bluuebunny/crossref_metadata_2025 huggingface dataset:
Sustained ERK-2 activation in rat glomerular mesangial cells: differential regulation by protein phosphatases
Authors: Schramek H. , Schumacher M. , Pfaller W.
Published: December 2017

The duration of extracellular signal-regulated protein kinase (ERK) activation is critical for cell signaling decisions and probably determines whether a stimulus elicits proliferation or differentiation. We studied the intracellular signals regulating sustained ERK-2 activity in glomerular mesangial cells (GMC), utilizing combination of GMC mitogens of different potency. Incubation of GMC with both endothelin-1 (ET-1) and platelet-derived growth factor BB (PDGF-BB) led to a long-lasting, monophasic increase in ERK-2 activity. In contrast, when ET-1 was administered together with epidermal growth factor (EGF), a less pronounced and shorter activation occurred. Long-term stimulation of ERK-2 was accompanied by an increase in p45 MEK activity, which again was more pronounced when ET-1 was administered together with PDGF-BB compared with EGF. In the presence of actinomycin D (Act D), an inhibitor of RNA synthesis, ERK-2 activity induced by ET-1 and PDGF-BB but not by ET-1 and EGF remained elevated more than sixfold throughout the whole incubation period of 6 h. The effect of Act D on ET-1- and PDGF-BB-induced ERK-2 activation was mimicked by the protein phosphatase inhibitor sodium orthovanadate. In addition, vanadate also unmarked an ET-1- and EGF-induced ERK-2 activity after 6 h. The serine/threonine phosphatase inhibitor okadaic acid (OA) did neither alter agonist-induced ERK-2 activity after 6 h (0.5 nM OA) nor after 10 min or 1 h (250 nM). Together these results suggest that, in GMC, long-term activation of the mitogen-activated protein kinase ERK-2 is differentially regulated, depending on the combination of agonists administered. ET-1- and PDGF-BB-induced long-term activation of ERK-2 is regulated by a vanadate-sensitive protein phosphatase(s) and by a transcriptionally regulated protein(s). In contrast, ET-1- and EGF-induced sustained ERK-2 stimulation is regulated by a vanadate-sensitive protein phosphatase(s) but not by a transcriptionally regulated protein. Agonist-specific and time-dependent stimulation of ERK-2-regulating protein phosphatases may be critical for the length of ERK-2 activation in GMC and could thus be of pathophysiological significance in glomerular diseases associated with alterations in cell proliferation or cell differentiation.
Separate regulation of Na+ and anion transport by IMCD: location, aldosterone, hypertonicity, TGF-beta 1, and cAMP
Authors: Husted R. F. , Stokes J. B.
Published: December 2017

We have investigated some of the factors known or suspected to influence ion transport by the rat inner medullary collecting duct and have analyzed their actions on active Na+ absorption and active anion secretion by primary cultures. Cells from the terminal 1-2 mm (tip) of the papilla had a lower basal rate of Na+ absorption (2.0 microA/cm2) than cells from the more proximal portions (6.5 microA/cm2). Aldosterone increased Na+ transport approximately sevenfold in the tip cells and approximately threefold in the proximal cells. The magnitude of anion secretion in response to adenosine 3',5'-cyclic monophosphate (cAMP) agonists was similar in the two regions and was unaffected by aldosterone. The morphology of monolayers from both regions was also similar. In monolayers cultured from the entire inner medulla, hypertonic (100 mosM) urea, NaCl, or sucrose reduced Na+ transport but had no significant effect on anion secretion. Transforming growth factor-beta 1, known to blunt the effect of steroids on Na+ transport, had no effect on anion secretion. Finally, cAMP had no effect on Na+ transport, a result that contrasts with its effect on Na+ transport by other epithelial cells demonstrating steroid-responsive, electrogenic Na+ transport. These results demonstrate some potential differences in the magnitude of Na+ transport by position along the inner medulla. They further demonstrate separate regulation of Na+ and anion transport.
Development of urinary concentrating capacity: role of aquaporin-2
Authors: Yasui M. , Marples D. , Belusa R. , Eklof A. C. , Celsi G. , Nielsen S. , Aperia A.
Published: December 2017

The capacity to concentrate urine develops progressively during postnatal life in most mammalian species. Here we have examined whether low expression of the arginine vasopressin (AVP)-activated water channel aquaporin-2 (AQP-2) may be a limiting factor for the concentrating capacity in the infant rats. Urine osmolality in response to 24-h dehydration increased significantly from 10 to 40 days of age. The most rapid increase occurred during the weaning period, i.e., days 15-20. A similar developmental pattern was observed for AQP-2 mRNA levels in the renal medulla. AQP-2 protein levels also increased markedly from day 10 to 40. Immunohistochemistry revealed that AQP-2 was exclusively located in collecting duct principal cells both in infant and adult rats but that the signal was much weaker in infants. To further examine the relationship between urinary concentrating capacity and AQP-2 expression, we treated rats with a single injection of betamethasone, which is known to accelerate maturation in several organs. Twenty-four hours after treatment, there was an increase in urine osmolality, renal medullary AQP-2 mRNA, and AQP-2 protein levels in infant but not in adult rats. A single injection of a specific V2 agonist caused within 6 h significant increase of AQP-2 mRNA in both infant and adult. The expression of the mRNA of three other transporters involved in the concentrating process, medullary Na(+)-K(+)-ATPase alpha-subunit, Na-K-2Cl cotransporter, and epithelial chloride channel also increased during the weaning period and were upregulated by glucocorticoids. We conclude that there is a well-synchronized development of the many of the components that determine the concentrating capacity and that the low expression of AQP-2 is one of the limiting factors for low concentrating capacity in infants.
Mechanisms of apoptosis and its potential role in renal tubular epithelial cell injury
Authors: Lieberthal W. , Levine J. S.
Published: December 2017

