bluuebunny/crossref_metadata_2025 huggingface dataset:
Betaine transport in rabbit renal brush-border membrane vesicles
Authors: Wunz T. M. , Wright S. H.
Published: December 2017

Transport of the organic osmolyte betaine was characterized in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex. Inwardly directed gradients of either Na+ or H+ supported concentrative uptake in a manner consistent with the presence of parallel Na(+)-betaine and H(+)-betaine cotransport processes. Concentrative uptake occurred in the presence of membrane potential alone, indicating that betaine transport is electrogenic. Accumulation of betaine was not dependent on chloride in the medium. Whereas L-proline inhibited both the H(+)- and Na(+)-sensitive components of betaine transport, glycine blocked the H(+)-sensitive pathway and had little effect on Na(+)-sensitive betaine transport. Both pathways were adequately described by Michaelis-Menten kinetics. Under Na(+)-gradient conditions (pH equilibrium), the maximal rate of total betaine transport (Jmax) = 50.8 +/- 13.3 nmol.mg-1.min-1 and the concentration of total betaine producing half-maximal uptake (Kt) = 4.1 +/- 0.5 mM. Under H(+)-gradient conditions (Na+ free), Jmax = 102.5 +/- 10.5 nmol.mg-1.min-1 and Kt = 2.8 +/- 0.3 mM. Imposition of both Na+ and H+ gradients increased Jmax (142 +/- 25.5 nmol.mg-1.min-1) to a level significantly greater than that noted in the presence of a Na+ gradient alone. We conclude that betaine transport in renal BBMV involves two distinct transport pathways that are differentiated on the basis of sensitivity to either Na+ or H+ and by their specificity to proline and glycine.
Assessment of changes in intrarenal blood flow in conscious rats using laser-Doppler flowmetry
Authors: Lu S. , Mattson D. L. , Roman R. J. , Becker C. G. , Cowley A. W.
Published: December 2017

The present study was designed to develop, for the first time, a method that allows long-term repeated measurements of renal cortical blood flow (CBF) and medullary blood flow (MBF) in conscious unanesthetized rats. The use of fiber-optic probes (0.5 mm diam) for the chronic measurement of renal CBF and MBF was evaluated. Basal renal cortical and medullary laser-Doppler flow (LDF) signals and the responses to intravenous bolus injections of angiotensin II (ANG II, 12.5 ng) were determined every other day for 11 days in conscious Sprague-Dawley rats (n = 9). A recovery period of 5-7 days after surgery was required before stable signals were obtained from the implanted probes. Thereafter, the fiber-optic probes gave reproducible laser-Doppler measurements of CBF and MBF for 11 days. The CBF and MBF responses to intravenous bolus injections of ANG II (12.5 ng) were also constant during this period. Chronic implantation of the fiber-optic probes caused minimal tissue damage and did not significantly alter urine concentrating ability or renal function. These findings suggest that LDF technique with chronically implanted optical fibers provides a new tool for the continuous long-term monitoring of regional blood flow in the kidney of conscious rats.
Transport of organic anion in the OK kidney epithelial cell line
Authors: Hori R. , Okamura M. , Takayama A. , Hirozane K. , Takano M.
Published: December 2017

Transport of an organic anion, p-aminohippurate (PAH), was investigated in an established cell line derived from the American opossum kidney (OK). Transepithelial transport and accumulation of PAH were measured in OK cells cultured on permeable supports. Specifically mediated transcellular transport of PAH across OK cell monolayers occurred only from basal to apical side, and the accumulation of PAH in the cells was much larger from basal side than from apical side. Both the transcellular transport of PAH from basal to apical side and the accumulation from basal side were temperature dependent, saturable, and inhibited by organic anions such as probenecid but not by organic cations. In contrast, such a specific transport of PAH was not observed in LLC-PK1, another epithelial cell line derived from pig kidney. These findings suggest that OK cells can transport PAH by the organic anion transport system unidirectionally from basal to apical side across the cell monolayers and that OK cells might be a useful model for the study of organic anion secretion in the renal proximal tubules.
Renal renin, angiotensinogen, and ANG I-converting-enzyme gene expression: influence of dietary protein
Authors: Martinez-Maldonado M. , Benabe J. E. , Wilcox J. N. , Wang S. , Luo C.
Published: December 2017

