bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutral endopeptidase inhibition potentiates the natriuretic actions of adrenomedullin
Authors: Lisy Ondrej , Jougasaki Michihisa , Schirger John A. , Chen Horng H. , Barclay Paul T. , Burnett John C.
Published: December 2017

Adrenomedullin (ADM) is a potent renal vasodilating and natriuretic peptide possessing a six amino acid disulfide ring. Neutral endopeptidase 24.11 (NEP) is localized in greatest abundance in the kidney and cleaves endogenous peptides like atrial natriuretic peptide, which also possesses a disulfide ring. We hypothesized that NEP inhibition potentiates the natriuretic actions of exogenous ADM in anesthetized dogs ( n = 6). We therefore investigated renal function in which one kidney received intrarenal infusion of ADM (1 ng ⋅ kg−1⋅ min−1) while the contralateral kidney served as control before and during the systemic infusion of a NEP inhibitor (Candoxatrilat, 8 μg ⋅ kg−1⋅ min−1; Pfizer). In response to ADM, glomerular filtration rate (GFR) in the ADM kidney did not change, whereas renal blood flow, urine flow (UV), and urinary sodium excretion (UNaV) increased from baseline. Proximal and distal fractional reabsorption of sodium decreased in the ADM-infused kidney. In response to systemic NEP inhibition, UNaV and UV increased further in the ADM kidney. Indeed, ΔUNaV and ΔUV were markedly greater in the ADM kidney compared with the control kidney. Plasma ADM was unchanged during ADM infusion but increased during NEP inhibition. In conclusion, the present investigation is the first to demonstrate that NEP inhibition potentiates the natriuretic and diuretic responses to intrarenal ADM. This potentiation occurs secondary to a decrease in tubular sodium reabsorption. Lastly, the increase in plasma ADM during systemic NEP inhibition supports the conclusion that ADM is a substrate for NEP.
Regulation of human mesangial cell collagen expression by transforming growth factor-β1
Authors: Poncelet Anne-Christine , Schnaper H. William
Published: December 2017

Transforming growth factor (TGF)-β1 has been implicated in glomerular extracellular matrix accumulation. Since the spectrum and mechanism of changes in collagen turnover have not been fully characterized, we evaluated effects of TGF-β1 on collagen expression by human mesangial cells. TGF-β1 induced increased α1(I), α1(III), and α1(IV) collagen mRNA expression. Greater mRNA expression of matrix metalloproteinase (MMP)-2 was compensated by increased tissue inhibitor of metalloproteinases (TIMP)-2 mRNA. There was no change in TIMP-1 or membrane-type MMP mRNA expression, whereas MMP-1 mRNA decreased. Types I and IV collagen protein accumulated in both the cell layer and medium. Changes in collagen mRNA and protein occurred within 4 and 8 h, respectively. MMP-2 and TIMP-1 and -2 activities showed little change. Cycloheximide markedly decreased collagen detection within 4 h and reversed late, but not early, changes in α1(I) collagen mRNA. In this system, increased synthesis may be more significant than degradation for collagen accumulation, but collagen is short-lived in culture. Diverse TGF-β1 actions on collagen turnover may be either immediate or mediated through synthesis of regulatory molecules.
Aquaporin-2 expression in primary cultured rat inner medullary collecting duct cells
Authors: Maric Kenan , Oksche Alexander , Rosenthal Walter
Published: December 2017

Cultured renal epithelial cells rapidly downregulate expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2). Our aim was to define conditions that favor maintenance of AQP-2 expression in vitro without genetic manipulation. We show here that primary cultures of rat inner medullary collecting duct (IMCD) cells retain AQP-2 expression for at least 6 days when grown with dibutyryl cAMP (DBcAMP) supplementation. We also found that coating the culture dishes with type IV collagen, rather than rat-tail collagen, retards AQP-2 downregulation. Immunofluorescence and biochemical studies indicate a shuttling of AQP-2-bearing vesicles after stimulation with vasopressin or forskolin. Rab3 proteins, known to be involved in regulated exocytosis, were detected only in cells grown in the presence of DBcAMP. Using the adenylyl cyclase assay, we confirmed the functional integrity of the vasopressin V2receptor in a broken cell preparation. Our data show that cAMP supplementation is sufficient for the maintenance of AQP-2 expression in primary cultured cells. The model system established here allows the study of the regulation of genes encoding the antidiuretic machinery at the cellular level.
Human renal and systemic hemodynamic, natriuretic, and neurohumoral responses to different doses ofl-NAME
Authors: Broere A. , Van Den Meiracker A. H. , Boomsma F. , Derkx F. H. M. , Man In’T Veld A. J. , Schalekamp M. A. D. H.
Published: December 2017