Cells can die by two distinct pathways: apoptosis or necrosis. Necrosis is associated with rapid metabolic collapse that leads to cell swelling, early loss of plasma membrane integrity, and ultimate cell rupture. Cytosolic contents leak from the necrotic cell causing injury and inflammation to surrounding tissue. In contrast, apoptosis is an energy-requiring, gene-directed process, which, when activated, results in cell "suicide." The morphological and biochemical characteristics of cells dying by apoptosis differ markedly from those of cells dying by necrosis. During apoptosis, cells decrease in size and round up. The nuclear chromatin undergoes condensation and fragmentation. The apoptotic cell then breaks apart into many plasma membrane-bound vesicles called "apoptotic bodies," which contain fragments of condensed chromatin and morphologically intact organelles such as mitochondria. Apoptotic cells and bodies are rapidly phagocytosed, thereby protecting surrounding tissues from injury. The rapid and efficient clearance of apoptotic cells makes apoptosis extremely difficult to detect in tissue sections. Recent studies show that multiple cytotoxic stimuli well known to cause necrosis can lead to apoptosis instead when cells are exposed to the same noxious agents at lower concentrations. This insight has led to an interest in the role of apoptosis in the pathogenesis of renal diseases that result primarily from injury to renal tubular epithelial cells. These diseases include acute and chronic renal failure from exposure of the kidney to ischemia or to cytotoxic agents. In this review we discuss some relevant aspects of the differences between necrotic and apoptotic cell death. We also present evidence to support the hypothesis that apoptosis is an important pathogenic mechanism in those forms of acute and chronic renal failure in which the renal tubular epithelial cell is the primary target of ischemic or toxic injury.
Urodilatin is involved in sodium homeostasis and exerts sodium-state-dependent natriuretic and diuretic effects
Authors: Meyer M. , Richter R. , Brunkhorst R. , Wrenger E. , Schulz-Knappe P. , Kist A. , Mentz P. , Brabant E. G. , Koch K. M. , Rechkemmer G. , Forssmann W. G.
Published: December 2017

Urodilatin is involved in sodium homeostasis exerts sodium-state-dependent natriuretic and diuretic cts. Eight male volunteers participated in a study consisting of three consecutive phases of 7 days each. The volunteers a sodium diet with 52, 172.6, and 347.8 mmol um/day. Sodium excretion increased from 57.4 +/- 3.7 via .8 +/- 4.6 (P < 0.001) to 322.5 +/- 10.2 mmol/24 h (P < 0.001) at the end of each sodium diet. Urinary urodilatin excretion increased from 24.8 +/- 3.0 via 35.5 +/- 9.0 (P = 0.07) to 49.0 = mol/min (P < 0.01). At the end of each diet, urodilatin was infused for 2 h at 20 ng.kg body wt-1.min-1. Natriuresis increased after low- (4.1 to 52.9 mmol/h, P < 0.001), normal (6.9 to 44.9 mmol/h, P < 0.05), and high-sodium diet (20.1 to 102.9 mmol/h, P < 0.001). Diuresis increased from 174 to 709 (P < 0.001), 395 to 1,026 (P < 0.05), and 266 to 1,339 ml/h < 0.001). The present results indicate that endogenous urodilatin plays an important role in sodium homeostasis and that renal response to exogenous urodilatin is modulated by sodium balance.
Arachidonic acid inhibits activity of cloned renal K+ channel, ROMK1
Authors: Macica C. M. , Yang Y. , Hebert S. C. , Wang W. H.
Published: December 2017

Arachidonic acid (AA) has been shown to inhibit the activity of the low-conductance ATP-sensitive K+ channel in the apical membrane of the cortical collecting duct [W. Wang, A. Cassola, and G. Giebisch. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F554-F559, 1992]. ROMK1, a K+ channel derived from the rat renal outer medulla, shares many biophysical properties of the native low-conductance K+ channel, which is localized to the apical membranes of the cortical collecting duct and thick ascending limb. This study was designed to determine whether the ROMK channel maintains the property of AA sensitivity of the native low-conductance K+ channel. Experiments were conducted in Xenopus oocytes injected with cRNA encoding the ROMK1 channel by use of patch-clamp techniques. We have confirmed previous reports that the cloned ROMK1 has similar channel kinetics, high open probability, and inward slope conductance as the native low-conductance K+ channel, respectively. Addition of 5 microM AA to an inside-out patch resulted in reversible inhibition of channel activity at a concentration similar to the inhibitor constant for AA on the native K+ channel. The effect of AA on channel activity was preserved in the presence of 10 microM indomethacin, a cyclooxygenase inhibitor, 4 microM cinnamyl-3,4-dihydroxycyanocinnamate, a lipoxygenase inhibitor, and 4 microM 17-octadecynoic acid, an inhibitor of cytochrome P-450 monooxygenases, thus indicating that the effect of AA was not mediated by metabolites of AA. The effect did not appear to be the result of changes in membrane fluidity, since 5 microM eicosatetraynoic acid, an AA analogue that is a potent modulator of membrane fluidity, had no effect. Furthermore, the addition of AA to the outside of the patch also had no effect on channel activity. These results indicate that, like the native low-conductance channel, AA is able to directly inhibit ROMK1 channel activity.
Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells
Authors: Firestein B. L. , Xing M. , Hughes R. J. , Corvera C. U. , Insel P. A.
Published: December 2017