Low-protein (LP) feeding (6%) of rats results in renal hemodynamic changes that are abolished by converting enzyme and nonpeptide AT1 inhibitors, suggesting a role for intrarenal angiotensin II (ANG II). Dietary protein is a stimulus for the expression of renal renin mRNA in intact and partially nephrectomized rats. In the present study, LP increased renal renin immunoreactivity and renin mRNA as assessed by in situ hybridization and Northern blot analysis; whole kidney expression of angiotensinogen mRNA was unaltered. Whole kidney, cortical, and medullary ANG I-converting-enzyme (ACE) mRNA was also increased in LP vs. normal protein. The changes occurred despite a reduced protein synthetic rate (RNA-to-DNA ratio) in the kidney of LP, which did not change expression of renal glyceraldehyde-3-phosphate dehydrogenase mRNA. These studies show for the first time that LP diet increases the expression of renal renin and ACE mRNA in intact rats; these may be responsible for the renal hemodynamic changes of LP.
Role of cardiac innervation in atrial natriuretic peptide secretion in transplanted heart recipients
Authors: Geny B. , Piquard F. , Follenius M. , Mettauer B. , Schaefer A. , Canguilhem B. , Eisenmann B. , Haberey P.
Published: December 2017

To investigate whether cardiac innervation modulates atrial natriuretic peptide (ANP) secretion, we performed acute volume expansion on eight normal and eight matched (age, weight, and total blood volume) transplanted denervated heart patients (Htx), while monitoring fluid-regulating hormone, systemic blood pressure, and echocardiographic atrial area changes. At rest, plasma ANP and guanosine 3',5'-cyclic monophosphate (cGMP) were lower in control subjects than in Htx (45 +/- 16 vs. 103 +/- 35 pg/l and 0.9 +/- 0.3 vs. 3.5 +/- 1.4 pM, respectively; P < 0.001). Plasma active renin, aldosterone, and catecholamines did not differ significantly in the two populations, whereas arginine vasopressin and cortisol were higher in controls (P < 0.01 and P < 0.005). Although volume expansion (+15%) and atrial stretch were similar in the two groups, plasma ANP and cGMP increased significantly only in the Htx group (103 +/- 35 to 189 +/- 69 pg/l and 3.5 +/- 1.4 to 5.8 +/- 1.4 pM, respectively; P < 0.001). The decrease observed for the other hormones was not significant except for arginine vasopressin and cortisol (P < 0.05 and P < 0.001) in the control group. These results support the hypothesis of an inhibitory role of cardiac innervation in biologically active ANP secretion in humans, at rest and after acute volume expansion.
Role of muscarinic receptors in renal response to acetylcholine
Authors: Yun J. C. , Oriji G. , Gill J. R. , Coleman B. R. , Peters J. , Keiser H.
Published: December 2017

Renal arterial infusion of acetylcholine (ACh) (40 micrograms/min) in control dogs produced an ipsilateral increase in renal plasma flow (RPF) and in sodium excretion (UNaV) without a change in glomerular filtration rate (GFR). The increase in RPF and UNaV was maintained during the infusion of ACh. In indomethacin (Indo)-treated dogs (5 mg/kg) ACh produced a transient rise in RPF and UNaV, followed by a progressive decline in RPF and UNaV. The profound renal vasoconstriction was accompanied by a decline in GFR. To determine the role of the muscarinic receptor in the renal vasodilation and in vasoconstriction produced by ACh in Indo-treated dogs, atropine at 6, 60, and 600 micrograms/min was infused into the renal artery before and during the infusion of ACh. In Indo-treated dogs, all dosages of atropine prevented renal vasoconstriction by ACh. Renal arterial infusion of atropine at 600 micrograms/min completely inhibited the renal vasodilation produced by ACh. Atropine infused at 60 micrograms/min partially inhibited, whereas 6 micrograms/min atropine failed to inhibit, the renal vasodilation produced by ACh. Our data suggest that the renal vasodilator and vasoconstrictor effects of ACh in Indo-treated dogs are mediated by two separate types of muscarinic receptors.
Activation of Na/H exchanger in mesangial cells is associated with translocation of PKC isoforms
Authors: Saxena R. , Saksa B. A. , Fields A. P. , Ganz M. B.
Published: December 2017