Experimental evidence indicates that the renal circulation is more sensitive to the effects of nitric oxide (NO) synthesis inhibition than other vascular beds. To explore whether in men the NO-mediated vasodilator tone is greater in the renal than in the systemic circulation, the effects of three different intravenous infusions of NG-nitro-l-arginine methyl ester (l-NAME; 1, 5, and 25 μg ⋅ kg−1⋅ min−1for 30 min) or placebo on mean arterial pressure (MAP), systemic vascular resistance (SVR), renal blood flow (RBF), renal vascular resistance (RVR), glomerular filtration rate (GFR), and fractional sodium and lithium excretion (FENaand FELi) were studied in 12 healthy subjects, each receiving randomly two of the four treatments on two different occasions. MAP was measured continuously by means of the Finapres device, and stroke volume was calculated by a model flow method. GFR and RBF were estimated from the clearances of radiolabeled thalamate and hippuran. Systemic and renal hemodynamics were followed for 2 h after start of infusions. During placebo, renal and systemic hemodynamics and FENaand FELiremained stable. With the low and intermediate l-NAME doses, maximal increments in SVR and RVR were similar: 20.4 ± 19.6 and 23.5 ± 16.0%, respectively, with the low dose and 31.4 ± 26.7 and 31.2 ± 14.4%, respectively, with the intermediate dose (means ± SD). With the high l-NAME dose, the increment in RVR was greater than the increment in SVR. Despite a decrease in RBF, FENaand FELidid not change with the low l-NAME dose, but they decreased by 31.2 ± 11.0 and 20.2 ± 6.3%, respectively, with the intermediate dose and by 70.8 ± 8.1 and 31.5 ± 15.9% with the highl-NAME dose, respectively. It is concluded that in men the renal circulation is not more sensitive to the effects of NO synthesis inhibition than the systemic circulation and that the threshold for NO synthesis inhibition to produce antinatriuresis is higher than the threshold level to cause renal vasoconstriction.
Ultrastructural localization of Na-K-2Cl cotransporter in thick ascending limb and macula densa of rat kidney
Authors: Nielsen Søren , Maunsbach Arvid B. , Ecelbarger Carolyn A. , Knepper Mark A.
Published: December 2017

A bumetanide-sensitive Na-K-2Cl cotransporter, BSC-1, is believed to mediate the apical component of transcellular NaCl absorption in the thick ascending limb (TAL) of Henle’s loop. To study its ultrastructural localization in kidney, we used an affinity-purified, peptide-derived polyclonal antibody against rat BSC-1. Immunoblots from rat kidney cortex and outer medulla revealed a solitary 161-kDa band in membrane fractions. Immunocytochemistry of 1-μm cryosections demonstrated strong BSC-1 labeling of the apical and subapical regions of medullary and cortical TAL cells. Notably, macula densa cells also exhibited distinct labeling. Distal convoluted tubules and other renal tubule segments were unlabeled. Immunoelectron microscopy demonstrated that BSC-1 labeling was associated with the apical plasma membrane and with subapical intracellular vesicles in medullary and cortical TAL and in macula densa cells. Smooth-surfaced TAL cells, in particular, had extensive BSC-1 labeling of intracellular vesicles. These results support the view that BSC-1 provides the apical pathway for NaCl transport across the TAL and that an extensive intracellular reservoir of BSC-1 is present in a subpopulation of TAL cells. Furthermore, the BSC-1 localization in the apical plasma membrane of macula densa cells is consistent with its proposed role in tubuloglomerular feedback.
Extracellular matrix-related genes in kidney after ischemic injury: potential role for TGF-β in repair
Authors: Basile David P. , Martin Daniel R. , Hammerman Marc R.
Published: December 2017

The renal expression of transforming growth factor-β1 (TGF-β1) is enhanced following induction of ischemic injury in rat. In cultured renal cells, TGF-β stimulates the synthesis of extracellular matrix. To link TGF-β1 expression with the regulation of extracellular matrix postischemia, we characterized the expression of several genes known to regulate extracellular matrix synthesis at various times during recovery from acute ischemic renal injury in rat. Levels of mRNA for plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloprotease-1 (TIMP-1), α1(IV) collagen, and fibronectin-EIIIA (FN-EIIIA) mRNAs were significantly enhanced in kidneys within 12 h to 3 days after injury and remained elevated at 7–28 days postischemia relative to levels in kidneys of sham-operated controls. PAI-1 mRNA and peptide were localized in regenerating proximal tubules at 3 and 7 days postischemic injury. α1(IV) Collagen and FN-EIIIA mRNAs were expressed primarily in regenerating proximal tubule cells. Immunoreactivity for FN-EIIIA was enhanced in the tubular basement membrane (TBM) of regenerating proximal tubules, and α1(IV) collagen immunoreactivity was detected in thickened tubulointerstitial spaces. In contrast, TIMP-1 immunoreactivity was enhanced in distal nephron structures postischemia. Immunoneutralization of TGF-β in vivo attenuated the increases in FN-EIIIA, α1(IV) collagen, PAI-1, and TIMP-1 mRNAs by 52%, 73%, 43%, and 27%, respectively. These data are consistent with TGF-β expression postischemic injury participating in renal regeneration of extracellular matrix homeostasis in the proximal TBM.
Inhibition of adenosine-1 receptor-mediated preglomerular vasoconstriction in AT1Areceptor-deficient mice
Authors: Traynor Timothy , Yang Tianxin , Huang Yuning G. , Arend Lois , Oliverio Michael I. , Coffman Thomas , Briggs Josie P. , Schnermann Jürgen
Published: December 2017

The effect of the adenosine type 1 receptor agonist N 6-cyclohexyladenosine (CHA) on glomerular vascular reactivity was studied in male angiotensin II type 1A (AT1A) receptor knockout mice (9). Vascular reactivity was assessed as the response of stop-flow pressure (PSF) to infusion of CHA into loops of Henle using micropuncture techniques. In AT1A +/+ mice at ambient arterial blood pressure (96.7 ± 2.8 mmHg), the presence of CHA (10−5 M) in the perfusate increased PSF responses from 6.8 ± 0.6 to 14.3 ± 0.9 mmHg when the loop of Henle of the index nephron was perfused and from 0.7 ± 0.3 to 12.3 ± 1.0 mmHg when the loop of an adjacent nephron was perfused. At reduced arterial blood pressure (82.8 ± 1.3 mmHg), index nephron perfusion with CHA increased PSF responses from 4.5 ± 0.3 to 9.4 ± 0.4 mmHg. In AT1A −/− mice with a mean arterial blood pressure of 80 ± 1.9 mmHg, CHA increased PSF responses only from 0.1 ± 0.3 to 3.6 ± 0.54 mmHg during index nephron perfusion and from 0.25 ± 0.2 to 2.7 ± 0.55 mmHg during adjacent nephron perfusion, significantly less than in wild-type animals ( P < 0.001). Responses to CHA were intermediate in AT1A +/− mice. Thus AT1A receptor knockout mice show a markedly reduced constrictor response to CHA both in the presence and absence of simultaneous activation of the tubuloglomerular feedback system. These data support the notion of a functional interaction between adenosine and angiotensin II in the regulation of afferent arteriolar tone.
Kid-1 expression is high in differentiated renal proximal tubule cells and suppressed in cyst epithelia
Authors: Witzgall Ralph , Obermüller Nicholas , Bölitz Ulrike , Calvet James P. , Cowley Benjamin D. , Walker Cheryl , Kriz Wilhelm , Gretz Norbert , Bonventre Joseph V.
Published: December 2017