We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.
Prostaglandins stimulate renin secretion and renin mRNA in mouse renal juxtaglomerular cells
Authors: Jensen B. L. , Schmid C. , Kurtz A.
Published: December 2017

This study examined 1) effects of prostaglandins (PG) on renin secretion and renin gene expression from isolated juxtaglomerular granular cells and 2) expression of cyclooxygenases in juxtaglomerular structures. Incubation of granular cell cultures with PGE2, -I2, -F2 alpha, and thromboxane B2 identified PGI2 and PGE2 as stimulators of renin secretion; the effects were dose and time dependent. PGE2 also increased renin mRNA accumulation time and dose dependent. PGE2 and PGI2 activated adenylate cyclase concentration dependent in granular cells. PGE2 stimulations of renin secretion and renin mRNA were nonadditive to those of forskolin and were inhibited by endothelin. The findings are compatible with cellular actions through adenosine 3',5'-cyclic monophosphate (cAMP). On total RNA harvested from whole kidneys, from microdisected glomeruli with attached afferent arterioles and from mesangial cells in primary culture, reverse transcription-polymerase chain reaction revealed significant expression of cyclooxygenase I and II. By direct interaction with PG receptors on renal juxtaglomerular cells, PGE2 and PGI2 can act as potent and rapid stimulators of renin secretion and renin mRNA probably through cAMP-dependent pathways.
Specific effect of maleate on an apical membrane glycoprotein (gp330) in proximal tubule of rat kidneys
Authors: Bergeron M. , Mayers P. , Brown D.
Published: December 2017

Maleate treatment of rats induces transport defects similar to those seen in the Fanconi syndrome (glycosuria, aminoaciduria, phosphaturia, proteinuria, etc.) and causes an accumulation of apical vesicles in proximal tubule epithelial cells. Because the apical membrane glycoprotein, gp330, is a receptor associated with the apical endocytotic and recycling apparatus in these cells, we examined the effect of maleate on the distribution of this protein and other brush border markers. Rats received sodium maleate (400 mg/kg ip) and were killed at various times between 45 min and 3 h; kidneys were perfusion fixed with paraformaldehyde-lysine-periodate before processing for immunofluorescence and immunoelectron microscopy. In control rats, staining with a polyclonal or monoclonal gp330 antibody showed a uniform distribution on the brush border and in coated pits of all proximal tubule cells. In the S3 segments, the immunofluorescence labeling of the microvilli was generally uniform but at times showed spike labeling, suggesting that gp330 sheds easily from the apical membrane. After maleate treatment, the staining intensity of the brush border was decreased in all proximal tubule segments, and cytoplasmic streaks as well as an intense vacuolar staining were seen. In the S3 segment, a remarkable mosaic pattern of staining was observed, with the brush border of some cells being completely negative, while adjacent cells showed an apparently normal staining pattern. These results were confirmed at the electron microscope level, using the protein A-gold technique. Maleate had no effect on the distribution or staining intensity of four other brush border markers, dipeptidyl peptidase IV, and various lectins (Helix pomatia lectin, peanut lectin, elderberry bark lectin). The urinary excretion of gp330 occurs in normal rats and was already increased as early as 1 h after maleate injection and remained at a twofold increment between 6 and 24 h. These data suggest that the generalized membrane transport derangement seen in this experimental Fanconi syndrome could occur via a specific effect on gp330, which seems to block endocytosis and the recycling apparatus at the late endosome level and inhibits the formation of new dense apical tubules.
Heavy metals inhibit Pi-induced currents through human brush-border NaPi-3 cotransporter in Xenopus oocytes
Authors: Wagner C. A. , Waldegger S. , Osswald H. , Biber J. , Murer H. , Busch A. E. , Lang F.
Published: December 2017

Heavy metal intoxication with Hg2+, Pb2+ and Cd2+ commonly leads to phosphaturia. In this study, we examined the effects of these heavy metals on Pi-induced currents (Ip) through NaPi-3, the human renal cotransporter for Na+ and Pi. Hg2+ inhibited Ip in a dose- and time-dependent fashion. Hg2+ decreased the extrapolated maximal current but did not alter the apparent affinity for Pi. This inhibition was also observed with the membrane-permeable oxidizing agent 2,2'-dithio-bis(5-nitropyridine) (DTNP) but not with the membrane-impermeable 5,5'-dithiobis(2-nitrobenzoic acid). Hg(2+)- and DTNP-mediated inhibition of Ip was reversible only in the presence of the reducing agent 2,3-dihydroxybutane-1,4-dithiol. Cd2+ and Pb2+ also inhibited Ip. However, while CD2+ did not significantly alter the apparent affinity for Pi, the apparent concentration needed for half-maximal current (Km) for Pi was increased by Pb2+. In contrast to Hg2+, the inhibition of Ip by Cd2+ and Pb2+ was rapidly reversible upon washout. In the presence of the Na(+)-K(+)-adenosinetriphosphatase inhibitor ouabain, Ip was not reduced, and the effects of the heavy metals were maintained. In summary, the three heavy metals Hg2+, Cd2+, and Pb2+ inhibit Ip through the Na+/Pi cotransporter NaPi-3 by distinct mechanisms. Heavy metal-mediated inhibition of NaPi-3 may be responsible for the phosphaturia observed after intoxication with these compounds.
Effect of age on blood acid-base composition in adult humans: role of age-related renal functional decline
Authors: Frassetto L. A. , Morris R. C. , Sebastian A.
Published: December 2017