We investigated the potential coupling of the Na/H exchanger to protein kinase C (PKC) activation. Mesangial cells (MC), passage 3-8, were exposed to either serotonin (5-HT), arginine vasopressin (AVP), or fibroblast growth factor (FGF). We assessed the effect of these agonists on recovery from an acid load (NH4+/NH3) in the nominal absence of CO2/HCO3. 5-HT, AVP, and FGF significantly enhanced the rate of recovery from 24.2 +/- 4.5 x 10(-4) intracellular pH units/s to 67.7 +/- 5.7, 70.5 +/- 5.1, and 55.7 +/- 6.8, respectively (n > 6, P < 0.0001). The increase in activity was abolished by ethylisopropylamiloride and removal of extracellular Na+, thereby suggesting that activation of Na/H exchanger activity was responsible for enhanced recovery by 5-HT, AVP, and FGF. MC were then pretreated with phorbol ester [phorbol 12-myristate 13-acetate (PMA); 10 microM for 40 h] and acid loaded. 5-HT and AVP did not enhance recovery of PMA-pretreated cells (23.3 +/- 6.8 and 24.9 +/- 4.7, n > 4, not significant), whereas FGF stimulated activity (48.2 +/- 5.5, n > 4, P < 0.001). Similar results were found when MC were pretreated with the PKC antagonist, sphingosine. Specific antibodies were raised against alpha-, beta I-, beta II-, and gamma-isoforms of PKC. We observed that MC express the alpha-, gamma-, and beta I-isoforms, but not the beta II-isoform of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)
Hormonal regulation of inner medullary collecting duct sodium transport
Authors: Zeidel M. L.
Published: December 2017

The inner medullary collecting duct (IMCD) is the final arbiter of renal Na+ excretion, and Na+ transport in this segment is controlled by a wide variety of hormones and renal autacoids. This review examines the mechanisms of IMCD Na+ transport and its regulation using results obtained from micropuncture and microcatheterization studies in the intact animal, as well as data from isolated perfused tubules, freshly prepared cell suspensions, and cultured IMCD cells. Where appropriate, results from closely related tissues such as the cortical collecting duct and model urinary epithelia are examined. Na+ reabsorption in this segment occurs predominantly via apical amiloride-sensitive Na+ channels and basolateral Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase). Although there is some evidence for the activities of other transporters such as Na(+)-K(+)-2Cl- and Na-Cl cotransporters and Na+/H+ exchanger, their role in Na+ homeostasis remains undefined. Mineralocorticoids augment the activities of both apical Na+ channels and basolateral Na(+)-K(+)-ATPase by a variety of complex mechanisms. Prostaglandin E2 inhibits Na(+)-K(+)-ATPase and appears to mediate the actions of several peptide hormones, including endothelin, interleukin-1, and atrial natriuretic peptide [ANP-(31-67)]. Several peptides in the ANP family [ANP-(99-126), urodilatin, and brain natriuretic peptide] bind to guanylate cyclase-linked receptors, leading to inhibition of apical Na+ channel function. These mechanisms of regulation of IMCD Na+ transport likely play important roles in total body Na+ balance in health and disease.
Proliferative synergy of ANG II and EGF in porcine aortic vascular smooth muscle cells
Authors: Bagby S. P. , Kirk E. A. , Mitchell L. H. , O'Reilly M. M. , Holden W. E. , Stenberg P. E. , Bakke A. C.
Published: December 2017

To test growth effects of angiotensin II (ANG II) in porcine vascular smooth muscle cells (VSMC) and potential ANG II synergy with epidermal growth factor (EGF), we exposed subconfluent, near-quiescent porcine aortic VSMC to ANG II, EGF, or ANG II + EGF (each 10(-9) M) in Dulbecco's modified Eagle's-Ham's F-12 medium with insulin + 0.4% fetal calf serum (FCS) selected for minimal ANG II-degrading capacity. Cell number and DNA and protein synthesis (by [3H]-thymidine and [35S]methionine incorporation, respectively) were determined serially over 1-6 days. ANG II alone induced an early 20% increase and then a plateau in cell number over the 0.4% FCS control (P < 0.01; n = 8), thus without sustained increase in proliferation rate. Yet ANG II alone did not increase fractional DNA or protein synthesis (each as cpm/10(3) cells) and, by flow cytometry, reduced S phase fraction without increase in cell size. EGF alone induced brisk DNA synthesis yet minimal cell division over days 0-4, thus late-cycle arrest. ANG II + EGF, despite no increase in fractional DNA or protein synthesis rates over EGF alone, induced significant indomethacin-resistant dose-dependent (P < 0.001) increase in cell proliferation rate over EGF alone with a median effective dose of 5 x 10(-10) M ANG II, thus proliferative synergy. We propose that 1) ANG II induces a subpopulation of cells arrested in or beyond S phase to proceed through mitosis but does not influence G1 traversal or S phase entry and 2) ANG II + EGF achieve proliferative synergy by complementary actions at sequential cell cycle loci, with EGF supporting progression from G0/G1 to S phase and ANG II inducing completion of mitosis by cells already in or beyond S phase ("late-cycle completion").
Glomerular angiotensin II receptor subtypes during development of rat kidney
Authors: Ciuffo G. M. , Viswanathan M. , Seltzer A. M. , Tsutsumi K. , Saavedra J. M.
Published: December 2017

