cells
Renal cortical ceramide patterns during ischemic and toxic injury: assessments by HPLC-mass spectrometry
Authors: Kalhorn Thomas , Zager Richard A.
Published: December 2017

Ceramides are a class of signaling molecules that can acutely accumulate in tissues as part of a “stress response.” They are classically measured by the diacylglycerol kinase assay, which, in general, measures total ceramide rather than individual moieties within the diverse ceramide family. The present study was undertaken to 1) adapt current HPLC-mass spectrometry technology for measuring individual renal ceramides, and 2) use this technique to more fully characterize the nature of the renal ceramide “stress” reaction. Renal cortical tissues were obtained from CD-1 mice under control conditions and 2 or 18 h after renal injury (ischemia-reperfusion and glycerol-mediated myohemoglobinuria). C24, C22, and C16 ceramides were identified in normal renal cortex, constituting 70, 10, and 20% of the total ceramide pool, respectively. Within each of these families, heterogeneity was apparent because of differing degrees of unsaturation (0–3 double bonds) in the constituent fatty acid of ceramide. Renal injury dramatically changed ceramide profiles: 1) total ceramide increased by ∼300%; 2) although all ceramides participated in this reaction, they did so to differing degrees; 3) this caused pronounced changes in ceramide distribution patterns; 4) injury induced a striking shift toward unsaturated (vs. saturated) fatty acids within the C22 and C24 (but not the C16) ceramide pools; and 5) the extent of these qualitative changes differed according to the etiology of the initiating renal damage. Thus we conclude that ceramide stress response involves major qualitative (and not simply quantitative) changes in ceramide expression that are partially disease dependent. These findings underscore the fact that simply measuring total renal ceramide content (e.g., by diacylglycerol kinase assay) substantially oversimplifies the nature and, hence, the potential implications of the ceramide stress reaction.
20-HETE agonists and antagonists in the renal circulation
Authors: Alonso-Galicia Magdalena , Falck John R. , Reddy Komandla Malla , Roman Richard J.
Published: December 2017

The present study examined the effects of a series of 20-hydroxyeicosatetraenoic acid (20-HETE) derivatives on the diameter of renal arterioles to determine the structural requirements of the vasoconstrictor response to 20-HETE. The vascular responses to 5-, 8-, 12-, 15-, 19-, 20-, 21-HETEs, arachidonic acid (AA), and saturated, partially saturated, dimethyl, carboxyl, and 19-carbon derivatives of 20-HETE (10−8 to 10−6 M) were assessed in rat renal interlobular arteries (65–125 μm). 20-HETE, 21-HETE, dimethyl-20-HETE, and a partially saturated derivative of 20-HETE, 20-hydroxyeicosa-5( Z),14( Z)-dienoic acid, reduced vessel diameter by 19 ± 3, 17 ± 3, 16 ± 2, and 28 ± 2%, respectively. In contrast, 5-, 8-, 12-, 15-, and 19-HETE, AA, saturated, partially saturated, carboxyl, and the 19-carbon derivatives of 20-HETE had no effect on vessel diameter. Pretreatment with 5-, 15-, and 19-HETE, the 19-carbon derivative or 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid (1 μM) completely blocked the vasoconstrictor response to 20-HETE in renal arterioles. Pretreatment with AA, carboxyl, saturated 19-carbon, and saturated 20-HETE derivatives (1 μM) partially blocked the response, whereas 8- and 12-HETE (1 μM) had no effect on the vasoconstrictor response to 20-HETE. These findings suggest that 20-HETE agonists and antagonists require a carboxyl or an ionizable group on carbon 1 and a double bond near the 14 or 15 carbon. 20-HETE agonists also require a functional group capable of hydrogen bonding on carbon 20 or 21, whereas antagonists lack this reactive group.
Estradiol suppresses mesangial cell type I collagen synthesis via activation of the MAP kinase cascade
Authors: Neugarten Joel , Medve Ildiko , Lei Jun , Silbiger Sharon R.
Published: December 2017

We have previously shown that estradiol suppresses the synthesis of type I collagen by murine mesangial cells grown in the presence of serum via activation of the transcription factor activator protein-1 (AP-1). We hypothesized that estradiol upregulates AP-1 via activation of the mitogen-activated protein (MAP) kinase cascade, a signal transduction pathway that regulates AP-1 activity. Estradiol (10−10 to 10−7 M) upregulated the MAP kinase pathway in murine mesangial cells grown in the presence of serum in a dose-dependent manner. Activation was evident by 1 min, peaked at 10 min, and was completely dissipated by 2 h. In contrast, estradiol had no significant effect on total (phosphorylated + unphosphorylated) p44 extracellular signal-related protein kinase (ERK) or p42 ERK. Nuclear extracts isolated from mesangial cells treated with estradiol showed increased binding to a consensus sequence AP-1 binding oligonucleotide in gel shift assays. In contrast, nuclear extracts from cells exposed to PD-98059, a highly selective inhibitor of MAP kinase-ERK kinase 1 (MEK1) and MEK2, showed reduced binding. In addition, PD-98059 antagonizes the enhanced binding induced by estradiol. Estradiol (10−9M) suppressed mesangial cell type I collagen synthesis (37.8 ± 2.4%, expressed as a percentage of control values, P < 0.001 vs. control). In contrast, PD-98059 increased type I collagen synthesis (344.6 ± 98.8, P < 0.01) and reversed the suppression of type I collagen synthesis induced by estradiol. The effects of estradiol, PD-98059, and PD-98059 plus estradiol on type I collagen protein synthesis were closely paralleled by their effects on steady-state levels of mRNA for the α1 chain of type I collagen. These data suggest that estradiol suppresses type I collagen synthesis via upregulation of the MAP kinase cascade, leading to stimulation of AP-1 activity.
Cyclin kinase inhibitor p21CIP1/WAF1limits interstitial cell proliferation following ureteric obstruction
Authors: Hughes Jeremy , Brown Paul , Shankland Stuart J.
Published: December 2017