In 64 apparently healthy adult humans (ages 17-74 yr) ingesting controlled diets, we investigated the separate and combined effects of age, glomerular filtration rate (GFR, index of age-related renal functional decline), renal net acid excretion [NAE, index of endogenous acid production (EAP)], and blood PCO2 (PbCO2, index of respiratory set point) on steady-state blood hydrogen ion ([H+]b) and plasma bicarbonate concentration ([HCO3-]p). Independent predictors of [H+]b and [HCO3-]p were PbCO2, NAE, and either age or GFR, but not both, because the two were highly correlated (inversely). [H+]b increased with increasing PbCO2, NAE, and age and with decreasing GFR. [HCO3-]p decreased with increasing NAE and age but increased with increasing PbCO2 and GFR. Age (or GFR) at constant NAE had greater effect on both [H+]b and [HCO3-]p than did NAE at constant age (or GFR). Neither PbCO2 nor NAE correlated with age or GFR. Thus two metabolic factors, diet-dependent EAP and age (or GFR), operate independently to determine blood acid-base composition in adult humans. Otherwise healthy adults manifest a low-grade diet-dependent metabolic acidosis, the severity of which increases with age at constant EAP, apparently due in part to the normal age-related decline of renal function.
Differential regulation of renal regional blood flow by endothelin-1
Authors: Gurbanov K. , Rubinstein I. , Hoffman A. , Abassi Z. , Better O. S. , Winaver J.
Published: December 2017

The present study evaluated the effects and mechanisms of action of endothelin-1 (ET-1) on medullary and cortical blood flow (MBF and CBF, respectively). CBF and MBF were measured simultaneously by laser-Doppler flowmetry in anesthetized male Wistar rats. Bolus injection of ET-1 (1.0 nmol/kg iv) produced a sustained decrease in CBF (delta = -30%) and a transient increase in MBF (delta = +35%). The medullary vasodilation induced by ET-1 was observed with doses lower than that required to produce cortical vasoconstriction; was completely blocked by bosentan, a mixed ETA/B-receptor antagonist; and was mimicked by IRL-1620, a specific ETB-receptor agonist. In contrast, BQ-123, an ETA-receptor antagonist, failed to inhibit the ET-1-dependent medullary vasodilation but effectively blocked the cortical vasoconstriction induced by the peptide. Finally, inhibition of nitric oxide (NO) synthase completely abolished, whereas cylooxygenase inhibition attenuated, the effect of ET-1 on MBF. The data demonstrate that ET-1 exerts opposite effects on renal cortical and medullary circulation, i.e., ETA-receptor-mediated cortical vasoconstriction and ETB-mediated medullary vasodilation. Furthermore, the medullary vasodilation induced by ET-1 is dependent on the NO system and, to a lesser extent, on prostaglandin generation.
Hyperosmolality inhibits sodium absorption and chloride secretion in mIMCD-K2 cells
Authors: Green R. B. , Slattery M. J. , Gianferrari E. , Kizer N. L. , McCoy D. E. , Stanton B. A.
Published: December 2017

Previously we demonstrated that a cell line derived from mouse inner medullary collecting duct (mIMCD-K2) absorbs Na+ and secretes Cl- by electrogenic mechanisms and that arginine vasopressin (AVP) stimulates Cl- secretion. The objective of the present study was to determine whether hyperosmolality, both acute (minutes) and chronic (weeks), affects electrogenic Na+ absorption IscNa and electrogenic Cl- secretion IscCl across the IMCD. To this end, we measured IscNa and IscCl across monolayers of mIMCD-K2 cells mounted in Ussing-type chambers. Osmolality was increased from 290 to 590 mosmol/kgH2O by adding 200 mosmol/kgH2O of NaCl and 100 mosmol/kgH2O of urea or 300 mosmol/kgH2O of sucrose to the bathing solutions. Acute and chronic hyperosmolality reduced basal IscNa and IscCl and the AVP-stimulated rise in IscCl. These findings indicate that osmolality is an important determinant of IscNa and IscCl across IMCD cells and that the osmolality of the interstitial fluid should be considered when evaluating the effects of hormones and other factors on Na+ and Cl- transport by the IMCD.
Functional differentiation of the human red blood cell and kidney urea transporters
Authors: Martial S. , Olives B. , Abrami L. , Couriaud C. , Bailly P. , You G. , Hediger M. A. , Cartron J. P. , Ripoche P. , Rousselet G.
Published: December 2017

The recent cloning of two urea transporters will allow to better understand their role in the urinary concentrating mechanism. This physiological approach needs to be sustained by a knowledge of their functional characteristics. We compared the pharmacological properties of the human red blood cell and kidney urea transporters (HUT11 and HUT2) in the Xenopus oocyte expression system. Both proteins allow the rapid transfer of urea but not of water. Both are inhibited by phloretin, although with different half-maximal inhibitory concentrations (IC50; 75 microM, for HUT11 and 230 microM for HUT2). Whereas para-chloromercuribenzene sulfonate inhibits HUT11 with an IC50 of 150 microM, it does not inhibit HUT2, whatever the concentration used. We demonstrate that thiourea diffuses through HUT11 with a Michaelis constant (Km) of 40 mM, but not through HUT2. In contrast, it inhibits urea transport through both proteins. This identification of a substrate binding site independent from the transport activity is the first step in the understanding of the molecular events underlying urea transport.
Vasopressin constricts outer medullary descending vasa recta isolated from rat kidneys
Authors: Turner M. R. , Pallone T. L.
Published: December 2017