The cDNA coding for the transcriptional repressor protein Kid-1 was cloned in a screen for zinc finger proteins, which are regulated during renal development and after renal ischemia. Kid-1 mRNA levels increase in the course of postnatal renal development and decrease after acute renal injury caused by ischemia or administration of folic acid. We have raised a monoclonal anti-Kid-1 antibody and demonstrate that the Kid-1 protein is strongly expressed in the proximal tubule of the adult rat kidney. During nephron development, the Kid-1 protein appears after the S-shaped body stage concomitantly with the brush-border enzyme alkaline phosphatase. In two animal models of polycystic kidney disease, the expression of Kid-1 is downregulated. The loss of expression of Kid-1 in cyst wall cells correlates with the loss of alkaline phosphatase histochemical staining. Kid-1 mRNA levels are also reduced in rodent renal cell carcinomas, another condition characterized by epithelial cell dedifferentiation and increased proliferation. We propose that Kid-1 plays an important role during the differentiation of the proximal tubule.
Regulation of c-ret expression by retinoic acid in rat metanephros: implication in nephron mass control
Authors: Moreau Evelyne , Vilar José , Lelièvre-Pégorier Martine , Merlet-Bénichou Claudie , Gilbert Thierry
Published: December 2017

Vitamin A and its derivatives have been shown to promote kidney development in vitro in a dose-dependent fashion. To address the molecular mechanisms by which all- trans-retinoic acid (RA) may regulate the nephron mass, rat kidneys were removed on embryonic day 14( E14) and grown in organ culture under standard or RA-stimulated conditions. By using RT-PCR, we studied the expression of the glial cell line-derived neurotrophic factor (GDNF), its cell surface receptor-α (GDNFR-α), and the receptor tyrosine kinase c-ret, known to play a major role in renal organogenesis. Expression of GDNF and GDNFR-α transcripts was high at the time of explantation and remained unaffected in culture with or without RA. In contrast, c-ret mRNA level, which was low in E14 metanephros and dropped rapidly in vitro, was increased by RA in a dose-dependent manner. The same is true at the protein level. Exogenous GDNF barely promotes additional nephron formation in vitro. Thus the present data establish c-ret as a key target of retinoids during kidney organogenesis.
Regulation of sodium transport in M-1 cells
Authors: Nakhoul Nazih L. , Hering-Smith Kathleen S. , Gambala Cecilia T. , Hamm L. Lee
Published: December 2017

The M-1 cell line, derived from the mouse cortical collecting duct (CCD), is being used as a mammalian model of the CCD to study Na+ transport. The present studies aimed to further define the role of various hormones in affecting Na+ transport in M-1 cells grown in defined media. M-1 cells on permeable support, in serum-free media, developed amiloride-sensitive current 4–5 days after seeding. As expected for the involvement of epithelial Na+ channels, α-, β-, and γ-subunits of the epithelial Na+ channel were identified by RT-PCR. Either dexamethasone (Dex, 10–100 nM) or aldosterone (Aldo, 10−6–10−7M) for 24 h stimulated transport. Cells grown in the presence of Aldo and Dex had higher transport than with Dex alone. Spironolactone added to Dex media decreased transport. The acute effects of hormones reported to inhibit Na+ transport in CCD were also examined. Epidermal growth factor, phorbol esters, and increased intracellular Ca2+ with thapsigargin did not alter transport. Arginine vasopressin caused a transient increase in transport (probably Cl− secretion), which was not amiloride sensitive. Also, the protease inhibitor aprotinin decreased Na+ transport; in aprotinin-treated cells, trypsin stimulated transport. This study demonstrates that adrenal steroids (Dex > Aldo) stimulate Na+ transport in M-1 cells. At least part of this response may represent activation of mineralocorticoid receptors based on an additive effect of Dex and Aldo, as well as inhibition by spironolactone. Responses to immediate-acting hormones is limited. However, an endogenous protease activity, which activates Na+ transport, is present in these cells.
Obstruction stimulates COX-2 expression in bladder smooth muscle cells via increased mechanical stretch
Authors: Park John M. , Yang Tianxin , Arend Lois J. , Schnermann Jürgen B. , Peters Craig A. , Freeman Michael R. , Briggs Josephine P.
Published: December 2017