Tubulointerstitial renal injury induced by unilateral ureteric obstruction (UUO) is characterized by marked cell proliferation and apoptosis. Proliferation requires cell cycle transit that is positively regulated by cyclins and cyclin-dependent kinases (CDKs) and inhibited by the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs: p21, p27, and p57). We have shown that the absence of p27 results in markedly increased tubular epithelial cell proliferation and apoptosis following UUO (V. Ophascharoensuk, M. L. Fero, J. Hughes, J. M. Roberts, and S. J. Shankland. Nat. Med.4: 575–580, 1998). Since p21 mRNA is upregulated following UUO, we hypothesized that p21 would also serve to limit cell proliferation and apoptosis. We performed UUO in p21 +/+ and p21 −/− mice. Cell proliferation [bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA)], apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method], interstitial myofibroblast accumulation (actin), macrophage infiltration (F4/80), and collagen I expression were quantified at days 3, 7, and 14. In contrast to p27 −/− mice, there was no difference in tubular epithelial cell proliferation or apoptosis between p21 −/− and p21 +/+ mice at any time point. However, interstitial cell proliferation at day 3 was significantly increased in p21 −/− mice [BrdU, 40.7 ± 1.9 cells/high-power field (cells/hpf) vs. 28.8 ± 2, P< 0.005], although, interestingly, no difference was seen in interstitial cell apoptosis. Actin/BrdU double staining demonstrated increased interstitial myofibroblast proliferation at day 3 in p21 −/− animals (10 ± 0.12 vs. 5.8 ± 0.11 cells/hpf, P < 0.05), which was followed by increased myofibroblast accumulation at day 7 in p21 −/− mice. No differences were detected in interstitial macrophage infiltration, collagen I deposition or transforming growth factor-β1 mRNA (in situ hybridization) expression. In conclusion p21, unlike p27, is not essential for the regulation of tubular epithelial cell proliferation and apoptosis following UUO, but p21 levels do serve to limit the magnitude of the early myofibroblast proliferation. This study demonstrates a differential role for the CKI p21 and p27 in this model.
Age-related progressive renal fibrosis in rats and its prevention with ACE inhibitors and taurine
Authors: Cruz Carmen Iglesias-De La , Ruiz-Torres Piedad , del Moral Raimundo García , Rodríguez-Puyol Manuel , Rodríguez-Puyol Diego
Published: December 2017

Our previous studies demonstrated an increased reactive oxygen species (ROS) production, as well as transforming growth factor-β1 (TGF-β1) expression in the rat kidney with aging. In the present study, we examined the effect of aging on extracellular matrix (ECM) accumulation and the effects of treatment with angiotensin-converting enzyme inhibitors (captopril and lisinopril) and taurine, an antioxidant amino acid. Age-related increases in types I and IV collagen and fibronectin mRNA expression were found at 24 and 30 mo of age. In contrast, type III collagen only increased in 30-mo-old rats. Captopril-, lisinopril-, and taurine-treated animals showed a statistically significant decrease in ECM protein expression at both ages. Moreover, treatment with taurine reduced the TGF-β1 mRNA levels in 24- and 30-mo-old rats by 40%. Taurine also completely blocked increases in type I and type IV collagen expression in mesangial cells in response to TGF-β1. Our results demonstrate a protective role from both converting enzyme inhibitors and taurine in the age-related progressive renal sclerosis. In addition, taking into account that taurine is considered as an antioxidant amino acid, present data suggest a role for ROS in age-related progressive renal fibrosis, perhaps through interactions with the TGF-β1 pathway.
UT-A2: a 55-kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin
Authors: Wade J. B. , Lee A. J. , Liu J. , Ecelbarger C. A. , Mitchell C. , Bradford A. D. , Terris J. , Kim G.-H. , Knepper M. A.
Published: December 2017