Arginine vasopressin (AVP) can selectively decrease blood flow in the renal medulla, but the sites of vasoconstriction are uncertain. We have examined the effects of vasopressin-receptor agonists and antagonists on the diameters of outer medullary descending vasa recta (OMDVR), isolated and perfused in vitro. AVP can constrict OMDVR, apparently via V1a-receptors. Ablumenal AVP (10(-10)-10(-6)M) or the selective V1a-receptor agonist [Phe2, Ile3, Orn8]-vasopressin (PO-VT, 10(-8) M) constricted OMDVR focally and (at higher AVP concentrations) transiently. The V1b agonist ideamino-Cys1,D-3-(pyridyl)Ala2,Arg8)vasopressin (DP-VP; 10(-8) M) and the V2 agonist [deamino-Cys1, D-Arg8]vasopressin (DDAVP; 10(-8) M) did not constrict OMDVR. The V1a antagonist [d(CH2)5(1), O-Me-Tyr2,Arg8]vasopressin (CTM-VP, 10(-10) 10(-8) M) inhibited vasoconstriction by AVP 10(-9 M), whereas the V2 antagonist [d(CH2)5(1), D-Ile2,Ile4 Arg8]vasopressin (II-VP) at low concentration (10(-10) M) did not. V2 stimulation seems to inhibit V1a constriction of OMDVR. DDAVP prevented constriction by PO-VT (10(-8) M) applied at the same time and dilated OMDVR preconstricted with PO-VT.
Nitric oxide prevents neutrophil-mediated acute renal failure
Authors: Linas S. , Whittenburg D. , Repine J. E.
Published: December 2017

The contribution of nitric oxide (NO) to ischemic acute renal failure is unclear. Because polymorphonuclear neutrophils (PMN) accentuate injury in kidneys subjected to ischemia-reperfusion and because NO has potent vascular and PMN effects, we examined the contribution of NO to PMN-mediated injury in isolated perfused rat kidneys. Nonischemic and ischemic kidneys were perfused by the isolated kidney technique in the presence or absence of PMN and NO agonists [sodium nitroprusside (SNP), L-arginine (L-Arg)] or a NO synthase inhibitor [N omega-nitro-L-arginine (L-NNA)]. In nonischemic kidneys, the NOS antagonist decreased perfusion flow rate by 25% without affecting glomerular filtration rate (GFR) or tubular sodium reabsorption (TNa), whereas NOS agonist treatment had no effects. After 20 min of ischemia/60 min reperfusion in the absence of PMN NO agonist treatment potentiated ischemia-reperfusion-induced loss of GFR and TNa, whereas adding the NO antagonist lessened glomerular and tubular injury. Reperfusion of ischemic kidneys with PMN resulted in PMN retention and potentiated ischemic injury. However, increases in PMN retention as well as decreases in GFR and TNa caused by PMN were prevented by SNP and worsened by L-NNA. Moreover, in nonischemic kidneys, activated PMN caused renal injury and PMN retention, which were prevented by SNP and worsened by L-NNA. In conclusion, 1) NO worsens ischemic injury in the absence of PMN, and 2) NO prevents the PMN component of ischemic renal injury by blocking PMN retention and the deleterious effects of activated PMN on glomerular and tubular function.
Surface exposure of phosphatidylserine increases calcium oxalate crystal attachment to IMCD cells
Authors: Bigelow M. W. , Wiessner J. H. , Kleinman J. G. , Mandel N. S.
Published: December 2017

The development of urolithiasis is a multifaceted process, starting at urine supersaturation and ending with the formation of mature renal calculi. The retention of microcrystals by the urothelial cell membrane is a critical event in the process. The current study examines calcium oxalate monohydrate (COM) crystal attachment to inner medullary collecting duct (IMCD) cells following selective changes in cell membrane phospholipid composition. Both primary culture of IMCD cells and a continuous IMCD cell line were used for these studies. Cell membrane composition was selectively altered by either exogenous addition of membrane phospholipids or using membrane lipid scrambling agents. Enrichment with anionic phospholipids was found to greatly increase attachment of crystals to the cells. This increased attachment correlated with the exposure of phosphatidylserine (PS) on the exofacial leaflet of the cell membrane as demonstrated by the use of the membrane scrambling agent A-23187. Furthermore, the increased COM attachment following PS exposure could be blocked by incubating the cells with the PS-specific binding protein, annexin V. These results support the hypothesis that exposure of PS head groups on the papillary epithelial cell surface may mediate stone crystal attachment to the kidney tubule cell epithelium in the renal papilla, possibly as an initiating event in urolithiasis.
Distribution of de novo synthesized betaine in rat kidney: role of renal synthesis on medullary betaine accumulation
Authors: Moeckel G. W. , Lien Y. H.
Published: December 2017