We used quantitative autoradiography to investigate distribution of angiotensin II (ANG II) receptor subtypes during development of the kidney in the rat. In fetal, newborn, and 3-day-old rats, immature glomeruli in the form of comma and S-shaped bodies, located in the nephrogenic zone of the renal cortex, expressed only the angiotensin AT2 receptor subtype. Conversely, the juxtamedullary glomeruli, in more advanced developmental stages, expressed only the AT1 subtype. Similarly, maturing and fully developed glomeruli, present in 1-, 2-, and 8-wk-old rats, expressed only AT1 receptors. In the kidney medulla, there was a similar change in ANG II receptor subtype expression, with the AT2 subtype expressed earlier and the AT1 subtype later during development. Our results demonstrate a selective expression of ANG II receptor subtypes during kidney development. We have found glomerular and medullary AT1 receptors only at developmental stages when kidney function has matured. Conversely, AT2 receptors are expressed only in immature structures, suggesting that they may have a role during kidney organogenesis.
Influence of endothelium-derived relaxing factor on renal microvessels and pressure-dependent vasodilation
Authors: Hoffend J. , Cavarape A. , Endlich K. , Steinhausen M.
Published: December 2017

The influence of endothelium-derived relaxing factor (EDRF) on renal microvessels and autoregulation was visualized in vivo, in the split hydronephrotic kidney of rats. EDRF synthesis was inhibited by local administration of 10(-5) M NG-nitro-L-arginine methyl ester (L-NAME). Diameters of arcuate arteries decreased by 17%. In cortical vessels efferent arterioles constricted more (13-16%) than interlobular arteries and afferent arterioles (7-12%). Cortical glomerular blood flow (GBF) decreased by 46% after L-NAME. A similar behavior of blood flow and vascular diameters was also observed in juxtamedullary (JM) arterioles. The responses to acetylcholine but not to sodium nitroprusside were attenuated after L-NAME. After local administration of L-arginine (10(-3) M) diameters of all vessels and GBF increased, vascular responses to L-NAME were blunted. Stepwise reduction of renal perfusion pressure revealed that autoregulation was preserved in cortical vessels after L-NAME. In JM arterioles, which do not autoregulate in female Wistar rats, autoregulation of GBF was enhanced after L-NAME. These data suggest that tonic formation of EDRF influences basal renal hemodynamics to a considerable extent. EDRF may also impair autoregulation of JM glomeruli without disturbing autoregulation of cortical glomeruli.
Quantitation and localization of laminin A, B1, and B2 chain RNA transcripts in developing kidney
Authors: Vanden Heuvel G. B. , Abrahamson D. R.
Published: December 2017

To quantitate expression of laminin A, B1, and B2 chains during kidney development, we prepared riboprobes of similar size (318-365 nucleotides) and carried out scanning densitometry of Northern blots from newborn mouse and rat kidneys. Much higher expression of B1 than A or B2 mRNAs was observed with an A-to-B1-to-B2 ratio of approximately 1:8:4 in mice and approximately 1:12:1 in rats. In addition to the 9.5-kb laminin A chain transcript, we also observed specific hybridization of the A chain probe to approximately 6-kb band in both newborn mice and rats. To localize these mRNAs, newborn rat kidneys were sieved to obtain separate glomerular and tubule fractions. Northern blots from these samples showed that most of the B1, B2, and 9.5-kb laminin A chain mRNAs were in glomerular fractions, whereas tubules contained more of the 6-kb A chain band. Analyses of poly(A)+ mRNA from newborn and adult kidneys showed severalfold decreases in adult RNA encoding the three laminin chains and the alpha 1-chain type IV collagen. However, only a 20% decline from newborn levels was seen for heparan sulfate proteoglycan (HSPG) core protein message. These quantitative results provide additional evidence that different laminin isoforms are present in separate kidney basement membranes and that HSPG core protein synthesis remains at relatively high levels in adult kidneys.
Renal mouse proximal tubular cells are more susceptible than MDCK cells to chemical anoxia
Authors: Sheridan A. M. , Schwartz J. H. , Kroshian V. M. , Tercyak A. M. , Laraia J. , Masino S. , Lieberthal W.
Published: December 2017