The renal urea transporter gene (UT-A) produces different transcripts in the inner medullary collecting ducts (UT-A1) and thin descending limbs of Henle's loop (UT-A2), coding for distinct proteins. Peptide-directed rabbit polyclonal antibodies were used to identify the UT-A2 protein in renal medulla of mouse and rat. In the inner stripe of outer medulla, an antibody directed to the COOH terminus of UT-A recognized a membrane protein of 55 kDa. The abundance of this 55-kDa protein was strongly increased in response to chronic infusion of the vasopressin analog 1-deamino-[8-d-arginine]vasopressin (DDAVP) in Brattleboro rats, consistent with previous evidence that UT-A2 mRNA abundance is markedly increased. Immunofluorescence labeling with the COOH-terminal antibody in Brattleboro rats revealed labeling in the lower portion of descending limbs from short-looped nephrons (in the aquaporin-1-negative portion of this segment). This UT-A labeling was increased in response to DDAVP. Increased labeling was also seen in descending limbs of long-looped nephrons in the base of the inner medulla. These results indicate that UT-A2 is expressed as a 55-kDa protein in portions of the thin descending limbs of Henle's loop and that the abundance of this protein is strongly upregulated by vasopressin.
High glucose induces the activity and expression of Na+/H+exchange in glomerular mesangial cells
Authors: Ganz Michael B. , Hawkins Karen , Reilly Robert F.
Published: December 2017

.—Changes in activity or expression of transporters may account for alterations in cell behavior in diabetes. We sought to ascertain if mesangial cells (MC) grown in different glucose concentrations exhibit changes in activity and expression of acid-extruding transporters, the Na+/H+and Na+-dependent Cl−/[Formula: see text]exchanger. pHiwas determined by the use of the fluorescent pH-sensitive dye BCECF. In MCs grown in 5 mM glucose (control), the Na+/H+exchanger was responsible for 31.8 ± 5.1% of steady-state pHi, whereas Na+-dependent Cl−/[Formula: see text]contributed 62.9 ± 4.0% ( n = 11). In MCs grown in high glucose for 2 wk, Na+/H+exchange contribution to acid-extrusion increased as follows: 42.3 ± 4.6% [ n = 8, 10 mM, not significant (NS)], 51.1 ± 5.1% ( n = 8, 20 mM, P < 0.01), and 64.8 ± 5.5% ( n = 7, 30 mM, P < 0.001). The Na+-dependent Cl−/[Formula: see text]exchanger contributed less [47.0 ± 4.6, 38.6 ± 5.8, and 21.1 ± 3.8%, for 10, 20, and 30 mM glucose, respectively ( n > 7)]. We sought to ascertain if the magnitude of the acute stimulated response to ANG II by the Na+/H+and Na+-dependent Cl−/[Formula: see text]exchanger is changed. Na+/H+exchanger (1.89-fold increase in 30 vs. 5 mM, P < 0.002), but not Na+-dependent Cl−/[Formula: see text]exchange (0.17-fold, NS), exhibited an enhanced response to ANG II (1 μM). Na+/H+exchange (NHE1) expression was significantly different (1.72-fold) after prolonged exposure to high glucose. These results suggest that the Na+/H+exchanger, but not Na+-dependent Cl−/[Formula: see text]exchanger, may play an early role in the response to hyperglycemia in the diabetic state.
Mixed descending- and ascending-type thin limbs of Henle's loop in mammalian renal inner medulla
Authors: Pannabecker Thomas L. , Dahlmann Anke , Brokl Olga H. , Dantzler William H.
Published: December 2017

Previous studies have generally indicated that the entire descending (DTL) and ascending thin limbs (ATL) of Henle's loops in the mammalian inner medulla exhibit structurally and functionally distinct properties. In the present study, we found that about 50% of Munich-Wistar rat inner medullary thin limbs, lying at positions distinctly above the bend, had segments exhibiting structural characteristics of DTL located immediately adjacent to segments exhibiting structural characteristics of ATL. Multiple DTL-type and ATL-type segments of variable length existed along a single straight portion of these mixed tubules. Inner medullary thin limbs with repeating, sequential expression of DTL-type and ATL-type regions were also numerous in Sprague-Dawley rats, mice, and rabbits with no evidence of sexual dimorphism. RT-PCR of microdissected segments showed that the water channel aquaporin-1 (AQP1) and the urea transporter UT-A2 were expressed in pure DTL, but not in pure ATL, and in DTL-type, but not in ATL-type, regions of mixed-type thin limbs. Immunocytochemistry revealed expression of AQP1 in cells of pure DTL, but not pure ATL, and in cells of DTL-type, but not ATL-type, regions of mixed-type thin limbs. In contrast, the chloride channel ClC-K1 was expressed in pure ATL, but not pure DTL, and in ATL-type, but not DTL-type, regions of mixed-type thin limbs. Discontinuous axial expression of AQP1, UT-A2, and ClC-K1 along the straight portion of single thin limbs indicates that these nephrons possess a more heterogeneous structure than previously recognized.
Effects of ammonia on bicarbonate transport in the cortical collecting duct
Authors: Frank Amy E. , Wingo Charles S. , Weiner I. David
Published: December 2017