The trimethylamine glycine-betaine is accumulated to high concentrations in medullary cells of mammalian kidneys, whereas betaine synthesis from choline is predominant in the renal cortex. We investigated the contribution of renal betaine synthesis to medullary betaine accumulation. De novo synthesis of betaine in situ was accomplished by injecting [14C]choline into the renal artery of male Sprague-Dawley rats. [14C]betaine was measured in the renal cortex and medulla, as well as in serum and urine samples. Betaine concentration in the cortex decreased from 3.5 +/- 1.3 at 5 min to 0.4 +/- 0.2 nmol/mg protein at 60 min, but it increased from 1.4 +/- 0.1 to 2.5 +/- 0.6 nmol/mg protein in the medulla. Serum and total urine [14C]betaine increased from 2.7 +/- 1.3 and 0.9 +/- 0.1 nmol/ml at 5 min to 5.3 +/- 0.3 and 2.1 +/- 0.4 nmol/ml at 60 min, respectively. Concentrations of newly synthesized betaine were not decreased by the ligation of the hepatic artery and portal vein, suggesting that most [14C]betaine was synthesized in the kidney. Coinjection with 5 mM dimethylamino-ethanol, a choline oxidase inhibitor, and 100 mM cold betaine reduced medullary betaine accumulation by 80 and 76%, respectively. Water deprivation for 60 h increased both cortical and medullary [14C]betaine, whereas furosemide diuresis decreased the medullary [14C]betaine concentration. We concluded that betaine synthesized in the kidney can be accumulated in the medulla and that the medullary concentrations of newly synthesized betaine are closely related to the hydration state of the animal.
Endothelial cell targeting during renal development: use of monoclonal antibodies
Authors: Oliver J. A. , Goldberg M. R. , Al-Awqati Q.
Published: December 2017

Development of the different renal capillary beds requires that the transformation of the metanephrogenic mesenchyme and ureteric bud into the different nephron segments be temporally and spatially coordinated with the migration and growth of the endothelial cells present in the renal anlage. This suggests that ureteric bud and/or metanephrogenic mesenchymal cells provide molecules which guide endothelial cells to their appropriate locations. We found that monoclonal antibody (MAb) 5B6-E4, obtained with mechanically dispersed cells of embryonic day 15 (E15) rat renal anlage, identifies an antigen that is temporally and spatially associated with endothelial cell location and migration during renal development. Furthermore, 5B6-E4 disrupts the close association between ureteric bud ampullae and endothelial cells in E14 renal anlages grown in vitro: whereas 43% of the ureteric bud ampullae were in contact with endothelial cells in control conditions, the presence of 20 microg/ml 5B6-E4 reduced this number to 22% (P < 0.02). These results suggest that the antigen recognized by 5B6-E4 is involved in endothelial cell targeting during renal development.
Effect of epinephrine on cAMP accumulation in cultured rat inner medullary collecting duct cells
Authors: Yasuda G. , Umemura S. , Jeffries W. B.
Published: December 2017

In a previous study we have reported the existence of alpha2- and beta-adrenoceptors in cultured rat inner medullary collecting duct (IMCD) cells. In this report, we examined the effect of epinephrine on intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation and evaluated whether alpha2-adrenoceptors interact with beta-receptors, vasopressin receptors, and prostaglandin (PG) E2 receptors by measuring cAMP generation. Epinephrine stimulated cAMP accumulation in a dose-dependent manner [half-maximal effective concentration (EC50) = 300 nM]. Rauwolscine (10 microM) enhanced epinephrine effects, shifting the dose-response curve for epinephrine to the left (EC50 = 120 nM); however, beta-antagonists inhibited epinephrine-induced cAMP accumulation. Epinephrine (10 microM) inhibited cAMP accumulation maximally induced by isoproterenol (10 microM); this effect was reversed by rauwolscine (10 microM). Epinephrine inhibited vasopressin (100 nM)-induced cAMP accumulation but failed to inhibit PGE2 (10 microM)-induced cAMP accumulation. We conclude that epinephrine acts as an alpha2- and beta-adrenoceptor agonist and that alpha2-adrenoceptors interact with beta-adrenoceptors and vasopressin receptors but not with PGE2 receptors on cAMP accumulation. This suggests that alpha2-adrenoceptors play a physiological role via interaction with different hormone receptors.
Met -/- kidneys express epithelial cells that chemotax and form tubules in response to EGF receptor ligands
Authors: Kjelsberg C. , Sakurai H. , Spokes K. , Birchmeier C. , Drummond I. , Nigam S. , Cantley L. G.
Published: December 2017

The growth factor/receptor combination of hepatocyte growth factor (HGF)/c-met has been postulated to be critical for mesenchymal-to-epithelial conversion and tubule formation in the developing kidney. We therefore isolated and immortalized cells from embryonic kidneys of met -/- transgenic mice to determine whether these cells were epithelial and able to chemotax and form tubules in vitro. The cells were immortalized with retrovirus expressing human papillomavirus 16 (HPV 16) E6/E7 genes. Two rapidly dividing clones were isolated and found to express the epithelial cell markers cytokeratin, zonula occludens-1, and E-cadherin but not to express the fibroblast marker vimentin. The met -/- cells were able to chemotax in response to epidermal growth factor and transforming growth factor-alpha (TGF-alpha) and form tubules in vitro in response to TGF-alpha but not HGF. These experiments suggest that the HGF/c-met axis is not essential for epithelial cell development in the embryonic kidney and demonstrate that other growth factors are capable of supporting early tubulogenesis.
Nitric oxide regulates HCO3- and Na+ transport by a cGMP-mediated mechanism in the kidney proximal tubule
Authors: Wang T.
Published: December 2017

