bluuebunny/crossref_metadata_2025 huggingface dataset:
20-Hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule
Authors: Lin F. , Rios A. , Falck J. R. , Belosludtsev Y. , Schwartzman M. L.
Published: December 2017

Hydroxyeicosatetraenoic acid (20-HETE) is a major cytochrome P-450-arachidonic acid metabolite in the rat kidney, and its synthesis along the nephron is specifically localized to the proximal tubule, where receptor density for epidermal growth factor (EGF) is the highest. EGF stimulated endogenous 20-HETE formation in a concentration and time-dependent manner, i.e., from 1.6 to 2.6 +/- 0.3 and 3.0 +/- 0.6 pmol 20-HETE.mg-1.min-1 at 10(-8) and 10(-7) M EGF, respectively. The effect of 20-HETE on proximal tubular cell proliferation was examined using primary cultures of rat proximal tubular cells and proximal tubular-derived cell lines, LLC-PK1 and opossum kidney OK. In both cell lines, 20-HETE increased thymidine incorporation into DNA with maximal effect at 10(-9) M. Addition of 20-HETE to serum-deprived LLC-PK1 or OK cells for 48 h caused a concentration-dependent increase in cell number with maximal effect at 10(-9) M. This effect was specific, as structurally similar eicosanoids such as 20-COOH-arachidonic acid, 19(R)-HETE, and 19(S)-HETE did not increase cell number. In 4-day primary cultures of proximal tubular cells, EGF (10(-9) M) and 20-HETE (10(-9) M) increased bromodeoxyuridine (BrdU) incorporation by 40 and 28%, respectively. Addition of both resulted in a twofold increase in BrdU incorporation. Although 20-HETE synthesis in cultured cells is greatly diminished with time, significant picomolar concentrations can be obtained in 4-day cultures. Addition of 17-octadecynoic acid (17-ODYA), an inhibitor of 20-HETE synthesis, significantly inhibited EGF-stimulated BrdU incorporation. The demonstrations that EGF stimulates proximal tubular 20-HETE production and that the latter is a potent mitogen to these cells suggests that 20-HETE may act as a mediator of the EGF effect on cellular growth in the proximal tubule.
Differential mRNA expression of insulin-like growth factor system during renal injury and hypertrophy
Authors: Hise M. K. , Li L. , Mantzouris N. , Rohan R. M.
Published: December 2017

The cellular effects of insulin-like growth factor I (IGF-I) are modified by a family of binding proteins (IGFBPs) that act as reservoirs in serum for the growth factor and are produced locally by tissues, including the kidney. Because regulation of these proteins may influence renal repair, either directly or by their interactions with IGF-I, we studied gene expression during the recovery from renal failure induced by folic acid and during the compensatory increase in renal function following uninephrectomy (UNX). Expression of IGF-I, the IGF-I receptor (IGF-IR), and all six IGFBPs was detected using an ribonuclease protection assay. IGFBP-5 was the most abundant binding protein mRNA present in kidney, whereas IGFBP-2 and -6 were the least abundant. During regeneration following folic acid-induced acute renal failure, IGF-I, IGFBP-3, and IGFBP-5 mRNAs declined in abundance approximately two- to threefold. On the other hand, IGF-IR, IGFBP-1, and IGFBP-2 were increased (approximately 2-, 6-, and 6-fold, respectively) in the first 24 h. IGFBP-1 mRNA remained elevated for at least 3 days. Despite the known increase in cellular RNA content following UNX, little difference in specific expression of mRNAs was observed. Because IGFBP-1 has been shown to stimulate cell migration and has previously been localized to the distal nephron, the site of greatest injury in the folic acid model, these data are compatible with the notion that this protein may function either directly to affect cellular repair or act as a reservoir for IGF-I under conditions of cellular damage.
Interleukin-1 beta induces interstitial collagenase gene expression and protein secretion in renal mesangial cells
Authors: Daphna-Iken D. , Morrison A. R.
Published: December 2017

Degradation and tissue remodeling of the extracellular matrix in the normal glomerulus occur through the coordinate action of neutral metalloproteinases, which are in turn regulated by specific inhibitors. Many of these proteins can be secreted by mesangial cells. In the current study, gene regulation of a rat matrix metalloproteinase, interstitial collagenase and its tissue inhibitor of metalloproteinase-1 (TIMP-1), was investigated by Northern blot analysis. Stimulation of rat mesangial cell (RMC) collagenase by interleukin 1 beta (IL-1 beta) produced an increase (> 45-fold) in mRNA which peaked at 12 h. Lesser effects on the TIMP-1 mRNA expression were observed in response to IL-1 beta. Indomethacin did not influence the effect of IL-1 beta on collagenase, and exogenous prostaglandin E2 had no significant effect either on basal or IL-1 beta-stimulated mRNA levels. Collagenase was secreted into the media and showed minimal gelatinolytic activity at 36-h stimulation with IL-1 beta by zymography. By Western immunoblotting, we demonstrated with 24 h of stimulation the secretion of the active form of collagenase, which further increased after 36 h with IL-1 beta compared with the control. When RMC were retreated with genistein and herbimycin A, both inhibited collagenase mRNA induction by IL-1 beta. These data suggest that IL-1 beta stimulates interstitial collagenase synthesis and activation and that a tyrosine kinase pathway is involved in the signal transduction mechanisms and is not dependent on endogenous prostaglandin biosynthesis. Recently, a third interstitial collagenase (collagenase-3) has been identified from breast carcinoma. This cDNA is 84% identical to the rat interstitial collagenase cDNA probe we have utilized in this study and thus may represent the rat homologue of the human collagenase-3 now called matrix metalloproteinase (MMP)-13.
In vivo and in vitro pressor effects of erythropoietin in rats
Authors: Vaziri N. D. , Zhou X. J. , Smith J. , Oveisi F. , Baldwin K. , Purdy R. E.
Published: December 2017