Both acidosis and hypokalemia stimulate renal ammoniagenesis, and both regulate urinary proton and potassium excretion. We hypothesized that ammonia might play an important role in this processing by stimulating H+-K+-ATPase-mediated ion transport. Rabbit cortical collecting ducts (CCD) were studied using in vitro microperfusion, bicarbonate reabsorption was measured using microcalorimetry, and intracellular pH (pHi) was measured using the fluorescent, pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Ammonia caused a concentration-dependent increase in net bicarbonate reabsorption that was inhibited by luminal addition of either of the H+-K+-ATPase inhibitors, Sch-28080 or ouabain. The stimulation of net bicarbonate reabsorption was not mediated through apical H+-ATPase, basolateral Na+-K+-ATPase, or luminal electronegativity. Although ammonia caused intracellular acidification, similar changes in pHiinduced by inhibiting basolateral Na+/H+exchange did not alter net bicarbonate reabsorption. We conclude that ammonia regulates CCD proton and potassium transport, at least in part, by stimulating apical H+-K+-ATPase.
Interstitial water and solute recovery by inner medullary vasa recta
Authors: Edwards Aurélie , Delong Mark J. , Pallone Thomas L.
Published: December 2017

A recent model of volume and solute microvascular exchange in the renal medulla was extended by simulating the deposition of NaCl, urea, and water into the medullary interstitium from the loops of Henle and collecting ducts with generation rates that undergo spatial variation within the inner medullary interstitium. To build an exponential osmolality gradient in the inner medulla, as suggested by Koepsell et al. (H. Koepsell, W. E. A. P. Nicholson, W. Kriz, and H. J. Höhling. Pflügers Arch. 350: 167–184, 1974), the ratio of the interstitial area-weighted generation rate of small solutes to that of water must increase along the corticomedullary axis. We satisfied this condition either by holding the area-weighted generation rate of water constant while increasing that of NaCl and urea or by reducing the input rate of water with medullary depth. The latter case, in particular, yielded higher solute concentrations at the papillary tip. Assuming that the fraction of the filtered load recovered by inner medullary vasa recta for water, NaCl, and urea is 1%, 1%, and 40%, respectively, papillary tip osmolality is 1,470 mosmol/kgH2O when urea generation and NaCl generation per unit volume of interstitium increase exponentially and linearly, respectively. The inner medullary osmolar gradient also increases further when 1) medullary blood flow is reduced, 2) hydraulic conductivity of descending vasa recta (DVR) is lowered, and 3) vasa recta permeability to NaCl and urea is maximized. The coupling between water and small solute transport, resulting from aquaporin-1-mediated transcellular flux in DVR, also enhances tip osmolality.
DDM-PGE2-mediated cytoprotection in renal epithelial cells by a thromboxane A2receptor coupled to NF-κB
Authors: Weber Thomas J. , Monks Terrence J. , Lau Serrine S.
Published: December 2017

The present studies were conducted to determine the pharmacological nature of a cytoprotective 11-deoxy-16,16-dimethyl-PGE2(DDM-PGE2) receptor in LLC-PK1cells. DDM-PGE2-mediated cytoprotection against 2,3,5-(trisglutathion- S-yl)hydroquinone (TGHQ)-mediated cytotoxicity can be reproduced using thromboxane A2(TXA2) receptor (TP) agonists (U46619 and IBOP), and the cytoprotective response to DDM-PGE2and TP agonists is inhibited by TP antagonists (SQ-29,548 and ISAP). Western blot analysis using an antipeptide antibody against the human platelet TP receptor (55 kDa) identified a particulate associated 54-kDa protein. DDM-PGE2-mediated 12- O-tetradecanoyl phorbol-13-acetate (TPA) responsive element (TRE) binding activity is not inhibited by cyclooxygenase inhibitors (aspirin and indomethacin) or a TXA2synthase inhibitor (sulfasalazine), suggesting that the biological response to DDM-PGE2is not dependent on de novo TXA2biosynthesis. Peak DDM-PGE2- and U46619-mediated TRE binding activity and nuclear factor-κB (NF-κB) binding activity are inhibited by SQ-29,548. The full cytoprotective response to DDM-PGE2requires an 8-h pulse with agonist. DDM-PGE2-mediated TRE and NF-κB binding activity remain elevated in the presence of agonist and rapidly decay following agonist washout, suggesting a direct correlation between DDM-PGE2-mediated cytoprotection and persistent DNA binding activities. TPA, a protein kinase C activator, induces cytoprotection and a persistent increase of NF-κB binding activity. DDM-PGE2-mediated cytoprotection and NF-κB binding activity but not TRE binding activity are inhibited by sulfasalazine. We conclude that the DDM-PGE2receptor is a TP receptor and that the cytoprotective response may be mediated in part by NF-κB.
Renal physiology of the mouse
Authors: Meneton Pierre , Ichikawa Iekuni , Inagami Tadashi , Schnermann Jürgen
Published: December 2017

As the transgenic and gene-targeting technology has become an invaluable experimental approach to study the function of gene products, the need has been expanded to assess the physiology in the mouse, which is virtually the only animal species to which that new genetic technology can apply. In this regard, renal physiologists have also received fruits of success from modern technology in several key areas, and areas are expanding in both depth and scope.
Asymmetrical targeting of type II Na-Picotransporters in renal and intestinal epithelial cell lines
Authors: Hernando N. , Sheikh S. , Karim-Jimenez Z. , Galliker H. , Forgo J. , Biber J. , Murer H.
Published: December 2017