To elucidate the mechanisms responsible for the resistance of continuous cell lines to anoxic injury, we have compared the effects of ATP depletion induced by chemical anoxia on primary cultures of mouse proximal tubular (MPT) cells and on Madin-Darby canine kidney (MDCK) cells. Inhibition of ATP production by cyanide and 2-deoxyglucose (CN+DOG) in the absence of dextrose reduced cell ATP content to < 5% of control values in MPT cells and caused progressive deterioration in mitochondrial function as well as loss of cell viability in these cells. Cell free fatty acid (FFA) content rose from 4.3 +/- 0.9 to 23.7 +/- 2.0 micrograms/mg of total lipid weight after 4 h of CN + DOG (P < 0.05). The mitochondrial injury and cell death induced by CN + DOG in MPT cells was ameliorated by the addition of fatty acid-free bovine albumin to the cell medium, which reduced cell FFA content during chemical anoxia from 25.0 +/- 3.0 to 10.4 +/- 2.0 micrograms/mg (P < 0.05). The phospholipase A2 (PLA2) inhibitor, mepacrine, also resulted in functional protection and reduction of cell FFA content from 20.2 +/- 2.3 to 15.9 +/- 1.7 micrograms/mg (P < 0.05). These data suggest a role for phospholipase activation and accumulation of toxic lipid metabolites in the pathophysiology of MPT cell injury. We then compared cell injury induced by CN + DOG in MPT and MDCK cells. Despite comparable reduction in cell ATP content in the two cell types, injury was far more severe in MPT than MDCK cells.(ABSTRACT TRUNCATED AT 250 WORDS)
PDGF-induced Egr-1 expression in rat mesangial cells is mediated through upstream serum response elements
Authors: Rupprecht H. D. , Sukhatme V. P. , Lacy J. , Sterzel R. B. , Coleman D. L.
Published: December 2017

Platelet-derived growth factor (PDGF) has been implicated in the process of mesangial cell (MC) proliferation in vitro and in vivo. To investigate early changes in gene expression that couple biochemical events with changes in phenotype in PDGF-stimulated cultured MC, we studied expression of the early growth response gene 1 (Egr-1), a member of the family of immediate early genes. Our findings show that protein tyrosine phosphorylation is required for induction of Egr-1 mRNA and proliferation by PDGF in MC. Nuclear run-off assays show that Egr-1 induction occurs at the transcriptional level. An 11.3-fold increase in Egr-1 transcription rate was observed as early as 5 min after PDGF stimulation of MC. Promoter deletion analysis revealed that the region critical for Egr-1 inducibility by PDGF contains serum response element (SRE) consensus sequences. Sequential deletion of the Egr-1 SREs led to a stepwise drop in promoter activity, suggesting that PDGF induces Egr-1 transcription through SREs in the Egr-1 promoter region. Interestingly, electrophoretic mobility shift assays, with an Egr-1 SRE as probe, demonstrate that protein-SRE complexes of differing size undergo modest quantitative changes following PDGF stimulation. These data in MC suggest that the upstream SREs mediate the transcriptional induction of Egr-1 by PDGF.
The urease inhibitor acetohydroxamic acid is transported by the urea pathway in rat terminal IMCD
Authors: Star R. A. , Gillin A. D. , Parikh V. J. , Sands J. M.
Published: December 2017