Hypertension (HTN) is a common complication of recombinant erythropoietin (EPO) therapy, but the mechanism of the EPO-associated HTN is uncertain. In the present study we examined the effects of EPO and the vehicle alone on rat caudal artery contractile response and basal and thrombin-stimulated platelet cytosolic Ca2+ concentration ([Ca2+]i) in vitro and on blood pressure (BP) and heart rate in vivo. At high concentrations (200 U/ml) EPO caused a small but consistent contraction in the caudal artery rings (P < 0.01) without affecting the response to either angiotensin II (ANG II) or the alpha 1-agonist methoxamine. Incubation with EPO significantly increased basal platelet [Ca2+]i (P < 0.01) and augmented the thrombin-induced rise of [Ca2+]i in Ca(2+)-free medium (P < 0.05). Long-term EPO administration led to a significant elevation of BP within 2 wk regardless of whether the hematocrit was allowed to rise or was kept constant by dietary iron deficiency. In contrast, single intravenous administration of high-dose EPO (400 and 5,000 U/kg), estimated to yield plasma concentrations comparable with those employed in vitro, failed to either alter BP or modify the BP response to ANG II during a 60-min observation period. This was associated with a significant rise in plasma guanosine 3',5'-cyclic monophosphate but no discernible change in plasma atrial natriuretic peptide, suggesting enhanced nitric oxide (NO) release. Thus, at high concentrations, EPO appears to possess a fast-acting pressor effect in vitro but not in vivo. The observed discrepancy may be due to enhanced NO release with EPO administration in vivo. However, HTN does occur with repeated EPO administration in a time-dependent and hematocrit-independent manner. This suggest that expression of the hypertensive effect of EPO in vivo involves a gradual conditioning process.
Hypoxia induces changes in phospholipase A2 in rat proximal tubules: evidence for multiple forms
Authors: Choi K. H. , Edelstein C. L. , Gengaro P. , Schrier R. W. , Nemenoff R. A.
Published: December 2017

Increased free fatty release during hypoxia is believed to contribute to cell injury. This phenomenon is likely to be mediated through activation of specific isoforms of phospholipase A2 (PLA2). In this study, PLA2 enzymatic activity was measured in cell-free extracts prepared from rat renal proximal tubules. Both soluble and membrane-associated PLA2 activity were detected. All PLA2 activity detected during normoxia was Ca2+ dependent. Fractionation of cytosolic extracts by gel filtration revealed three peaks of PLA2 activity. Exposure of tubules to hypoxia resulted in stable activation of soluble PLA2 activity, which correlated with disappearance of the highest molecular mass form (> 100 kDa) and appearance of a low-molecular-mass form (approximately 15 kDa) of PLA2. Hypoxia also resulted in release of a low-molecular-mass form of PLA2 into the extracellular medium. Pretreatment of tubules with glycine before hypoxia blocked this release of PLA2 but not activation of soluble PLA2 activity. These studies provide direct evidence for PLA2 activation during hypoxia and suggest that multiple mechanisms regulate free fatty acid release associated with hypoxia injury.
Flow dependence of chloride transport in rat S1 proximal tubules
Authors: Wong K. R. , Berry C. A. , Cogan M. G.
Published: December 2017

These studies examined whether the luminal flow dependency of chloride absorption in the S1 proximal tubule during glomerulotubular balance was due to change in active and/or passive transport of chloride. Using in vivo microperfusion in the Munich-Wistar rat and an essentially pure sodium chloride perfusate (devoid of bicarbonate and organic solutes), we found that an increase in luminal perfusion rate from 30 to 45 nl/min caused stimulation of total chloride absorption (active plus passive) by 87 peq.mm-1.min-1 (632 +/- 17 to 719 +/- 11, P < 0.001). When cyanide was added to this perfusate to eliminate active transport, the flow-induced change in passive transport was 58 peq.mm-1.min-1 (479 +/- 9 to 537 +/- 11, P < 0.001). The cyanide-inhibitable active transport component was therefore 29 peq.mm-1.min-1. With elimination of the transepithelial chloride gradient and, hence, passive transport by isethionate substitution, active transport increased by 63 peq.mm-1.min-1 (121 +/- 4 to 184 +/- 7, P < 0.001) as flow rate rose from 30 to 45nl/min. Removal of organic solutes from a glomerular ultrafiltrate-like perfusate had a minimal effect on flow-induced change in chloride transport (190 vs. 207 peq.mm-1.min-1). In conclusion, flow-dependent active and passive chloride transport in the S1 proximal tubule may both contribute to normal glomerulotubular balance.
Adenosine inhibits arginine vasopressin-stimulated chloride secretion in a mouse IMCD cell line (mIMCD-K2)
Authors: Moyer B. D. , McCoy D. E. , Lee B. , Kizer N. , Stanton B. A.
Published: December 2017