Targeting of newly synthesized transporters to either the apical or basolateral domains of polarized cells is crucial for the function of epithelia, such as in the renal proximal tubule or in the small intestine. Recently, different sodium-phosphate cotransporters have been identified. Type II cotransporters can be subdivided into two groups: type IIa and type IIb. Type IIa is predominantly expressed in renal proximal tubules, whereas type IIb is located on the intestinal and lung epithelia. To gain some insights into the polarized targeting of the type II cotransporters, we have transiently expressed type IIa and type IIb cotransporters in several epithelial cell lines: two lines derived from renal proximal cells (opossum kidney and LLC-PK1), one from renal distal cells (Madin-Darby canine kidney), and one from colonic epithelium (CaCo-2). We studied the expression of the transporters fused to the enhanced green fluorescent protein. Our data indicate that the polarized targeting is dependent on molecular determinants most probably located at the COOH terminus of the cotransporters as well as on the cellular context.
Role of Na+/H+exchanger NHE3 in nephron function: micropuncture studies with S3226, an inhibitor of NHE3
Authors: Vallon Volker , Schwark Jan-Robert , Richter Kerstin , Hropot Max
Published: December 2017

Na+/H+exchanger NHE3 is expressed in the luminal membrane of proximal tubule and thin and thick ascending limb of Henle's loop. To further define its role, the novel NHE3 inhibitor S3226 was employed in micropuncture experiments in nephrons with superficial glomeruli of anesthetized rats. Microperfusion of proximal convoluted tubule with S3226 revealed a dose-dependent inhibition of reabsorption (IC50of 4–5 μM) with a maximum inhibition of 30% for fluid and Na+. During microperfusion of Henle's loop (last superficial proximal to first superficial distal tubular loop), no effect of S3226 (10 or 30 μM) on the reabsorption of fluid or Na+was observed. Finally, S3226 (30 μM) left the tubuloglomerular feedback response unaltered as determined by the fall in proximal tubular stop-flow pressure in response to increasing loop of Henle perfusion rate. These studies indicate that NHE3 significantly contributes to fluid and Na+reabsorption in proximal convoluted tubule. NHE3 appears not to significantly contribute to fluid or Na+reabsorption in the loop of Henle (including the S3 segment of proximal tubule) or macula densa control of nephron filtration.
Regulation of stanniocalcin in MDCK cells by hypertonicity and extracellular calcium
Authors: Sheikh-Hamad David , Rouse Diane , Yang Yu
Published: December 2017

Differential display RT-PCR cloning method was applied to poly(A)+RNA isolated from Madin-Darby canine kidney (MDCK) cells in isotonic or hypertonic medium. A differentially expressed 360-bp PCR fragment was isolated, subcloned, sequenced, and used to screen an MDCK cDNA library constructed in λZapII. A composite sequence of two overlapping cDNA clones provided 1,053 bp of sequence that was 93% identical to human stanniocalcin and corresponded to the 3′-end of the mRNA. Although the fish homolog of this hormone inhibits calcium uptake by the gill and intestine, the function of mammalian stanniocalcin remains unknown. Stanniocalcin cDNA probe hybridizes to a 4.4-kb mRNA that is induced eightfold by hypertonicity, in a manner that is dependent on medium organic osmolytes. The mRNA induction correlates with increased total cellular content of the protein and its concomitant release to the medium, consistent with secretion for autocrine or paracrine activity. Furthermore, induction of the mRNA by hypertonicity is dependent on extracellular calcium and displays a threshold phenomenon. The data suggest that kidney stanniocalcin may have a role in the adaptation of kidney cells to osmotic stress, in a manner that is extracellular calcium dependent.
Macula densa Na+/H+exchange activities mediated by apical NHE2 and basolateral NHE4 isoforms
Authors: Peti-Peterdi János , Chambrey Régine , Bebok Zsuzsanna , Biemesderfer Daniel , St. John Patricia L. , Abrahamson Dale R. , Warnock David G. , Bell P. Darwin
Published: December 2017

Functional and immunohistochemical studies were performed to localize and identify Na+/H+exchanger (NHE) isoforms in macula densa cells. By using the isolated perfused thick ascending limb with attached glomerulus preparation dissected from rabbit kidney, intracellular pH (pHi) was measured with fluorescence microscopy by using 2′,7′-bis-(2-carboxyethyl)-5-(and -6) carboxyfluorescein. NHE activity was assayed by measuring the initial rate of Na+-dependent pHirecovery from an acid load imposed by prior lumen and bath Na+removal. Removal of Na+from the bath resulted in a significant, DIDS-insensitive, ethylisopropyl amiloride (EIPA)-inhibitable decrease in pHi. This basolateral transporter showed very low affinity for EIPA and Hoechst 694 (IC50= 9.0 and 247 μM, respectively, consistent with NHE4). The recently reported apical NHE was more sensitive to inhibition by these drugs (IC50= 0.86 and 7.6 μM, respectively, consistent with NHE2). Increasing osmolality, a known activator of NHE4, greatly stimulated basolateral NHE. Immunohistochemical studies using antibodies against NHE1–4 peptides demonstrated expression of NHE2 along the apical and NHE4 along the basolateral, membrane, whereas NHE1 and NHE3 were not detected. These results suggest that macula densa cells functionally and immunologically express NHE2 at the apical membrane and NHE4 at the basolateral membrane. These two isoforms likely participate in Na+transport, pHi, and cell volume regulation and may be involved in tubuloglomerular feedback signaling by these cells.
mLin-7 is localized to the basolateral surface of renal epithelia via its NH2terminus
Authors: Straight Samuel W. , Karnak David , Borg Jean-Paul , Kamberov Emmanuel , Dare Heidi , Margolis Ben , Wade James B.
Published: December 2017