Acetohydroxamic acid (AHA), a urea analogue, is used clinically to dissolve struvite stones because it inhibits the urease produced by Proteus mirabilis. To be effective, the concentration of AHA must be high in the collecting duct system and final urine. Because AHA is structurally similar to urea, we investigated whether AHA is transported by the urea carrier found in the terminal inner medullary collecting duct (IMCD) and the erythrocyte. We examined AHA transport under four conditions known to affect urea movement across the terminal IMCD, i.e., stimulation by vasopressin (AVP) and hyperosmolality, and inhibition by phloretin and urea analogues. The AHA permeability was determined with a 10 mM bath-to-lumen AHA gradient. AHA was measured by ultramicrocolorimetry. Addition of 1 nM AVP to the bath increased the AHA permeability of the perfused terminal IMCD. Increasing perfusate and bath osmolality from 290 to 690 mosmol/kgH2O (by adding NaCl) also increased tubule permeability to AHA. Addition of either 0.25 mM phloretin to the bath or 200 mM thiourea to the lumen reversibly inhibited the AVP-stimulated AHA permeability. AHA-induced osmotic lysis of erythrocytes was inhibited by phloretin or thionicotinamide; AHA inhibited the osmotic lysis induced by the urea analogue acetamide. Thus, in the rat terminal IMCD, both urea and AHA transport are stimulated by AVP and hyperosmolality, and both are inhibited by phloretin and thiourea. In erythrocytes, both urea and AHA transport are inhibited by phloretin or thionicotinamide. Thus AHA is transported by the urea carrier in the terminal IMCD and erythrocyte.(ABSTRACT TRUNCATED AT 250 WORDS)
Different mechanisms of renal Na-K-ATPase regulation by protein kinases in proximal and distal nephron
Authors: Satoh T. , Cohen H. T. , Katz A. I.
Published: December 2017

We recently reported a novel intracellular mechanism of Na-K-adenosinetriphosphatase (Na-K-ATPase) regulation in the cortical collecting duct (CCD) by agents that increase cell adenosine 3',5'-cyclic monophosphate (cAMP), which involves stimulation of protein kinase A (PKA) and phospholipase A2 (PLA2). We now determined whether this mechanism also operates in other nephron segments. In the medullary thick ascending limb (MTAL) dopamine, the DA1 agonist fenoldopam, forskolin, or dibutyryl-cAMP inhibited Na-K-ATPase activity, similar to results in CCD. In both segments this effect was blocked by 20-residue inhibitory peptide (IP20), a peptide inhibitor of PKA, but not by staurosporine, a protein kinase C (PKC) inhibitor. PKC activators phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, and 1,2-myristate 13-acetate, phorbol 12,13-dibutyrate, and 1,2-dioctanoylglycerol had no effect on Na-K pump activity in either CCD or MTAL. In contrast, all three PKC activators inhibited pump activity in the proximal convoluted tubule (PCT), an effect reproduced only by dopamine or by parathyroid hormone [PTH-(1-34)]. In PCT the pump inhibition by dopamine or PTH-(1-34) was abolished by staurosporine but not by IP20. The PLA2 inhibitor mepacrine prevented the effect of all agents, and arachidonic acid produced a dose-dependent pump inhibition in each of the three segments studied. We conclude that intracellular mechanisms of Na-K-ATPase regulation differ along the nephron, as they involve activation of PKA in CCD and MTAL and of PKC in PCT. These two pathways probably share a common mechanism in stimulating PLA2, arachidonic acid release, and production of eicosanoids in both the proximal and distal nephron.
Oxytocin affects apical sodium conductance in rabbit cortical collecting duct
Authors: Inoue T. , Naruse M. , Nakayama M. , Kurokawa K. , Sato T.
Published: December 2017