Studies were performed to investigate the regulatory mechanism of bladder cyclooxygenase-2 (COX-2) expression after outlet obstruction. In situ hybridization of murine bladder tissues using COX-2-specific riboprobes demonstrated that COX-2 expression was induced predominantly in the bladder smooth muscle cells after outlet obstruction. To study the effect of increased mechanical stretch on COX isoform expression, cultured rat bladder smooth muscle cells were grown on silicone elastomer-bottomed plates coated with collagen type I and were subjected to continuous cycles of stretch/relaxation for variable duration. COX-1 mRNA levels did not change with stretch. COX-2 expression increased in a time-dependent manner after stretch, with maximal mRNA and protein levels occurring after 4 h. PGE2levels increased more than 40-fold in the culture media after stretch, consistent with increased COX activity, and this was reduced to near completion in the presence of a COX-2 inhibitor, NS-398. Exposure to stretch over a 48-h period induced a 4.7 ± 0.6-fold increase in tritiated thymidine incorporation rate. This increase in DNA synthesis was markedly suppressed when the cells were stretched in the presence of NS-398. We conclude that in bladder obstruction COX-2 activation occurs predominantly in the smooth muscle cells in response to mechanical stretch. Our findings also suggest that stretch-activated COX-2 expression may participate in bladder smooth muscle cell proliferation and thereby play a role in pathological bladder wall thickening after obstruction.
Hypotonic saline infusion alters the renal response to amino acids in men
Authors: Claris-Appiani Aldo , Tirelli Amedea S. , Ardissino Gianluigi , Daccò Valeria , Moretto Eugenia , Corbetta Carlo , Guidi Laura , Assael Baroukh M.
Published: December 2017

We investigated the effects of hypotonic saline-induced modifications of extracellular volume and sodium handling on the renal and metabolic response to amino acids (AA). Renal hemodynamics (Inutest, p-aminohippurate clearance), plasma AA, and glucagon levels, as well as urea and sodium excretion, were studied in seven adult volunteers infused for 2 h, on six separate occasions, according to the following protocols: 1) high-AA solution (300 mg ⋅ min−1⋅ 1.73 m−2); 2) low-AA solution (150 mg ⋅ min−1⋅ 1.73 m−2); 3) low AA + 2,000 ml/1.73 m2of 0.23% saline solution; 4) high AA + 0.23% saline; 5) high AA + 0.45% saline; and 6) 0.45% saline alone. The glomerular filtration rate (GFR) rise induced by the high-AA solution was similar to that induced by the low-AA solution (ΔGFR = +24 ± 6 and +20.2 ± 7 ml ⋅ min−1⋅ 1.73 m−2, respectively), whereas the plasma AA and glucagon levels and urea excretion rate increases were related to AA dose. The addition of 0.23% saline to the low-AA solution and of 0.45% saline to the high-AA solution blunted the renal hemodynamic response (ΔGFR = +6.6 ± 10.1 and +11.4 ± 8.3 ml ⋅ min−1⋅ 1.73 m−2, respectively) without modifying the pattern of plasma AA and glucagon levels and urea excretion observed with the AA infusion alone. Urinary sodium excretion increased from baseline with each protocol and rose even further when saline was added to AA. A negative correlation ( r = −0.38, P < 0.05) was found between the changes from basal values in GFR and those in sodium excretion rate with high-AA infusion at different levels of sodium concentration. These data suggest that AA-induced hyperfiltration might be blunted by hypotonic saline infusion, possibly through an acute modification of renal sodium handling and extracellular volume.
Basolateral Na+ pump modulates apical Na+ and K+ conductances in rabbit cortical collecting ducts
Authors: Muto Shigeaki , Asano Yasushi , Seldin Donald , Giebisch Gerhard
Published: December 2017

Previous studies indicated that an acute elevation of peritubular K+ enhances K+ secretion and Na+ reabsorption in the isolated perfused cortical collecting duct (CCD) from rabbit kidneys [S. Muto, G. Giebisch, and S. Sansom. Am. J. Physiol. 255 ( Renal Fluid Electrolyte Physiol. 24): F108–F114, 1988]. To determine the underlying cellular mechanisms, we used microelectrode techniques to assess the membrane properties of collecting duct cells in isolated perfused CCDs of control and desoxycorticosterone acetate (DOCA)-treated rabbits following acute stimulation of the basolateral Na+-K+pump by rapidly increasing the bath solution from 2.5 to 8.5 mM K+. This induced in both groups of tubules, first, a short-lasting hyperpolarization and, second, a sustained phase of depolarization of transepithelial, basolateral, and apical membrane voltages. Whereas the transepithelial conductance ( G T) and fractional apical membrane resistance (f R A) remained unchanged during the initial phase of hyperpolarization, during the depolarization, G T increased and f R A decreased. Perfusion of the lumen with solutions containing either amiloride or Ba2+ attenuated the high K+-induced apical electrical changes, and basolateral strophanthidin abolished both apical and basolateral electrical responses during elevation of K+ in the bath. From these results we conclude the following: 1) acute elevation of basolateral K+activates the basolateral Na+-K+pump, which secondarily elevates the apical Na+ and K+ conductances; 2) DOCA pretreatment increases the basolateral K+ conductance and augments the response to the rise of K+ of both basolateral Na+-K+pump activity and apical cation conductances.
Type II Na-Picotransport is regulated transcriptionally by ambient bicarbonate/carbon dioxide tension in OK cells
Authors: Jehle Andreas W. , Hilfiker Helene , Pfister Markus F. , Biber Jürg , Lederer Eleanor , Krapf Reto , Murer Heini
Published: December 2017

The purpose of the present study was to determine whether isohydric changes in HCO3concentration and Pco2directly affect apical Na-dependent Pi(Na-Pi) cotransport in OK cells (opossum kidney cell line). Cells were kept at either 44 mM NaHCO3/10% CO2, pH 7.4 (high-HCO3/CO2condition), or 22 mM NaHCO3/5% CO2, pH 7.4 (low-HCO3/CO2condition) (for 14–24 h). Incubation in lower HCO3/CO2concentrations increased Na-Picotransport 1.5-fold. The increased Na-Picotransport was paralleled by a two- to threefold increased expression of the NaPi-4 transporter protein and a two- to threefold increase in NaPi-4 mRNA abundance. The increase in NaPi-4 mRNA could be completely prevented by incubation in the presence of a transcriptional inhibitor, suggesting that the increase in NaPi-4 mRNA results from an increased NaPi-4 mRNA transcription. In agreement, the NaPi-4 promoter activity was stimulated by 50% at lower HCO3/CO2concentrations. In conclusion, our data demonstrate that isohydric changes in HCO3concentration and Pco2exert a significant, direct cellular effect on Na-Picotransport and NaPi-4 protein expression in OK cells by affecting NaPi-4 mRNA transcription.
VACM-1 receptor is specifically expressed in rabbit vascular endothelium and renal collecting tubule
Authors: Burnatowska-Hledin Maria , Lazdins Inara B. , Listenberger Laura , Zhao Ping , Sharangpani Aditi , Folta Vicki , Card Betsy
Published: December 2017