In Caenorhabditis elegans, the basolateral localization of the Let-23 growth factor receptor tyrosine kinase requires the expression of three genes: lin-2, lin-7, and lin-10. Mammalian homologs of these three genes have been identified, and a complex of their protein products exists in mammalian neurons. In this paper, we examine the interaction of these mammalian proteins in renal epithelia. Coprecipitation experiments demonstrated that mLin-2/CASK binds to mLin-7, and immunofluorescent labeling showed that these proteins colocalized at the basolateral surface of Madin-Darby canine kidney cells and renal epithelia. Although labeling intensity varied markedly among different renal epithelial cells, those cells strongly expressing mLin-7 also showed intense mLin-2/CASK labeling. We have also demonstrated that mLin-2/CASK binding requires amino acids 12–32 of mLin-7 and have shown that this region of mLin-7 is also necessary for the targeting of mLin-7 to the basolateral surface. Furthermore, the overexpression of mLin-2/CASK mutants in Madin-Darby canine kidney cells caused endogenous mLin-7 to mislocalize. In summary, the NH2terminus of mLin-7 is crucial for its basolateral localization, likely through its interaction with mLin-2/CASK.
Postglomerular vasoconstriction induced by dopamine D3 receptor activation in anesthetized rats
Authors: Luippold Gerd , Schneider Swetlana , Vallon Volker , Osswald Hartmut , Mühlbauer Bernd
Published: December 2017

In the present study we investigated the renal hemodynamic effects of dopamine D3 receptor activation by R(+)-7-hydroxy-dipropylaminotetraline (7-OH-DPAT) in thiopental-anesthetized Sprague-Dawley rats. In clearance experiments infusion of 7-OH-DPAT (0.01–1.0 μg ⋅ kg− 1 ⋅ min− 1) dose-dependently elevated glomerular filtration rate (GFR) without affecting mean arterial blood pressure (MAP). In renal blood flow experiments 7-OH-DPAT infusion (1.0 μg ⋅ kg− 1 ⋅ min− 1) increased GFR by 16 ± 2%, associated with an unexpected fall in renal blood flow by 20 ± 3% and a significant elevation of renal vascular resistance by 18 ± 3%. The renal hemodynamic changes were not influenced by pretreatment with the D2-receptor antagonist S(−)-sulpiride but were completely abolished during D3 receptor inhibition by 5,6-dimethoxy-2-(di-n-propylamino)indane (U-99194A). In micropuncture experiments 7-OH-DPAT (1.0 μg ⋅ kg− 1 ⋅ min− 1) significantly elevated stop-flow pressure measured in the early proximal tubules and reduced hydrostatic pressure at the first branching point of the efferent arteriole without altering MAP. We conclude from these data that pharmacological activation of dopamine D3 receptors affects renal hemodynamics in anesthetized rats by preferential postglomerular vasoconstriction.
Regulation ofsgkby aldosterone and its effects on the epithelial Na+channel
Authors: Shigaev Alexander , Asher Carol , Latter Hedva , Garty Haim , Reuveny Eitan
Published: December 2017

Aldosterone is the major corticosteroid regulating Na+absorption in tight epithelia and acts primarily by activating the epithelial Na+channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514–2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgkmRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.
Renal interstitial Ca2+
Authors: Mupanomunda Maria M. , Tian Bing , Ishioka Norio , Bukoski Richard D.
Published: December 2017

Renal interstitial fluid Ca2+concentration ([Ca2+]isf) was measured in anesthetized Wistar rats by using in situ microdialysis. During perfusion of 20 cm of the proximal small intestine with Ca2+-free buffer, renal [Ca2+]isfwas 1.63 ± 0.19 mmol/l in the cortex ( n = 6) and 1.93 ± 0.12 mmol/l in the medulla ( n = 5, P = 0.223). When Ca2+in the intestinal lumen was increased to 3 mmol/l, no change was seen in total or ionized serum Ca2+(SCa), urinary Ca2+excretion (UCa), or Ca2+in a microdialysate of the kidney cortex. Increasing intestinal Ca2+further, to 6 mmol/l, was without effect on SCabut significantly increased UCaby 38% and microdialysate Ca2+by 36% (1.25 ± 0.0.09 vs. 1.70 ± 0.14 mmol/l, n = 4, P < 0.05). Intravenous infusion of 28 ng ⋅ kg−1⋅ min−1of parathyroid hormone for 1 h during perfusion of the intestinal lumen with 1 mmol/ Ca2+caused a 7–10% rise in SCa, a 40% fall in UCa, and a 32% increase in microdialysate Ca2+(1.32 ± 0.13 vs. 1.74 ± 0.13 mmol/l, n = 6, P < 0.05). Interlobar arteries with a mean diameter of 120 μm were studied by using a wire myograph to determine whether changes in extracellular Ca2+affect muscle tone. When precontracted with 5 μmol/l serotonin, the arteries relaxed in response to cumulative addition of Ca2+(1–5 mmol/l) with an ED50value for Ca2+of 3.30 ± 0.08 mmol/l, n = 3. These data demonstrate that [Ca2+]isfchanges dynamically during manipulation of whole-animal Ca2+homeostasis and that intrarenal arteries relax in response to extracellular Ca2+varied over the range measured in vivo.
5-HT2Areceptors stimulate mitogen-activated protein kinase via H2O2generation in rat renal mesangial cells
Authors: Greene Eddie L. , Houghton Odette , Collinsworth Georgiann , Garnovskaya Maria N. , Nagai Toshio , Sajjad Tahir , Bheemanathini Venugopala , Grewal Jasjit S. , Paul Richard V. , Raymond John R.
Published: December 2017