The effects of nitric oxide (NO) on blood pressure and renal hemodynamics are well established, but those of NO on renal tubule HCO3- and Na+ transport are not fully understood. In this study, we combined renal clearance and in situ microperfusion techniques to investigate the effects of NO on the renal excretion of Na (FE(Na%)) and the rates of renal tubule absorption of fluid (J(V)) and bicarbonate (J(HCO3)) in the rat kidney. Administration of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 6 mg/kg iv bolus) did not change mean blood pressure and glomerular filtration rate significantly. However, L-NAME significantly increased urine flow rate and FE(Na%), and these effects were maintained over a 60-min period. Addition of L-NAME markedly decreased both J(V) and J(HCO3) in the proximal tubule. In contrast, addition of 1 microM sodium nitroprusside (SNP) or S-nitroso-N-acetylpenicillamine (SNAP) significantly increased both J(V) and J(HCO3). Similar stimulation was also observed when 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 microM) was added to the luminal perfusate. The stimulatory effects of SNP and 8-BrcGMP on J(V) and J(HCO3) were not additive. The increments in J(V) and J(HCO3) due to SNP were abolished by the Na+/H+ exchange blocker ethylisopropylamiloride and the guanylate cyclase inhibitor methylene blue. These results indicate that NO stimulates proximal tubule Na+ and HCO3- transport through a cGMP-linked pathway in the kidney proximal tubule.
Targeting deletion of angiotensin type 1B receptor gene in the mouse
Authors: Chen X. , Li W. , Yoshida H. , Tsuchida S. , Nishimura H. , Takemoto F. , Okubo S. , Fogo A. , Matsusaka T. , Ichikawa I.
Published: December 2017

We null mutated the mouse angiotensin type 1B (AT1B) receptor gene (Agtr1b) by gene targeting. To identify the specific cell types carrying high Agtr1b gene transcriptional activities, the AT1B coding exon was replaced with a reporter gene, lacZ. In 6- to 8-wk-old Agtr1b -/- mice, high AT1B transcriptional activity was observed in adrenal zona glomerulosa cells and the testis, including mature and immature spermatic cells, whereas low activity was detected homogeneously in anterior pituitary cells and choroidal plexus vessel walls. A similar pattern was observed in Agtr1b +/- mice with less intensity. Microscopically, the anterior pituitary, heart, adrenal, zona glomerulosa, kidney, and the testis of Agtr1b -/- mice were intact and were indistinguishable from those of Agtr1b +/+ mice. Systemic blood pressure was comparable in Agtr1b -/- and Agtr1b +/+ mice. Moreover, plasma aldosterone level was comparable between the two mouse groups. No compensatory enhancement of AT1A mRNA was found in the kidney and adrenal gland of Agtr1b -/- mice. The observed absence of the abnormal phenotypes in Agtr1b -/- mice, which have been described for homozygous angiotensinogen null mutant mice, indicates that 1) AT1A receptors can take over the role of AT1B receptors in Agtr1b -/- mice or 2) functionally significant non-AT1, non-AT2 receptor(s) may exist for the action of angiotensin.
Chloride is essential for contraction of afferent arterioles after agonists and potassium
Authors: Jensen B. L. , Ellekvist P. , Skott O.
Published: December 2017

A depolarizing chloride efflux has been suggested to activate voltage-dependent calcium channels in renal afferent arteriolar smooth muscle cells in response to vasoconstrictors. To test this proposal, rabbit afferent arterioles were microperfused, and the contractile dose responses to norepinephrine, angiotensin II (ANG II), and potassium were measured after chloride depletion and compared with controls. Chloride depletion did not change arteriolar diameters, but the response to norepinephrine was markedly reduced when chloride was substituted with gluconate (n = 6) or isethionate (n = 6). Reintroduction of chloride fully restored the sensitivity to norepinephrine. Contractions after ANG II and potassium were totally abolished in the absence of chloride (n = 6). In additional experiments (n = 7), the arteriolar contraction to 100 mM potassium was abolished only 1 min after removal of extracellular chloride. We conclude that norepinephrine and ANG II use different mechanisms for contraction and that extracellular chloride is essential for contraction in afferent arterioles after activation of voltage-dependent calcium channels. We suggest that a chloride influx pathway is activated concomitantly with the voltage-dependent calcium channel to allow chloride influx to compensate for the cation influx.
Angiotensin I-converting enzyme activity in tubular fluid along the rat nephron
Authors: Casarini D. E. , Boim M. A. , Stella R. C. , Krieger-Azzolini M. H. , Krieger J. E. , Schor N.
Published: December 2017

The activity of angiotensin I-converting enzyme (ACE) was determined in tubular fluid collected from several portions of the rat nephron and urine and in total and efferent arteriolar blood using hippuryl-L-His-Leu as substrate. ACE activity decreased 30% from the pre- to the postglomerular arterioles (P < 0.001), suggesting a role of the glomerulus in ACE clearance. The enzyme activity was found to be present throughout the rat nephron. However, the highest activities were found in the proximal tubule and urine (0.692 +/- 0.007 and 1.05 +/- 0.015 pmol x microl(-1) x min(-1), respectively). Compared with other segments, ACE activity decreased from the initial portion of the proximal tubule to the distal nephron and increased again in the urine. Along the proximal tubule, ACE was secreted and degraded and/or reabsorbed and then secreted again into the collecting duct; no ACE activity was found in the late distal tubule, but a high level was detected in the urine, indicating a potential physiological role in the inactivation of the kinins formed by kallikrein beyond the connecting tubules. Moreover, the possible role of mesangial cells (MC) in the decrease of intraglomerular ACE was also evaluated. The analysis of ACE gene showed that MC in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on MC function.
Localization of cyclooxygenase-1 and -2 in adult and fetal human kidney: implication for renal function
Authors: Komhoff M. , Grone H. J. , Klein T. , Seyberth H. W. , Nusing R. M.
Published: December 2017