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a novel arginine vasopressin (AVP) receptor that shares sequence homology with a cullin multigene family but not with the AVP receptors. To characterize the VACM-1 receptor, we examined its tissue-specific expression using Northern blot, RT-PCR, and immunostaining analyses. Northern blot hybridization identified a 6.4-kb cRNA species that was expressed in the rabbit kidney medulla, brain, heart, and ovaries. In human tissue, VACM-1 mRNA is a larger (7.5 kb) cRNA found in the kidney, brain, heart, placenta, and skeletal muscle. VACM-1-specific RT-PCR products were detected in mRNA from rabbit kidney medulla, brain, heart, and mesenteric arteries. No expression of VACM-1 could be detected in rabbit aorta, gastrointestinal tract, or liver. Coimmunostaining with anti-VACM-1 antibodies (Ab) and a specific vascular endothelial cell marker, CD31 monoclonal Ab, localized VACM-1 expression to the vasculature in specific tissues. We identified the kidney cells expressing VACM-1 receptor by coimmunostaining with the following monoclonal Ab, which recognize epitopes in specific segments of the nephron: rct-30 Ab, reactive against the cortical and medullary collecting tubule (CT) cells; mr-omct Ab, reactive against the mitochondria-rich cells of the outer medullary CT; and an Ab specific against the loop of Henle segment. These studies indicated that the VACM-1 receptor is expressed only in the medullary CT. Kidney coimmunostaining with anti-VACM-1 and CD31 Ab identified VACM-1-receptor expression in glomeruli and medullary vascular bundles. These results demonstrate that the novel VACM-1 receptor, expressed in many organs, is localized to the endothelial cells. In the kidney, it is also expressed in the medullary CT cells. Thus VACM-1 may be involved in the regulation of endothelial permeability and water transport in the CT.
H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line
Authors: Campbell W. Grady , Weiner I. David , Wingo Charles S. , Cain Brian D.
Published: December 2017

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HKα). Intracellular measurements of RCCT-28A cells using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) indicated that the mechanism accounting for recovery from an acid load exhibited both K+dependence and sensitivity to Sch-28080 characteristic of H-K-ATPases. Recovery rates were 0.022 ± 0.005 pH units/min in the presence of K+, 0.004 ± 0.002 in the absence of K+, and 0.002 ± 0.002 in the presence of Sch-28080. The mRNAs encoding the HKα1subunit and the H-K-ATPase β-subunit (HKβ) were detected by RT-PCR. In addition, two HKα2species were found by RT-PCR and 5′ rapid amplification of cDNA ends (5′-RACE) in the rabbit renal cortex. One was homologous to HKα2cDNAs generated from other species, and the second was novel. The latter, referred to as HKα2c, encoded an apparent 61-residue amino-terminal extension that bore no homology to reported sequences. Antipeptide antibodies were designed on the basis of this extension, and these antibodies recognized a protein of the appropriate mass in both rabbit renal tissue samples and RCCT-28A cells. Such findings constitute very strong evidence for expression of the HKα2csubunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.
Contribution of cytochromeP-450 4A1 and 4A2 to vascular 20-hydroxyeicosatetraenoic acid synthesis in rat kidneys
Authors: Wang Mong-Heng , Guan Hui , Nguyen Xuandai , Zand Barbara A. , Nasjletti Alberto , Laniado-Schwartzman Michal
Published: December 2017

20-Hydroxyeicosatetraenoic acids (20-HETE), a biologically active cytochrome P-450 (CYP) metabolite of arachidonic acid in the rat kidney, can be catalyzed by CYP4A isoforms including CYP4A1, CYP4A2, and CYP4A3. To determine the contribution of CYP4A isoforms to renal 20-HETE synthesis, specific antisense oligonucleotides (ODNs) were developed, and their specificity was examined in vitro in Sf9 cells expressing CYP4A isoforms and in vivo in Sprague-Dawley rats. Administration of CYP4A2 antisense ODNs (167 nmol ⋅ kg body wt−1⋅ day−1iv for 5 days) decreased vascular 20-HETE synthesis by 48% with no effect on tubular synthesis, whereas administration of CYP4A1 antisense ODNs inhibited vascular and tubular 20-HETE synthesis by 52 and 40%, respectively. RT-PCR of microdissected renal microvessel RNA indicated the presence of CYP4A1, CYP4A2, and CYP4A3 mRNAs, and a CYP4A1-immunoreactive protein was detected by Western analysis of microvessel homogenates. Blood pressure measurements revealed a reduction of 17 ± 6 and 16 ± 4 mmHg in groups receiving CYP4A1 and CYP4A2 antisense ODNs, respectively. These studies implicate CYP4A1 as a major 20-HETE synthesizing activity in the rat kidney and further document the feasibility of using antisense ODNs to specifically inhibit 20-HETE synthesis and thereby investigate its role in the regulation of renal function and blood pressure.
Enhanced renal vascular responsiveness to angiotensin II in hypertensiveren-2 transgenic rats
Authors: Jacinto Severina M. , Mullins John J. , Mitchell Kenneth D.
Published: December 2017