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT2Areceptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT2Areceptor antagonist ketanserin. The peak effect was noted at 5–10 min, and half-maximal stimulation was achieved at 10–30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H2O2and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT2Areceptor → Gqprotein → phospholipase C → diacylglycerol → classical PKC → NAD(P)H oxidase → superoxide → superoxide dismutase → H2O2→ mitogen-activated extracellular signal-regulated kinase → ERK. These studies demonstrate a role for the 5-HT2Areceptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H2O2in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.
Pushing, pulling, dragging, and vibrating renal epithelia by using atomic force microscopy
Authors: Henderson Robert M. , Oberleithner Hans
Published: December 2017

Renal physiologists focus on events that take place on and around the surfaces of cells. Various techniques have been developed that follow transport functions at the molecular level, but until recently none of these techniques has been capable of making the behavior of molecular structures visible under physiological conditions. This apparent gap may be filled in the future by the application of atomic force microscopy. This technique produces an image not by optical means, but by “feeling” its way across a surface. Atomic force microscopy can, however, be modified in a number of ways, which means that besides producing a high-resolution image, it is possible to obtain several types of data on the interactions between the ultrastructural components of cell membranes (such as proteins) and other biologically active molecules (such as ATP). In this review we describe the recent use of the atomic force microscope in renal physiology, ranging from experiments in intact cells to those in isolated renal transport protein molecules, include examples of these extended applications of the technique, and point to uses that the microscope has recently found in other areas of biology that should prove fruitful in renal physiology in the near future.
Angiotensinogen and AT1antisense inhibition of osteopontin translation in rat proximal tubular cells
Authors: Ricardo Sharon D. , Franzoni David F. , Roesener Colleen D. , Crisman Jacqueline M. , Diamond Jonathan R.
Published: December 2017

Antisense oligonucleotide inhibition of angiotensinogen and ANG II type 1 receptor (AT1) mRNA translation in rat proximal tubules (PT) was examined to provide direct evidence for a role of the renin-angiotensin system (RAS) in upregulated osteopontin expression observed following mechanical cell stretch. Male Sprague-Dawley rats underwent unilateral ureteral obstruction (UUO) under Brevital anesthesia. In situ hybridization and Western blot analysis demonstrated angiotensinogen mRNA and angiotensin converting enzyme (ACE) protein localized to PTs and upregulated in obstructed kidneys, respectively, confirming an increased expression of renal RAS in vivo. In vitro studies were performed to provide mechanistic insight into ANG II-dependent osteopontin expression following mechanical cell stretch, which putatively mimics the increased PT luminal pressure post-UUO. A cationic transfection method was used to introduce either angiotensinogen or AT1antisense oligonucleotide into cultured rat PT cells prior to 1 h of cyclic mechanical cell stretch. Northern blot analysis revealed that PT cells subjected to cyclic mechanical stretch with/without prior transfection with a sense oligonucleotide exhibited increased osteopontin mRNA expression compared with unstretched cells. Blockade of either angiotensinogen or AT1mRNA translation by antisense oligonucleotide inhibition prior to cell stretch was found to significantly decrease osteopontin mRNA levels 2.4-fold ( P < 0.004) and 1.6-fold ( P < 0.001), respectively, compared with values observed in control unstretched cells. This study provides evidence that stretch-induced upregulation of osteopontin mRNA expression is mediated, in part, via production of ANG II. These results lend insight into upregulation of osteopontin via a local PT RAS leading to macrophage infiltration in the tubulointerstitium in experimental hydronephrosis.
Novel biochemical and functional insights into nuclear Ca2+transport through IP3Rs and RyRs in osteoblasts
Authors: Adebanjo Olugbenga A. , Biswas Gopa , Moonga Baljit S. , Anandatheerthavarada Hindupur K. , Sun Li , Bevis Peter J. R. , Sodam Bali R. , Lai F. Anthony , Avadhani Narayan G. , Zaidi Mone
Published: December 2017

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40showed bands corresponding, respectively, to full-length RyR-1 (∼500 kDa) and IP3R-1 (∼250 kDa). Band intensity was reduced by just ∼20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+via Ca2+-ATPase activation (1 mM ATP and ∼100 nM Ca2+). Adequate Ca2+loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3or cADP ribose resulted in a rapid and sustained [Ca2+]npelevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+pathway.
Arachidonic acid metabolic pathways regulating activity of renal Na+-K+-ATPase are age dependent
Authors: Li Dailin , Belusa Roger , Nowicki Susana , Aperia Anita
Published: December 2017