Previously, we demonstrated that a mouse inner medullary collecting duct cell line (mIMCD-K2) secretes Cl- by an electrogenic mechanism via cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995; D. Vandorpe, N. Kizer, F. Ciampolillo-Bates, B. Moyer, K. Karlson, W. B. Guggino, and B. A. Stanton. Am. J. Physiol. 269 (Cell Physiol. 38): C683-C689, 1995]. The objective of the present study was to determine whether adenosine, and adenosine A1 receptors (A1AR) specifically, regulate electrogenic Cl- secretion (IscCl) in mIMCD-K2 cells. Neither N6-cyclohexyladenosine (CHA), a specific A1AR agonist, nor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1AR antagonist, altered basal, unstimulated IscCl in monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. In contrast, DPCPX increased arginine vasopressin (AVP)-stimulated IscCl, an effect that was reversed by CHA. Adenosine deaminase (ADA), which oxidatively deaminates adenosine to inosine, increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of ADA on AVP-stimulated IscCl. These results suggest that adenosine, via A1AR, inhibits AVP-stimulated IscCl. To identify the source(s) of extracellular adenosine, we examined the effects of dipyridamole, an inhibitor of nucleoside transport, and alpha,beta-methyleneadenosine 5'-diphosphate (AOPCP), an inhibitor of ecto-5'-nucleotidase, on AVP-stimulated IscCl. Both compounds increased AVP-stimulated IscCl. CHA reversed the stimulatory effect of dipyridamole and AOPCP on IscCl. Neither ADA nor CHA had an effect on 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated IscCl. Moreover, U-73122, an inhibitor of phospholipase C, failed to attenuate the increase in AVP-stimulated IscCl elicited by dipyridamole and AOPCP or the decrease in AVP-stimulated IscCl elicited by CHA. We conclude that adenosine, released by a nucleoside transporter and formed extracellularly by the breakdown of AMP, binds to A1AR, and decreases AVP-stimulated IscCl in mIMCD-K2 cells by reducing intracellular cAMP levels.
Expression of the thiazide-sensitive Na-Cl cotransporter in rat and human kidney
Authors: Obermuller N. , Bernstein P. , Velazquez H. , Reilly R. , Moser D. , Ellison D. H. , Bachmann S.
Published: December 2017

An electroneutral thiazide-sensitive Na-Cl cotransport pathway (TSC) has been localized functionally to the distal convoluted tubule (DCT), although the TSC has also been detected in the connecting tubule (CNT), the cortical collecting duct, and the medullary collecting tubule as well. The present experiments were designed to localize expression of message for the TSC in rat and human kidney. A riboprobe, generated from the mouse TSC, was used for in situ hybridization. Simultaneous immunocytochemistry, using antibodies to Tamm-Horsfall protein, band 3, and the Na+/Ca2+ exchanger, permitted delineation of specific nephron segments. In rat, message for the TSC was highly expressed in DCT cells but not elsewhere. The transition from thick ascending limb to DCT was abrupt, whereas the transition to CNT was gradual. In the more distal region of rat DCT (DCT-2), which contained few intercalated cells, both TSC message and Na+/Ca2+ exchanger immunoreactivity were present. Treatment of rats with furosemide for 5 days increased expression of TSC message within the DCT but did not induce its expression elsewhere. In humans, expression of TSC message was also highest in cells of the DCT. In humans, however, expression extended well into the CNT. These experiments indicate that the TSC is expressed predominantly by DCT cells in both rat and humans, although expression extends into the CNT cells in humans. They also show that the TSC and Na+/Ca2+ exchanger are coexpressed by a subpopulation of DCT cells near the junction with the CNT.
Glucocorticoids regulate NHE-3 transcription in OKP cells
Authors: Baum M. , Amemiya M. , Dwarakanath V. , Alpern R. J. , Moe O. W.
Published: December 2017

OKP cells express NHE-3, an amiloride-resistant Na+/H+ antiporter, which is likely an isoform responsible for apical proton secretion by the proximal tubule. We have previously shown that an amiloride-resistant Na+/H+ antiporter in OKP cells is regulated by dexamethasone, a synthetic glucocorticoid. The purpose of the present study was to examine the mechanism for the glucocorticoid-mediated increase in Na+/H+ antiporter activity. Incubation of OKP cells with 10(-6) M dexamethasone resulted in a two- to threefold increase in NHE-3 mRNA abundance. This increase was seen after 4 h of incubation with dexamethasone, a time course similar to that found for Na+/H+ antiporter activity. To examine the mechanism for the increase in NHE-3 mRNA abundance, mRNA half-life and in vitro transcription experiments were performed. NHE-3 mRNA had a half-life of 8 h in control and dexamethasone-treated cells. The rate of in vitro transcription was 1.8-fold greater when OKP cells were treated with dexamethasone. These data suggest that the glucocorticoid-mediated increase in Na+/H+ antiporter activity is due to an increase in NHE-3 gene transcription.
Adrenal steroids stimulate thiazide-sensitive NaCl transport by rat renal distal tubules
Authors: Velazquez H. , Bartiss A. , Bernstein P. , Ellison D. H.
Published: December 2017

The current experiments were designed to test the hypothesis that adrenal steroids increase thiazide-sensitive Na and Cl transport by the mammalian renal distal convoluted tubule (DCT). Male Sprague-Dawley rats were adrenalectomized and received steroid hormones by osmotic pumps. Six groups of animals were studied as follows: group I, no hormones; group II, replacement levels of dexamethasone only; group III, replacement levels of aldosterone only; group IV, replacement levels of both hormones; group V; replacement levels of aldosterone and high levels of dexamethasone; and group VI, replacement levels of dexamethasone and high levels of aldosterone. Circulating levels of both hormones were found to be in the high physiological range when infused at the high rate. In vivo microperfusion of distal tubules was performed to determine rates of Na and Cl transport. Chlorothiazide was used to assess the magnitude of electroneutral Na-Cl cotransport. Both aldosterone and dexamethasone stimulated thiazide-sensitive Na and Cl transport by the distal tubule by more than fivefold. [3H]metolazone binding was measured to assess the number of thiazide-sensitive Na-Cl cotransporters in renal cortex. Each steroid also increased the number of [3H]metolazone binding sites in kidney cortex more than threefold. The results are consistent with the presence of both mineralocorticoid and glucocorticoid receptors in the mammalian DCT. Physiological changes in circulating levels of adrenal steroids may affect renal NaCl excretion in part by regulating the rate of electroneutral Na-Cl absorption by the DCT.
Expression of proximal tubular Na-Pi and Na-SO4 cotransporters in MDCK and LLC-PK1 cells by transfection
Authors: Quabius E. S. , Murer H. , Biber J.
Published: December 2017