The present study was performed to evaluate renal vascular responsiveness (RVR) to ANG II in hypertensive transgenic rats [TGR; strain TGR(mRen2)27] harboring the mouse ren-2 renin gene. Renal blood flow (RBF) responses to either intravenous or intrarenal arterial administration of ANG II were assessed in pentobarbital sodium-anesthetized female heterozygous TGR (9–12 wk old) and age-matched transgene-negative Hanover Sprague-Dawley rats (HanSD). Intravenous bolus injections of 15 and 30 ng ANG II elicited dose-dependent increases in mean arterial blood pressure (AP) and decreases in RBF in both TGR and HanSD. However, the magnitude of the increases in AP was greater in TGR than in HanSD (24 ± 1 vs. 17 ± 2 mmHg and 33 ± 2 vs. 25 ± 1 mmHg, respectively, P < 0.05 in both cases). Similarly, the magnitude of the decrease in RBF elicited by intravenous administration of 15 ng of ANG II was greater in TGR than HanSD (−62 ± 3 vs. −52 ± 5%, P < 0.05). Intrarenal arterial administration of 1.5 and 3 ng ANG II did not alter mean AP in either group but elicited larger decreases in RBF in TGR than in HanSD (−24 ± 2 vs. −13 ± 1% and −41 ± 5 vs. −30 ± 2%, respectively, P< 0.05 in both cases). In contrast, intrarenal arterial administration of norepinephrine (40 and 80 ng) elicited smaller decreases in RBF in TGR than in HanSD (−24 ± 3 vs. −40 ± 6% and −51 ± 9 vs. −71 ± 8%, respectively, P < 0.05 in both cases), indicating that TGR do not exhibit a generalized increase in RVR to endogenous vasoconstrictors. Furthermore, the enhanced RVR to ANG II does not appear to reflect an impaired RVR to endogenous vasodilator factors since intrarenal administration of bradykinin and acetylcholine elicited larger increases in RBF in TGR than in HanSD. The present findings indicate that hypertensive TGR exhibit exaggerated renal and peripheral vascular responses to ANG II, which likely contributes to an increased renal and peripheral vascular resistance and thereby to the hypertension in TGR.
A nucleoside-sensitive organic cation transporter in opossum kidney cells
Authors: Chen Rong , Pan Bih Fang , Sakurai Mamoru , Nelson J. Arly
Published: December 2017

Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323–2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2′-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevisoocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.
Tumor necrosis factor-α and ceramide induce cell death through different mechanisms in rat mesangial cells
Authors: Guo Yan-Lin , Kang Baobin , Yang Li-Jun , Williamson John R.
Published: December 2017

It has been proposed that ceramide acts as a cellular messenger to mediate tumor necrosis factor-α (TNF-α)-induced apoptosis. Based on this hypothesis, it was postulated that resistance of some cells to TNF-α cytotoxicity was due to an insufficient production of ceramide on stimulation by TNF-α. The present study was initiated to investigate whether this was the case in mesangial cells, which normally are insensitive to TNF-α-induced apoptosis. Our results indicate that although C2ceramide was toxic to mesangial cells, the cell death it induced differed both morphologically and biochemically from that induced by TNF-α in the presence of cycloheximide (CHX). The most apparent effect of C2ceramide was to cause cells to swell, followed by disruption of the cell membrane. It is evident that C2ceramide caused cell death by necrosis, whereas TNF-α in the presence of CHX killed the cells by apoptosis. C2ceramide did not mimic the effects of TNF-α on the activation of c-Jun NH2-terminal protein kinase and nuclear factor-κB transcription factor. Although mitogen-activated protein kinase [extracellular signal-related kinase (ERK)] was activated by both C2ceramide and TNF-α, such activation appeared to be mediated by different mechanisms as judged from the kinetics of ERK activation. Furthermore, the cleavage of cytosolic phospholipase A2during cell death induced by C2ceramide and by TNF-α in the presence of CHX showed distinctive patterns. The present study provides evidence that apoptosis and necrosis use distinctive signaling machinery to cause cell death.
Resetting of exaggerated tubuloglomerular feedback activity in acutely volume-expanded young SHR
Authors: Brännström Kristina , Arendshorst William J.
Published: December 2017

One purpose of the present study was to evaluate the ability of 7-wk-old spontaneously hypertensive rats (SHR) to reset tubuloglomerular feedback (TGF) activity in response to acute volume expansion (VE). Second, we evaluated the contribution of ANG II, via its action on AT1 receptors, to TGF control of glomerular function during VE. TGF was assessed by micropuncture methods and proximal tubular stop-flow pressure (SFP) determinations in SHR, Wistar-Kyoto rats (WKY), and Sprague-Dawley rats (SD). During euvolemia SHR exhibited enhanced TGF activity. In the same animals acute VE was achieved by infusion of saline (5 ml ⋅ h−1 ⋅ 100 g body wt−1). VE led to resetting of TGF in all three strains. Maximal SFP responses, elicited by a 30–40 nl/min loop of Henle perfusion rate, decreased from 19 to 12 mmHg in SHR and, on average, from 11 to 5 mmHg in WKY and SD ( P < 0.001). Tubular flow rate producing a half-maximal response (turning point) shifted to higher flow rates during VE, from 12 to 14 nl/min in SHR and from 15 to 19 nl/min in WKY. Administration of the AT1 receptor blocker candesartan (0.05 mg/kg iv) during sustained VE decreased TGF-mediated reductions in SFP in SHR and slightly increased the turning point in WKY. Nevertheless, other parameters of TGF activity were unaffected by AT1 receptor blockade. In conclusion, young SHR possess the ability to reset TGF activity in response to VE to a degree similar to compensatory adjustments in WKY. However, TGF remains enhanced in SHR during VE. ANG II and its action on AT1 receptors are in part responsible for the exaggerated SFP responses in young SHR during VE.
ATP-mediated Ca2+signaling in preglomerular smooth muscle cells
Authors: Inscho Edward W. , Schroeder Alan C. , Deichmann Paul C. , Imig John D.
Published: December 2017