The physiological role of oxytocin (OT) in the kidney is still unclear, although autoradiographic data have shown the existence of OT receptors in the rat kidney. We examined the effect of OT in the microperfused rabbit cortical collecting duct (CCD) by using conventional cable analysis and microscope photometry. On addition of 10(-9) M OT to the bath, the lumen-negative transepithelial voltage (VT) transiently increased and the transepithelial resistance (RT) and the fractional resistance of the apical membrane (FRA) (1st phase) both decreased. After this initial change, the lumen-negative VT gradually decreased below its baseline level and RT and FRA (second phase) both increased. These electrical changes were dose dependent and were prevented by the addition of 10(-5) M amiloride to the lumen. Although responses to OT were not prevented by 10(-9) M arginine vasopressin (AVP) or 10(-6) M of a V1-receptor antagonist (OPC-21268) or V2-receptor antagonist (OPC-31260), they were inhibited by the addition of the specific OT antagonist des-Gly-NH2-[d(CH2)3,Tyr(Me),Thr]OVT. Additional studies of intracellular free calcium ([Ca2+]i) revealed that 10(-8)-10(-6) M OT caused an increase in [Ca2+]i in CCD in a dose-dependent manner. Also, pretreatment with 2 x 10(-8) M bis-(aminophenoxy)ethane-tetraacetic acid-acetoxymethyl ester, an intracellular Ca2+ chelator, abolished the electrical and [Ca2+]i responses to OT. Pretreatment with 5 x 10(-4) M 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) partially prevented the electrical responses to OT, thus reducing the decrease in lumen-negative VT below its basal level and the increase in RT after the 1st phase. These data show that OT affects the apical Na+ conductance of collecting duct cells through OT receptors distinct from the AVP receptors and that the effect of OT may, at least in part, be brought about by a mechanism(s) dependent on the increase in [Ca2+]i and cAMP production.
Glutamate activation of neurons in CV-reactive areas of cat brain stem affects urinary bladder motility
Authors: Chen S. Y. , Wang S. D. , Cheng C. L. , Kuo J. S. , De Groat W. C. , Chai C. Y.
Published: December 2017

To investigate the interaction between cardiovascular (CV)-reactive areas in the brain stem and urinary bladder (UB) motility, 48 adult cats of either sex were anesthetized intraperitoneally with alpha-chloralose (40 mg/kg) and urethan (400 mg/kg). The changes of UB motility and systemic arterial blood pressure (SAP) were produced by microinjection of sodium glutamate (0.5 M, 100-200 nl) into the pressor, depressor, or vagobradycardiac areas of the brain stem. Stimulation of these CV-reactive areas increased or decreased UB motility. Areas that produced an increase in UB motility listed in decreasing order of effectiveness are locus ceruleus-parabrachial nucleus in the pons, dorsal medulla, dorsal motor nucleus of vagus, and ventrolateral medulla. Areas eliciting a decrease in UB motility listed in decreasing order are gigantocellular tegmental field, parvocellular reticular nucleus, and ambiguus nucleus. Stimulation of other pressor sites in medulla also increased UB motility. Activation of the paramedian reticular nucleus, which consistently produced depressor responses, and activation of raphe nuclei, which produced depressor or pressor responses, consistently decreased UB motility. The integrity of the vagus nerve was not essential for the UB response to brain stimulation. These findings indicate that neuronal mechanisms for controlling UB and CV functions coexist at many sites in the brain stem. At those sites that commonly produce changes in UB motility, the type of UB response (excitation or inhibition) was in the same direction as the change of SAP. However, at some sites responses were inverse. It is not known whether the responses of the UB and CV system are controlled by common or separate populations of neurons at these sites.
Transforming growth factor-beta is a renin secretagogue at picomolar concentrations
Authors: Antonipillai I. , Le T. H. , Soceneantu L. , Horton R.
Published: December 2017

Transforming growth factor-beta s (TGF-beta s), a family of peptides, have many actions including modulating cellular growth, differentiation, and influencing steroidogenesis. Because both TGF-beta and renin are present in renal juxtaglomerular cells, we have examined the effects of these peptides on renin secretion using static incubations of rat renal cortical slices. We report here an effect of both TGF-beta 1 and -beta 2 on renin secretion. At low concentrations, both TGF-beta 1 (4 x 10(-12) M) and -beta 2 (8 x 10(-12) M) stimulate basal renin secretion (control, 100 +/- 4%; TGF-beta 1, 123 +/- 4%; TGF-beta 2, 124 +/- 5%; both P < 0.02 compared with control). However, at higher concentrations (2 x 10(-10) M), both peptides do not alter basal renin release. Our previous studies show that both prostaglandins and lipoxygenase (LO) products of arachidonic acid play an important dual regulatory role in renin secretion; therefore, we have examined the effects of both cyclooxygenase (CO) and LO inhibition in TGF-beta action.(ABSTRACT TRUNCATED AT 250 WORDS)
Conservation of structure in ATP-depleted proximal tubules: role of calcium, polyphosphoinositides, and glycine
Authors: Garza-Quintero R. , Weinberg J. M. , Ortega-Lopez J. , Davis J. A. , Venkatachalam M. A.
Published: December 2017