Locally formed arachidonic acid (AA) metabolites are important as modulators of many aspects of renal tubular function, including regulation of the activity of tubular Na+-K+-ATPase. Here we examined the ontogeny of the AA metabolic pathways regulating proximal convoluted tubular (PCT) Na+-K+-ATPase activity in infant and adult rats. Eicosatetraynoic acid, an inhibitor of all AA-metabolizing pathways, abolished this effect. AA inhibition of PCT Na+-K+-ATPase was blocked by the 12-lipoxygenase inhibitor baicalein in infant but not in adult rats and by the specific cytochrome P-450 fatty acid ω-hydroxylase inhibitor 17-octadecynoic acid in adult but not in infant rats. The lipoxygenase metabolite 12(S)-hydroxyeicosatetraenoic acid (HETE) and the cytochrome P-450 metabolite 20-HETE both inhibited PCT Na+-K+-ATPase in a protein kinase C-dependent manner, but the effect was significantly more pronounced in infant PCT. Lipoxygenase mRNA was only detected in infant cortex. Expression of renal isoforms of cytochrome P-450 mRNA was more prominent in adult cortex. In summary, the AA metabolic pathways that modulated the activity of rat renal proximal tubular Na+-K+-ATPase are age dependent.
Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia
Authors: Bush Kevin T. , Tsukamoto Tatsuo , Nigam Sanjay K.
Published: December 2017

Ischemic epithelial cells are characterized by disruption of intercellular junctions and loss of apical-basolateral protein polarity, which are normally dependent on the integrity of the adherens junction (AJ). Biochemical analysis of both whole ischemic kidneys and ATP-depleted Madin-Darby canine kidney (MDCK) cells demonstrated a striking loss of E-cadherin (the transmembrane protein of the AJ) with the appearance and accumulation of an ∼80-kDa fragment reactive with anti-E-cadherin antibodies on Western blots of ATP-depleted MDCK cells. This apparent ischemia-induced degradation of E-cadherin was not blocked by either inhibitors of the major proteolytic pathways (i.e., proteasome, lysosome, or calpain), or by chelation of intracellular calcium, suggesting the involvement of a protease capable of functioning at low ATP and low calcium levels. Immunocytochemistry revealed the movement of several proteins normally comprising the AJ, including E-cadherin and β-catenin, away from lateral portions of the plasma membrane to intracellular sites. Moreover, rate-zonal centrifugation and immunoprecipitation with anti-E-cadherin and anti-β-catenin antibodies indicated that ATP depletion disrupted normal E-cadherin-catenin interactions, resulting in the dissociation of α- and γ-catenin from E-cadherin and β-catenin-containing complexes. Because the generation and maintenance of polarized epithelial cells are dependent upon E-cadherin-mediated cell-cell adhesion and normal AJ function, we propose that the rapid degradation of E-cadherin and dissolution of the AJ is a key step in the development of the ischemic epithelial cell phenotype. Furthermore, we hypothesize that the reassembly of the AJ after ischemia/ATP depletion may require a novel bioassembly mechanism involving recombination of newly synthesized and sorted E-cadherin with preexisting pools of catenins that have (temporally) redistributed intracellularly.
KCNA10: a novel ion channel functionally related to both voltage-gated potassium and CNG cation channels
Authors: Lang Rainer , Lee George , Liu Weimin , Tian Shulan , Rafi Hamid , Orias Marcelo , Segal Alan S. , Desir Gary V.
Published: December 2017

Our laboratory previously cloned a novel rabbit gene ( Kcn1), expressed in kidney, heart, and aorta, and predicted to encode a protein with 58% amino acid identity with the K channel Shaker Kv1.3 (Yao X et al. Proc Natl Acad Sci USA 92: 11711–11715, 1995). Because Kcn1 did not express well (peak current in Xenopus laevis oocytes of 0.3 μA at +60 mV), the human homolog (KCNA10) was isolated, and its expression was optimized in oocytes. KCNA10 mediates voltage-gated K+currents that exhibit minimal steady-state inactivation. Ensemble currents of 5–10 μA at +40 mV were consistently recorded from injected oocytes. Channels are closed at the holding potential of −80 mV but are progressively activated by depolarizations more positive than −30 mV, with half-activation at +3.5 ± 2.5 mV. The channel displays an unusual inhibitor profile because, in addition to being blocked by classical K channel blockers (barium tetraethylammonium and 4-aminopyridine), it is also sensitive to inhibitors of cyclic nucleotide-gated (CNG) cation channels (verapamil and pimozide). Tail-current analysis shows a reversal potential shift of 47 mV/decade change in K concentration, indicating a K-to-Na selectivity ratio of at least 15:1. The phorbol ester phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibited whole cell current by 42%. Analysis of single-channel currents reveals a conductance of ∼11 pS. We conclude KCNA10 is a novel human voltage-gated K channel with features common to both K-selective and CNG cation channels. Given its distribution in renal blood vessels and heart, we speculate that KCNA10 may be involved in regulating the tone of renal vascular smooth muscle and may also participate in the cardiac action potential.