To gain insight into the roles of cyclooxygenase (COX)-1 and -2 in human kidney, we analyzed their expressions and localization in adult and fetal normal kidney. Immunohistology showed expression of COX-1 in collecting duct cells, interstitial cells, endothelial cells, and smooth muscle cells of pre- and postglomerular vessels. Expression of COX-2 immunoreactive protein could be localized to endothelial and smooth muscle cells of arteries and veins and intraglomerularly in podocytes. In contrast to the rat, COX isoforms were not detected in the macula densa. These data were confirmed by in situ mRNA analysis using digoxigenin-labeled riboprobes. In fetal kidney, COX-1 was primarily expressed in podocytes and collecting duct cells. Expression levels of COX-1 in both cell types increased markedly from subcapsular to juxtamedullary cortex. Glomerular staining of COX-2 was detectable in podocytes only at the endstage of renal development. In summary, the localization of COX-2 suggests that this enzyme may be primarily involved in the regulation of renal perfusion and glomerular hemodynamics. The expression of COX-1 in podocytes of the fetal kidney and its absence in adult glomeruli suggests that this isoform might be involved in glomerulogenesis.
Receptor-mediated endocytosis of immunoglobulin light chains by renal proximal tubule cells
Authors: Batuman V. , Guan S.
Published: December 2017

We examined the binding, endocytosis, and degradation of immunoglobulin light chains by primary cultures from rat renal kidneys and immortalized human proximal tubule cells. Both the association and dissociation of light chain were rapid and plateaued within 30 min at 4 degrees C. Up to 10(-3) M bovine serum albumin did not inhibit light chain binding to cells. Internalization studies with 125I-labeled kappa- and lambda-light chains by cells using the acid wash technique showed that up to 80% of total cell-associated binding at equilibrium (30 min) is rapidly internalized at 22 degrees C. Comparison of binding and internalization of light chains with transferrin, a ligand known to undergo receptor-mediated endocytosis, showed that both ligands displayed saturable kinetics. In contrast, endocytosis of sucrose, a marker for fluid-phase endocytosis, was unsaturable and nearly 200-fold less efficient than light chain internalization. Scatchard analysis of binding experiments done at 4 degrees C with trace 125I-labeled lambda-light chain in presence of 0 to 3.0 x 10(-3) M cold light chain revealed a single class of binding sites with a dissociation constant of 5.0 +/- 0.8 x 10(-5) and a maximal binding capacity of 1.6 +/- 0.3 x 10(-9) mol/mg cell protein. Hypertonic medium, a maneuver which interferes with the formation of the clathrin lattice, reduced endocytosis of light chain significantly but did not affect endocytosis of sucrose. Chloroquine and bafilomycin A, agents that interfere with vesicular acidification, also significantly suppressed light chain endocytosis. Using acid precipitation method, we observed that endocytosis of 125I-labeled lambda-light chain results in degradation by the rat renal proximal tubule cells. Degradation was maximum at 37 degrees C, significantly reduced at 22 degrees C, and absent at 4 degrees C. Excess light chain inhibited degradation of radiolabel, whereas excess albumin had no effect. These studies document the presence of binding sites for light chains on proximal tubule cells that mediate endocytosis of light chains by proximal tubule cells. The present data suggest that receptor-mediated endocytosis of light chains leads to delivery of this ligand to degradative sites through acidified vesicles.
Biosynthesis and homeostatic roles of nitric oxide in the normal kidney
Authors: Kone B. C. , Baylis C.
Published: December 2017

Nitric oxide (NO) is an important molecular mediator of numerous physiological processes in virtually every organ. In the kidney, NO plays prominent roles in the homeostatic regulation of glomerular, vascular, and tubular function. Differential expression and regulation of the NO synthase (NOS) gene family contribute to this diversity of action. This review explores recent advances in the molecular and cell biology of the NOS isoforms and relates these findings to functions of NO in the control of normal renal hemodynamics, the glomerular microcirculation, and renal salt excretion. Newly recognized molecular diversity of the NOS gene products, factors governing NOS isozyme gene expression and catalytic activity, and the intrarenal distribution of the NOS isoforms are examined. Physiological data regarding the complex roles of NO in the control of renal hemodynamics and the glomerular microcirculation are analyzed, and the effects of chronic NOS inhibition on glomerular function and structure are presented. The contributions of NO to renal salt excretion as well as functional and molecular biological evidence for adaptive changes in NOS isoform expression during variations in dietary salt balance are discussed. Current investigative challenges and goals for future research of renal NO biology are presented.
Evidence that aquaporin-1 mediates NaCl-induced water flux across descending vasa recta
Authors: Pallone T. L. , Kishore B. K. , Nielsen S. , Agre P. , Knepper M. A.
Published: December 2017

Outer medullary descending vasa recta (OMDVR) were perfused in vitro, and volume efflux was measured by driving water movement with transmural gradients of NaCl or albumin. Consistent with mediation by water channels, p-chloromercuribenzenesulfonic acid (pCMBS) markedly inhibited volume flux induced by NaCl. Dithiothreitol reversed the inhibition, pCMBS did not significantly alter water flux induced by albumin. Osmotic water permeability (Pf) of the pCMBS-sensitive pathway of glutaraldehyde-fixed and nonfixed OMDVR was 1,102 +/- 449 and 1,257 +/- 718 microns/s (means +/- SD), respectively. pCMBS reduced Pf to near zero, whereas diffusional water permeability in the same vessels was only slightly inhibited. Immunoreactive aquaporin-1 (AQP1) measured by enzyme-linked immunosorbent assay in collagenase-treated and untreated OMDVR was 5.2 +/- 1.0 and 4.2 +/- 0.4 fmol/mm, respectively, values that account well for the experimental Pf. We conclude that OMDVR water flux driven by NaCl gradients is most likely mediated by the AQP1 water channel and that NaCl and urea gradients drive water efflux in vivo by this route.