Two cD-NAs coding for proximal tubular Na-Pi cotransport (NaPi-2) and Na-SO4 cotransport (NaSi-1) have been transfected by the use of a dexamethasone-inducible vector (pLK-neo) into MDCK and LLC-PK1 cells. By reverse transcription-polymerase chain reaction, expression of corresponding mRNAs was observed after stimulation with dexamethasone only. Similarly, expression of the NaPi-2 protein was detected only after induction with dexamethasone. In transfected Madin-Darby canine kidney (MDCK) cells, dexamethasone induced a large increase of Na-Pi or Na-SO4 cotransport, whereas, in transfected LLC-PK1, cell transport was only minimally expressed. In MDCK cells grown on filter supports, transfected Na-Pi-cotransport activity was equally expressed at both cell surfaces; dual location of expressed NaPi-2 protein was also observed by immunohistochemistry. In contrast, transfected Na-SO4 cotransport activity was predominantly expressed at the apical cell surface of MDCK cells. The results demonstrate that 1), in MDCK cells, the sorting behavior of two proximal tubular cotransport systems seems to be different: apical for Na-SO4 cotransport (NaSi-1) and dual location for Na-Pi cotransport (NaPi-2); and 2) LLC-PK1 cells seem not to be a suitable system to functionally express sodium-dependent cotransport systems for phosphate and sulfate.
Inhibition of Na+ and Ca2+ reabsorption by P2u purinoceptors requires PKC but not Ca2+ signaling
Authors: Koster H. P. , Hartog A. , van Os C. H. , Bindels R. J.
Published: December 2017

Rabbit connecting tubule and cortical collecting duct cells were isolated by immunodissection and cultured to confluence on permeable filters and on glass coverslips. Extracellular ATP dose-dependently reduced transcellular Na+ and Ca2+ transport (half-maximal inhibitory concentration, IC50, of 0.5 +/- 0.2 and 3.2 +/- 0.5 microM), with a maximal inhibition of 57 +/- 5 and 43 +/- 4%, respectively. Purinergic receptor agonists inhibited transport with the following rank order of potency: UTP = ATP > ADP; this suggests involvement of P2u purinoceptors. ATP also caused a dose-dependent (50% effective dose, EC50, of 1.5 +/- 0.2 microM) transient increase in intracellular Ca2+ concentration ([Ca2+]i), which decreased to a sustained elevated level. In the absence of extracellular Ca2+, a similar Ca2+ transient occurred, but the sustained response was abolished. Preloading the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) completely prevented the ATP-induced Ca2+ transients, but not the ATP-induced inhibition of Na+ and Ca2+ absorption. Activation of protein kinase C (PKC) by the cell-permeable diacylglycerol analogue, 1,2-dioctanoyl-en-glycerol, mimicked ATP-induced inhibition of Na+ and Ca2+ absorption. The inhibitory effects of ATP were no longer observed after culturing cells in the presence of phorbol ester (12-O-tetradecanoylphorbol-13-acetate) for 5 days, which resulted in downregulation of cellular PKC activity.
Coexistence of two types of Ca(2+)-activated K+ channels in rat renal arterioles
Authors: Gebremedhin D. , Kaldunski M. , Jacobs E. R. , Harder D. R. , Roman R. J.
Published: December 2017

Single-channel K+ currents were characterized in vascular smooth muscle cells freshly isolated from preglomerular arterioles (15–40 microns OD) of the rat. Under conditions of symmetrical K+ (145 mM), two types of single-channel K+ currents with unitary slope conductances of 68 +/- 2.6 and 251 +/- 4.9 pS were recorded from excised inside-out membrane patches. The open state probability (NPo) of these two types of K+ channels was voltage sensitive and the channels were highly selective for K+ over Na+. Elevation of intracellular calcium concentration ([Ca2+]i) from 0.1 to 0.5 microM on the cytoplasmic face of inside-out patches increased the frequency of opening and the NPo of both the 68-pS and the 251-pS K+ channels. Application of ATP (0.1–1 mM) to the internal surface of inside-out patches had no effect on the activities of both channel types. Internally applied Ba2+ (1 mM) blocked both of these channels. Externally applied tetraethylammonium (0.1–0.3 mM) or charybdotoxin (50 nM) blocked both the 68-pS and the 251-pS K+ channels. Externally applied apamin (50 nM), however, selectively blocked the 68-pS K+ channel but had no effect on the frequency of opening of the 251-pS K+ channel. Apamin also reduced macroscopic K+ current recorded from voltage-clamped rat renal arteriolar muscle cells by 25–30%. These results indicate the coexistence of two types of Ca(2+)-activated K+ channels in the membranes of vascular smooth muscle cell isolated from renal preglomerular arterioles of the rat that differ in unitary conductances and pharmacological properties.
Participation of adenosine in the renal hemodynamic abnormalities of hypothyroidism
Authors: Franco M. , Bobadilla N. A. , Suarez J. , Tapia E. , Sanchez L. , Herrera-Acosta J.
Published: December 2017

To investigate the participation of adenosine (ADO) in the abnormalities of renal function associated with hypothyroidism, glomerular hemodynamics were evaluated in normal (Nl) and 2-wk thyroidectomized (Htx) rats. Studies were performed before and during intravenous infusion of the ADO blocker 1,3-dipropyl-8-p-sulfophenyl xanthine (PSPX, 20 mM, 1.2 ml/h), intra-aortic ADO (100 nmol.kg-1.min-1), or vehicle. In addition, single-nephron glomerular filtration rate (SNGFR) was measured during the infusion of different intrarenal ADO doses (1, 10, and 35 nmol.kg-1.min-1); plasma and renal content of ADO were measured by high-performance liquid chromatography in additional groups. Decreased SNGFR, glomerular blood flow (QA), and ultrafiltration coefficient (Kf) were found in Htx rats. PSPX did not modify glomerular hemodynamics in Nl rats; in contrast, in Htx rats, the antagonist increased SNGFR, QA, and Kf, with a fall in afferent (RA) and efferent (RE) resistances. ADO infusion in Nl rats produced renal vasoconstriction characterized by a fall in SNGFR, QA, and Kf, with an increased RA and RE. Paradoxically, in Htx rats, ADO increased SNGFR, QA, and Kf, decreasing RA and RE. However, when ADO was infused through the renal artery, it induced a 20% reduction of SNGFR at 1 nmol.kg-1.min-1 that rose to control values at 10 nmol.kg-1.min-1 and increased to 38.3% to 35.kg-1.min-1. Renal ADO content was markedly low in Htx rats (4.37 +/- 0.79 and 115.46 +/- 14.9 nmol/g wet wt for Htx and Nl rats, respectively). Renal vasodilation induced by PSPX in Htx rats suggests predominant activation of A1 receptors in this condition. The vasodilatory response to exogenous ADO suggests additional activation of A2 receptors.
Distribution and regulation of guanylyl cyclase type B in the rat nephron
Authors: Dean A. D. , Vehaskari V. M. , Ritter D. , Greenwald J. E.
Published: December 2017