We performed studies to determine the effect of extracellular ATP on the intracellular Ca2+concentration ([Ca2+]i) in freshly isolated microvascular smooth muscle cells (MVSMC). Suspensions of preglomerular MVSMC were prepared by enzymatic digestion and loaded with fura 2. Single cells were studied using a microscope-based fluorescence spectrophotometer during superfusion of a physiological salt solution with 1.8 mM Ca2+and during exposure to similar solutions containing ATP. Under control conditions, baseline [Ca2+]iaveraged 107 ± 6 nM ( n = 86 cells from 34 animals). ATP administration elicited concentration-dependent increases in [Ca2+]i. Exposure to ATP concentrations of 1, 10, and 100 μM increased intracellular Ca2+to peak concentrations of 133 ± 20, 338 ± 37, and 367 ± 35 nM, respectively ( P < 0.05 vs. respective baseline). Steady-state [Ca2+]iincreased to 113 ± 15, 150 ± 16 ( P < 0.05 vs. baseline), and 180 ± 12 nM ( P < 0.05 vs. baseline) for the same groups. The [Ca2+]iresponse to ATP was also assessed in the absence of extracellular Ca2+and during blockade of L-type Ca2+channels with diltiazem. In these studies, exposure to 100 μM ATP induced a transient peak increase in [Ca2+]iwith the plateau phase being totally abolished under Ca2+-free conditions and markedly attenuated during Ca2+channel blockade, respectively. These data indicate that ATP-mediated P2-receptor activation increases [Ca2+]iin freshly isolated preglomerular MVSMC by stimulating Ca2+release from intracellular stores, in addition to stimulating the influx of extracellular Ca2+through voltage-gated L-type Ca2+channels.
Agonist-induced translocation of Gq/11α immunoreactivity directly from plasma membrane in MDCK cells
Authors: Arthur John M. , Collinsworth Georgiann P. , Gettys Thomas W. , Raymond John R.
Published: December 2017

Both Gsα and Gqα are palmitoylated and both can move from a crude membrane fraction to a soluble fraction in response to stimulation with agonists. This response may be mediated through depalmitoylation. Previous studies have not demonstrated that endogenous guanine nucleotide-binding regulatory protein (G protein) α-subunits are released directly from the plasma membrane. We have examined the effect of agonist stimulation on the location of Gq/11α immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 μM) caused Gq/11α, but not Giα, to rapidly translocate from purified plasma membranes to the supernatant. AlF and GTP also caused translocation of Gq/11α immunoreactivity from purified plasma membranes. BK caused translocation of Gq/11α immunoreactivity in intact cells from the basal and lateral plasma membranes to an intracellular compartment as assessed by confocal microscopy. Thus Gq/11α is released directly from the plasma membrane to an intracellular location in response to activation by an agonist and direct activation of G proteins. G protein translocation may be a mechanism for desensitization or for signaling specificity.
Ischemia activates actin depolymerizing factor: role in proximal tubule microvillar actin alterations
Authors: Schwartz Niles , Hosford Melanie , Sandoval Ruben M. , Wagner Mark C. , Atkinson Simon J. , Bamburg James , Molitoris Bruce A.
Published: December 2017

Apical membrane of renal proximal tubule cells is extremely sensitive to ischemia, with structural alterations occurring within 5 min. These changes are felt secondary to actin cytoskeletal disruption, yet the mechanism responsible is unknown. Actin depolymerizing factor (ADF), a 19-kDa actin-binding protein, has recently been shown to play an important role in regulation of actin filament dynamics. Because ADF is known to mediate pH-dependent F-actin binding, depolymerization, and severing, and because ADF activation occurs by dephosphorylation, we questioned whether ADF played a role in microvilli microfilament disruption during ischemia. To test our hypothesis, we induced renal ischemia in the rat with the clamp model. Initial immunofluorescence and Western blot studies on cortical tissue documented the presence of ADF in proximal tubule cells. Under physiological conditions, ADF was distributed homogeneously throughout the cytoplasm, primarily in the Triton X-100-soluble fraction, and both phosphorylated (pADF) and nonphosphorylated forms were identified. During ischemia, marked alterations occurred. Intraluminal vesicle/bleb structures contained extremely high concentrations of ADF along with G-actin, but not F-actin. Western blot showed a rapidly occurring duration-dependent dephosphorylation of ADF. At 0–30 min of ischemia, total ADF levels were unchanged, whereas pADF decreased significantly to 72% and 19% of control levels, at 5 and 15 min, respectively. Urine collected under physiological conditions did not contain ADF or actin, whereas urine collected after 30 min of ischemia contained both ADF and actin. Reperfusion was associated with normalization of cellular pADF levels, pADF intracellular distribution, and repair of apical microvilli. These data suggest that activation of ADF during ischemia via dephosphorylation is, in part, responsible for apical actin disruption resulting in microvillar destruction and formation of intraluminal vesicles.
Nitric oxide inhibits transcription of the Na+-K+-ATPase α1-subunit gene in an MTAL cell line
Authors: Kone Bruce C. , Higham Sandra
Published: December 2017