C-type natriuretic peptide (CNP) has been localized to the proximal and distal nephron. In this study, we examined the distribution and regulation of the CNP receptor, guanylyl cyclase type B (GC-B), in the rat kidney. GC-B mRNA was detected most frequently in microdissected glomeruli, thin and thick limbs of the loop of Henle, and outer and inner medullary collecting ducts by reverse transcription-polymerase chain reaction (RT-PCR). This pattern of expression is supported by immunofluorescent staining, using anti-GC-B-specific antiserum. Nearly equivalent levels of GC-B and guanylyl cyclase type A (GC-A) mRNAs were found by quantitative RT-PCR (5,662 +/- 1,622 and 5,187 +/- 1,204 molecules of cDNA/microgram total RNA, respectively; means +/- SE, n = 6). Renal inner medulla GC-B mRNA levels, but not renal CNP mRNA levels, were 3.2-fold greater in hypervolemic and 2.3-fold less in hypovolemic rats compared with euvolemic controls. Immunohistochemical staining also supports a greater GC-B expression with increased volume status. These data link hydration status and GC-B expression and suggest an additional and novel mechanism for regulating intravascular volume.
Pressure threshold for fluid loss from the peritoneal cavity
Authors: Flessner M. F. , Schwab A.
Published: December 2017

Ascites or dialysis fluid in the peritoneal cavity causes fluid loss from the cavity to the body. Experiments in animals and in humans have shown that the fluid loss rate increases with large increments in the intraperitoneal hydrostatic pressure (Pip). We hypothesized that there is a low-threshold Pip above which this fluid loss occurs. Because the full Pip force is exerted across the abdominal wall (AW), we further hypothesized that fluid movement into the abdominal wall would vary directly with the Pip. To address these questions, we dialyzed rats for 3 h in the supine position at constant levels of Pip with isotonic and hypertonic dialysis solutions containing a protein marker of fluid movement. We measured total fluid loss, AW fluid-marker concentration, and lymph flow. With variation of Pip from 0 to 8 cmH2O, we found that 1) lymph flows (0.61 +/- 0.03 ml/h) were not dependent on Pip, 2) measured isotonic fluid loss rate varied from 0.29 +/- 0.06 ml/h at 0 cmH2O to 0.62 +/- 0.02 at 2 cmH2O and then rose in a linear fashion to 5.06 +/- 0.10 ml/h at 8 cmH2O, 3) fluid movement into the AW paralleled the measured fluid loss rate, and 4) protein clearance from the cavity overestimated the true fluid loss because of adsorption of the marker to the peritoneal surface. We conclude that, although peritoneal lymph flow is not dependent on intraperitoneal hydrostatic or osmotic pressure, fluid loss from the cavity and fluid loss to the abdominal wall are directly proportional to Pip > 2 cmH2O. We also note that protein markers of fluid movement require correction for tissue surface adsorption for accurate results.
Na+/Ca2+ exchanger in epithelial cells of the porcine cortical thick ascending limb
Authors: Dai L. J. , Ritchie G. , Bapty B. , Raymond L. , Quamme G. A.
Published: December 2017

Intracellular Ca2+ concentration ([Ca2+]i) plays an important role in the signal transduction processes within cortical thick ascending limb (CTAL) cells. Control of [Ca2+]i was investigated in isolated CTAL cells with microfluorescent techniques. CTAL cells pretreated with ouabain to elevate intracellular Na+ concentration ([Na+]i) had basal [Ca2+]i of 86 +/- 2 nM. Removal of extracellular Na (Nao+) or voltage depolarization with KCl (in the presence of Nao+) resulted in a rapid and reversible maximal elevation of [Ca2+]i (1,023 +/- 72 nM, n = 28), which was dependent on the presence of external Ca2+ (Cao2+). The rise in [Ca2+]i was inhibited with La3+, Mg2+, amiloride, and bepridil. The increments of [Ca2+]i with either removal of Nao+ or voltage depolarization were dependent on pretreatment with ouabain and increases in [Na+]i. The presence of a Na+/Ca2+ exchanger was, confirmed with hybridization techniques, and the isoform was identified by sequencing the alternative splicing site within the intracellular loop. A gene transcript that encodes a portion of the intracellular loop of the renal Na+/Ca2+ exchanger was amplified from cortical tissue and single CTAL cells by reverse transcription-polymerase chain reaction, using primers flanking the alternative splicing site. Southern hybridization and DNA sequencing demonstrated the isoform contained exons B and D, which is characteristic of one isoform (NACA3) of the renal Na+/Ca2+ exchanger. The results provide both functional and molecular evidence for a Na+/Ca2+ exchanger in thick ascending limb cells of the porcine kidney.
NH+4 augments net acid secretion by a ouabain-sensitive mechanism in isolated perfused inner medullary collecting ducts
Authors: Wall S. M.
Published: December 2017

We have shown that NH4+ and K+ compete for extracellular binding on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the rat terminal inner medullary collecting duct (tIMCD). The present study explored whether the Na(+)-K(+)-ATPase modulates transepithelial net acid flux [JH+ = total CO2 absorption (JtCO2) + total ammonia secretion (JtAM)]. Tubules from the tIMCD were dissected from deoxycorticosterone (DOC)-treated rats and perfused in vitro. Perfusate and bath were identical physiological saline solutions containing 25 mM NaHCO3 + 6 mM NH4Cl or were NH4Cl or were NH4Cl free. With NH4+ present, the fall in total CO2 from perfusate to collected fluid (delta tCO2, 2.5 +/- 0.4 mM; n = 6) was accompanied by an increase in collected total ammonia concentration (0.2 +/- 0.1 mM). However, in the absence of NH4Cl, delta tCO2 was only 0.9 +/- 0.2 mM (P < 0.05, n = 5). To determine the mechanism of this NH4Cl-induced increase in net acid secretion, the effect of Na+ pump inhibition on net acid secretion was explored. With NH4Cl present, JCO2 was 3.8 +/- 0.5 pmol.mm-1.min-1 (ouabain absent) but declined to 1.6 +/- 0.3 pmol.mm-1.min-1 with ouabain addition to the bath (n = 7, P < 0.05). Furthermore, in the presence of NH4Cl, intracellular pH (pHi) increased from 7.05 +/- 0.02 to 7.15 +/- 0.02 (P < 0.05, n = 5) with ouabain addition and returned to 7.06 +/- 0.03 (P < 0.05) with ouabain removal. However, in the absence of NH4Cl, ouabain failed to reduce JtCO2 (P = NS, n = 5), and an increase in pHi was not observed (n = 4, P = NS). In conclusion, NH4+ augments net acid secretion likely by serving as a proton source for bicarbonate absorption and titration of other luminal buffers. This ammonium pathway is dependent on the basolateral membrane Na(+)-K(+)-ATPase.
Interleukin-4 modulates collagen synthesis by human mesangial cells in a type-specific manner
Authors: Nakazato Y. , Okada H. , Tajima S. , Hayashida T. , Kanno Y. , Suzuki H. , Saruta T.
Published: December 2017

Several studies have suggested an involvement of interleukin (IL)-4 in the pathophysiology of glomerulonephritis (GN). To elucidate its possible role in GN, we have investigated the effect of IL-4 on collagen accumulation by human mesangial cells (MC). After incubation with IL-1 alpha and/or IL-4 for 48 h, type I, III, and IV collagens in both soluble and cell-associated forms were identified by Western blotting. IL-1 alpha stimulated type I and IV collagen synthesis, lacking significant effect on type III collagen synthesis. In contrast, IL-4 stimulated type III collagen synthesis without affecting type I and type IV synthesis. Enzyme-linked immunosorbent assay confirmed the dose-dependent effect of IL-4 on collagen type III secretion (2.0-fold at 10 ng/ml). Importantly, IL-1 alpha-stimulated type I and IV collagen synthesis was suppressed by concomitant IL-4 treatment. Northern analysis of type I and III procollagen mRNAs displayed consistent results. These data indicate that IL-4 selectively stimulates type III collagen synthesis but also suppresses IL-1 alpha-stimulated type I and IV collagen synthesis. Therefore IL-4 could potentially contribute to the pathological changes in glomerular diseases in cooperate with other cytokines.
Rat calcium-sensing receptor is regulated by vitamin D but not by calcium
Authors: Brown A. J. , Zhong M. , Finch J. , Ritter C. , McCracken R. , Morrissey J. , Slatopolsky E.
Published: December 2017

Parathyroid hormone (PTH) secretion is regulated by extracellular calcium acting through a cell surface calcium receptor (CaR). We have examined the potential regulation of the CaR in the parathyroid glands (PTG) and kidney by calcium and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Rats fed vitamin D-deficient (-D) diets containing 0.02, 0.4, or 2.0% Ca had a wide range of serum ionized Ca (2.5-5.2 mg/dl) and PTH (22-590 pg/ml) concentrations. PTG CaR mRNA did not vary significantly with ionized calcium or PTH, indicating that hypocalcemia and hyperparathyroidism may not alter CaR expression. However, PTG CaR mRNA was 40% lower in the -D rats than in age-matched rats fed a vitamin D-replete (+D) diet (P < 0.002). Repletion of -D rats with 1,25-(OH)2D3 produced a dose-dependent increase in PTG CaR mRNA. Treatment of +D rats with 100 ng of 1,25-(OH)2D3 increased CaR mRNA by 33% (P < 0.05) and 54% (P < 0.002) in the PTG and by 89% (P < 0.02) and 91% (P < 0.02) in the kidney in two independent experiments. PTG CaR peaked at 16 h (150% of control, P < 0.05) after 1,25-(OH)2D3 administration but returned to normal by 24 h. This upregulation of CaR expression by 1,25-(OH)2D3 may be involved in the suppressive effects of vitamin D compounds on PTH secretion.
H(+)-K(+)-ATPase in rabbit cortical collecting duct B-type intercalated cell
Authors: Weiner I. D. , Milton A. E.
Published: December 2017

The role of H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) in the cortical collecting duct (CCD) B-type intercalated cell (B cell) is unclear. This study examined whether H(+)-K(+)-ATPase contributes to B cell intracellular pH (pHi) regulation and, if so, whether it is present at the apical or basolateral membrane. B cell Na(+)-independent pHi recovery from an acid load was only partially inhibited by peritubular N-ethylmaleimide (NEM). Complete inhibition required combining peritubular NEM either with luminal Sch-28080 or with luminal K+ removal. In contrast, neither peritubular Sch-28080 nor peritubular K+ removal altered pHi regulation. Tomato lectin, which binds to the gastric H(+)-K(+)-ATPase beta-subunit, labeled the B cell apical membrane. We conclude that the rabbit CCD B cell possesses an apical H(+)-K(+)-ATPase that plays an important role in pHi recovery from an in vitro acid load.
Direct demonstration of aquaporin-2 water channel recycling in stably transfected LLC-PK1 epithelial cells
Authors: Katsura T. , Ausiello D. A. , Brown D.
Published: December 2017