Nitric oxide (NO) has been implicated as an autocrine modulator of active sodium transport. To determine whether tonic exposure to NO influences active sodium transport in epithelial cells, we established transfected medullary thick ascending limb of Henle (MTAL) cell lines that overexpressed NO synthase-2 (NOS2) and analyzed the effects of deficient or continuous NO production [with or without NG-nitro-l-arginine methyl ester (l-NAME) in the culture medium, respectively] on Na+-K+-ATPase function and expression. The NOS2-transfected cells exhibited high-level NOS2 expression and NO generation, which did not affect cell viability or cloning efficiency. NOS2-transfected cells were grown in the presence of vehicle, NG-nitro-d-arginine methyl ester (d-NAME), orl-NAME for 16 h, after which86Rb+uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The NOS2-transfected cells allowed to produce NO continuously (vehicle ord-NAME) exhibited lower rates of ouabain-sensitive86Rb+uptake (∼65%), lower levels of Na+-K+-ATPase α1-subunit mRNA (∼60%), and reduced rates of de novo Na+-K+-ATPase α1-subunit transcription compared withl-NAME-treated cells. These results have uncovered a novel effect of NO to inhibit transcription of the Na+-K+-ATPase α1-subunit gene.
The Cre/loxP system and gene targeting in the kidney
Authors: Stricklett Peter K. , Nelson Raoul D. , Kohan Donald E.
Published: December 2017

The Cre/loxP and Flp/FRT systems mediate site-specific DNA recombination and are being increasingly utilized to study gene function in vivo. These systems allow targeted gene disruption in a single cell type in vivo, thereby permitting study of the physiological and pathophysiological impact of a given gene product derived from a particular cell type. In the kidney, the Cre/loxP system has been employed to achieve gene deletion selectively within principal cells of the collecting duct. Disruption of target genes in the collecting duct, such as endothelin-1 or polycystic kidney disease-1 (PKD1), could lead to important insights into the biological roles of these gene products. With selection of the appropriate renal cell-specific promoters, these recombination systems could be used to target gene disruption to virtually any renal cell type. Although transgenic studies utilizing these recombination systems are promising, they are in their relative infancy and can be time consuming and expensive and yield unanticipated results. It is anticipated that continued experience with these systems will produce an important tool for analyzing gene function in renal health and disease.
Thromboxane A2contributes to the enhanced tubuloglomerular feedback activity in young SHR
Authors: Brännström Kristina , Arendshorst William J.
Published: December 2017

We performed micropuncture studies to determine the role of thromboxane A2in the exaggerated tubuloglomerular feedback (TGF) activity in young spontaneously hypertensive rats (SHR). Glomerular function was assessed by changes in proximal tubular stop-flow pressure (SFP) produced by different rates of orthograde perfusion through Henle’s loop. Seven-week-old SHR exhibited an exaggerated TGF activity compared with Wistar-Kyoto rats (WKY) during euvolemia, confirming earlier studies. During control periods, the feedback-induced maximal SFP response (ΔSFP) was greater in SHR (18–19 vs. 12–13 mmHg in WKY), whereas basal SFP and proximal tubular free-flow pressure were similar in both strains. In one series, the thromboxane A2agonist U-46619 was added to the tubular perfusate for a final concentration of 10−6M. In WKY, ΔSFP was increased by 100% to 26 mmHg. In contrast, ΔSFP in young SHR was unaffected by the thromboxane A2agonist. In other animals, the thromboxane synthase inhibitor pirmagrel (50 mg/kg) was injected intravenously to inhibit thromboxane production. In SHR, pirmagrel decreased ΔSFP by 8.5 mmHg and reduced reactivity. Less attenuation was observed in WKY; ΔSFP was reduced by 3 mmHg, whereas reactivity was unchanged. In other studies, tubular perfusion with the thromboxane receptor inhibitor SQ-29548 (10−6M) reduced ΔSFP more in SHR (7 vs. 3 mmHg in WKY) and also decreased reactivity more in SHR (2.3 vs. 0.5 mmHg ⋅ nl−1⋅ min−1). Coperfusion of SQ-29548 and U-46619 resulted in an 85% block of the effect of U-46619 on ΔSFP. Tubular perfusion with the agonist U-46619 during thromboxane synthase inhibition markedly enhanced ΔSFP in both strains, with a greater effect in WKY. These results suggest that elevated levels of thromboxane A2in young SHR contribute to the exaggerated TGF control of glomerular function in SHR during the developmental phase of hypertension.
Endothelin mediates renal vasodilation and hyperfiltration during pregnancy in chronically instrumented conscious rats
Authors: Conrad Kirk P. , Gandley Robin E. , Ogawa Tatsuhiro , Nakanishi Satoshi , Danielson Lee A.
Published: December 2017

Profound vasodilation of the kidneys and other nonreproductive organs transpires during early pregnancy. Because nitric oxide (NO) was found to mediate renal vasodilation and hyperfiltration in conscious pregnant rats, and endogenous endothelin (ET) was suggested to be vasodilatory in the renal circulation of nonpregnant rats, we tested whether endothelin mediates the NO-dependent changes in the renal circulation during pregnancy. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious pregnant and virgin rats before and during infusion of 30 μg/min RES-701-1 (a selective ETBreceptor subtype antagonist). Baseline GFR and ERPF were significantly increased by 35% in gravid rats relative to virgin controls. During infusion of RES-701-1, the pregnant rats responded more robustly, showing a greater decline in both GFR and ERPF such that renal function converged in the two groups of rats. ERPF also converged in pregnant and virgin rats during infusion of SB-209760, a nonselective ETA/Breceptor subtype antagonist. Combined infusion of Nω-nitro-l-arginine methyl ester [l-NAME, an NO synthase (NOS) inhibitor] and RES-701-1 reduced GFR and ERPF to levels comparable to those reached with either agent given alone, suggesting inhibition of a common vasodilatory pathway. RES-701-1 and SB-209670 significantly lowered the cGMP content of small renal arteries from gravid and virgin rats in vitro, strengthening the link between the renal endothelial ETBreceptor subtype and NO. Importantly, we showed that RES-701-1 is not a direct inhibitor of NOS. We conclude that endothelin mediates the NO-dependent changes in the renal circulation of conscious rats during pregnancy.