Vasopressin-dependent membrane insertion of aquaporin-2 (AQP-2) in collecting duct principal cells has been demonstrated in vivo and in vitro. However, the hypothesis that the AQP-2 molecule recycles between intracellular vesicles and the plasma membrane in response to hormonal stimulation and withdrawal remains to be demonstrated directly. In the present study, we examined AQP-2 recycling between intracellular vesicles and the plasma membrane in the absence of de novo protein synthesis using LLC-PK1 cells transfected with an AQP-2-c-myc construct. Cells were treated with cycloheximide for 30 min prior to vasopressin stimulation, and all subsequent treatments were performed in the continued presence of cycloheximide. Complete inhibition of AQP-2 biosynthesis by cycloheximide was verified by immuno-precipitation. Immunofluorescence revealed that AQP-2 was located on intracellular vesicles in nonstimulated cells but was relocated to the plasma membrane after vasopressin treatment, even in the presence of cycloheximide. After vasopressin washout, AQP-2 was retrieved to intracellular vesicles and was relocated to the plasma membrane after restimulation with forskolin. Subsequent forskolin washout resulted in AQP-2 endocytosis, and a second stimulation with forskolin resulted in relocation to the plasma membrane. These data, obtained in the absence of de novo protein synthesis, clearly indicate that AQP-2 can be recycled multiple times between intracellular vesicles and the plasma membrane.
Effects of chronic hypokalemia on renal expression of the "gastric" H(+)-K(+)-ATPase alpha-subunit gene
Authors: Ahn K. Y. , Turner P. B. , Madsen K. M. , Kone B. C.
Published: December 2017

Chronic potassium restriction leads to active potassium reabsorption in the late distal nephron and collecting duct, segments known to express "gastric" H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) alpha-subunit mRNA. In this study, the cellular distribution and relative abundance of mRNA encoding this isoform were examined in kidneys of normal and potassium-deprived (2 wk) rats. In situ hybridization with isoform-specific cRNA probes demonstrated prominent expression of this gene in the connecting segment (CNT), entire collecting duct, and renal papillary surface epithelium in a comparable distribution in both groups of rats. Hypertrophy of the outer medullary collecting ducts in the inner stripe of potassium-restricted rats was observed. Competitive polymerase chain reaction analysis revealed twofold greater levels of gastric H(+)-K(+)-ATPase alpha-subunit mRNA (normalized to the level of beta-actin mRNA) in the cortex, but roughly comparable levels in the outer and inner medulla, of potassium-restricted rats compared with controls. These data suggest that chronic potassium restriction results in modestly enhanced renal cortical expression of the gastric H(+)-K(+)-ATPase alpha-subunit gene and that this isoform may participate in potassium conversation by the CNT and cortical collecting duct during potassium deprivation.
Role of apoptotic and nonapoptotic cell death in removal of intercalated cells from developing rat kidney
Authors: Kim J. , Cha J. H. , Tisher C. C. , Madsen K. M.
Published: December 2017

In the developing rat kidney, both type A and type B intercalated cells are present throughout the medullary collecting duct (MCD), as well as the papillary surface epithelium. After birth, intercalated cells gradually disappear from the papillary surface epithelium and the terminal MCD, and type B cells disappear from the entire MCD. The purpose of this study was to establish the mechanism(s) by which intercalated cells are deleted from the MCD during development. Kidneys from 14-, 16-, 18-, and 20-day-old fetuses and 1-, 3-, 7-, and 14-day-old pups were preserved for light microscopic immunohistochemistry and electron microscopy. Intercalated cells were identified by immunostaining for H(+)-adenosinetriphosphatase (H(+)-ATPase) and band 3 protein. Apoptosis was identified by nick end labeling of DNA fragments, staining with the vital dye toluidine blue, and transmission electron microscopy. Two distinct mechanisms of elimination of intercalated cells were detected. Cells with apical labeling for H(+)-ATPase and basolateral labeling for band 3 protein protruded into the lumen of the MCD as if they were being extruded from the epithelium, and many had lost contact with the basement membrane. Extrusion of the cells with basolateral H(+)-ATPase or with no labeling for H(+)-ATPase was never observed. Apoptosis was observed in the MCD from shortly before birth to 7 days after birth, gradually progressing from the papillary tip toward the outer medulla. Staining for apoptosis was present in H(+)-ATPase-positive apoptotic bodies, located in cells that were negative for H(+)-ATPase. Staining was also occasionally observed in apoptotic cells with basolateral H(+)-ATPase but never in cells with apical H(+)-ATPase. Electron microscopy confirmed the presence of apoptotic intercalated cells in the MCD and demonstrated that apoptotic bodies were located in inner medullary collecting duct (IMCD) cells and principal cells. These results demonstrate that intercalated cells are deleted from the MCD by two distinct mechanisms, one involving apoptosis and subsequent phagocytosis by neighboring principal cells or IMCD cells. Elimination by extrusion affects only type A intercalated cells, whereas deletion by apoptosis appears to occur only in type B intercalated cells.
Differentiated phenotype of glomerular mesangial cells in nodular culture
Authors: Kitamura M. , Mitarai T. , Nagasawa R. , Maruyama N.
Published: December 2017

Prolonged culture of glomerular mesangial cells forms nodular structures composed of cells and surrounding extracellular matrix (ECM), which may mimic the situation in the glomerular mesangium of the kidney. The aim of this study was to investigate whether nodule-associated cells (NAC) exhibit a different phenotype to nodule-unassociated cells (NUC) in vitro. As phenotypic markers for rat mesangial cells, we examined mitogenic activity, expression of alpha-smooth muscle actin, and production of ECM constituents. Autoradiographic and immunohistochemical analyses revealed that NAC showed far less mitogenesis that NUC, like mesangial cells in the normal glomerulus. Immunofluorescence study and Northern blot analysis showed that alpha-smooth muscle actin, a marker of mesangial cell activation/dedifferentiation, was strongly expressed in NUC but faint in NAC. When nodules were dissolved by trypsinization, the dispersed NAC regained both active mitogenesis and alpha-smooth muscle actin expression, suggesting that the altered phenotype was reversible. Northern blot analysis revealed that the ratio of type IV collagen versus type I collagen expression, a marker of mesangial cell differentiation, was elevated in NAC compared with NUC. This phenotypic shift toward differentiation was associated with upregulation of transforming growth factor-beta 1. These findings demonstrate that mesangial cells in nodules exhibit a phenotype which is distinct from that of cells in two-dimensional cultures. We hypothesize that, as a differentiated feature, cultured mesangial cells have the ability to create an appropriate three-dimensional cyto-architecture that resembles the glomerular mesangium.
K depletion and NH4Cl acidosis increase apical insertion of studded membrane in type A intercalated cells
Authors: Narbaitz R. , Murugesan S. , Levine D. Z.
Published: December 2017

To examine the effects of K depletion and metabolic acidosis on the prevalence and distribution of H(+)-adenosinetriphosphatase (H(+)-ATPase)-related studs at the luminal plasma membrane of A-type intercalated cells (A-ICs), we conducted a quantitative electron microscopical study on the cortical collecting ducts (CCDs) of control, NH4Cl-loaded, and K-depleted rats. The percentage of A-ICs was slightly increased in the K-depleted but not in the acidotic rats. A-ICs were considered "active" when they presented a semicontinuous row of 9- to 10-nm studs at the cytoplasmic face of the apical membrane and "inactive" when all of the apical membrane was devoid of studs. The percentage of active A-ICs was greatly increased in acidotic (87.2%) and K-depleted (79.3%) rats compared with controls (41.6%). These results give a quantitative expression to the general view that acidosis elicits insertion of studded membrane in the apical plasma membrane of A-ICs. Furthermore, we show, for the first time, that an increase in the membrane insertion of H(+)-ATPase is also part of the response to K depletion.
Expression of subunits of the metalloendopeptidase meprin in renal cortex in experimental hydronephrosis
Authors: Ricardo S. D. , Bond J. S. , Johnson G. D. , Kaspar J. , Diamond J. R.
Published: December 2017

Meprin A is a metalloendopeptidase in the proximal tubular epithelium of rodents that is capable of hydrolyzing a great variety of peptides and proteins. The aim of the present investigation was to investigate effects of ureteral ligation on the expression of meprin subunits. Ureteral ligation resulted in marked decreases in the expression of both alpha- and beta-meprin subunits within 12 h of ureteral obstruction. Even greater downregulation of expression of meprin alpha- and beta-mRNA was noted at 24, 48, and 96 h after ureteral ligation. The greatest decrease in meprin mRNA expression in obstructed kidneys over contralateral unobstructed control kidneys (CUK) occurred at 24 h postunilateral ureteral obstruction (post-UUO) for the meprin alpha-subunit (20-fold decrease compared with controls) and at 48 h for the meprin beta-subunit (90-fold decrease). On immunolabeling, the intensity for the two meprin subunits at the corticomedullary junction was dramatically decreased at 24 to 96 h after ureteral ligation in contrast to the CUK specimens. Results of in situ hybridization indicated that the CUK specimens expressed meprin beta-mRNA at the corticomedullary junction, whereas the obstructed kidneys exhibited a decrease in mRNA signal for meprin beta-subunit. There was a steady increase in the interstitial macrophage number in UUO rat kidneys over the 96 h of evaluation post-UUO. ED-1-positive macrophages were observed almost exclusively in the peritubular cortical interstitial space in a ringlike pattern with a preponderance of macrophage clusters around glomeruli. Unexpectedly, after reversal of UUO, the interstitial macrophage number remained higher than controls, despite the demonstrable decompression of the renal pelvis and caliceal system. In summary, this investigation demonstrates downregulation of meprin alpha and beta within hours of UUO and indicates a novel tubular response to ureteral obstruction.
Electrodiffusive transport of Mg across renal membrane vesicles of the rainbow trout Oncorhynchus mykiss
Authors: Freire C. A. , Kinne R. K. , Kinne-Saffran E. , Beyenbach K. W.
Published: December 2017

The mechanism of tubular Mg transport was investigated in membrane vesicles (MV) of trout kidneys prepared by differential centrifugation with sucrose. MV consisted largely of brush-border membranes, as indicated by high enrichments of brush-border membrane enzymes. Although measured transport of 28 Mg included a binding component, most membrane transport was into or out of an osmotically active space. There was no evidence for amiloride-sensitive Na/Mg exchange, nor was Mg uptake affected by the carboxyl group reagents trimethyloxonium tetrafluoroborate, glycine methyl ester.HCl-1-ethyl-3- (3-dimethyl-aminopropyl)carbodiimide, and N,N'-dicyclohexyl carbodiimide or the Ca channel modulators D-600, verapamil, diltiazem, and BAY K 8644. However, Mg uptake increased in the presence of inside-negative voltages generated by inward gradients of the permeant anions NO3, SCN, and Cl or by outward gradients of K (plus valinomycin). Alkaline-earth cations displayed the selectivity sequence VII (Mg > Ca > Sr > Ba) for cis-inhibition of 28 Mg uptake. Mg efflux was trans-inhibited by La and Gd, and Mg uptake was cis-inhibited by Mn. The sulfhydryl group reagents p- chloromercuribenzoic acid and p-chloromercuriphenylsulfonate stimulated Mg uptake and efflux. These results reveal an electrodiffusive pathway for Mg transport in trout renal MV.