Increases of intracellular free Ca2+ (Caf) may mediate phospholipid hydrolysis and disintegration in energy-compromised cells; on the other hand, glycine and related amino acids preserve structure. We have examined the effects of increased Caf on phospholipids and structure in ATP-depleted cells, as well as how these actions may be modified by glycine. Incubation of isolated proximal tubules with antimycin A led to ATP depletion, delayed increases of Caf to micromolar levels, polyphosphoinositide (PPI) hydrolysis by phospholipase C, and generalized disintegration of cell structure. Glycine inhibited PPI hydrolysis and preserved cell structure in entirety but did not apparently modify the Caf increases. When overwhelming increases of Caf were induced by the additional presence of a Ca2+ ionophore, glycine did not inhibit either the hydrolysis of PPI or disruption of mitochondria and microvilli. However, the cells remained integrated and unbroken. Incubation in low-Ca2+ medium prevented Caf increases, inhibited PPI hydrolysis, and preserved the structure of mitochondria and microvilli. Nevertheless, there was lethal damage by disintegration of all other membranes. This damage was prevented specifically and completely by glycine. Thus compartments of cells were shown to be differentially susceptible to injury from increased Caf or lack of glycine. Although damage by either factor occurs by distinct mechanisms, glycine also appears to have effects that suppress the deleterious effects of Ca2+ so long as Caf increases are not overwhelming. Our results also suggest that the PPI have a major structural role, which may be compromised by Caf increase during ATP depletion.
Luminal and basolateral uptake and receptor binding of IGF-I in rabbit renal proximal tubules
Authors: Flyvbjerg A. , Nielsen S. , Sheikh M. I. , Jacobsen C. , Orskov H. , Christensen E. I.
Published: December 2017

The aim of the present study was to quantify and compare the luminal and basolateral binding and uptake of 125I-labeled insulin-like growth factor I (IGF-I) by means of 1) isolated, perfused, proximal tubules combined with electron microscope autoradiography and 2) luminal and basolateral membrane vesicles from rabbit proximal tubules. 125I-IGF-I was added to isolated perfused proximal tubules for 30 min in concentrations of 1.6-3.9 micrograms/l to either the perfusate or the bath. The luminal and basolateral uptake in 30 min averaged 447 and 410 fg/mm, respectively. About 20% of the luminally absorbed IGF-I was digested. Addition of excess unlabeled IGF-I (10(-7) M) to the bath produced complete inhibition of the basolateral binding/uptake, whereas no inhibition of the luminal uptake was seen. Electron microscope autoradiography showed that IGF-I after luminal endocytic uptake to a large extent was transported into lysosomes. After basolateral exposure the major portion of the grains was found over the basolateral cell membrane; however, a significant amount was located over endocytic vacuoles and lysosomes in both apical and basal parts of the cells. In both luminal and basolateral membrane vesicles, single-class, high-affinity binding sites for IGF-I were found with dissociation constants of 6.3 and 5.7 nM, respectively. Specific binding capacities averaged 2.7 and 25.7 pmol IGF-I/mg protein in luminal and basolateral vesicles. The biochemical data suggest an asymmetric distribution of specific IGF-I receptors in the luminal and basolateral membranes, with a greater abundance of receptors in the latter. The extensive basolateral endocytic binding/uptake of IGF-I compared with that of the luminal in isolated perfused tubules differs considerably from the processing of other peptide hormones.
Adrenergic regulation of renin secretion and renal hemodynamics during deliberate hypotension in humans
Authors: Zayas V. M. , Blumenfeld J. D. , Bading B. , McDonald M. , James G. D. , Lin Y. F. , Sharrock N. E. , Sealey J. E. , Laragh J. H.
Published: December 2017

To assess the relative contributions of neural and nonneural stimuli of renin secretion, the effects of an alpha 1-agonist, phenylephrine (Phe), or a beta-agonist, epinephrine (Epi), on plasma renin activity (PRA), renal blood flow (RBF), and glomerular filtration rate (GFR) were compared during sympathetic blockade with epidural hypotensive anesthesia [mean arterial blood pressure (MAP) = 60 and 50 mmHg]. Controls (NaCl) received saline alone to maintain MAP at 50 mmHg. Epi increased PRA (ng.ml-1.h-1) from 0.9 +/- 0.6 to 3.0 +/- 1.5 at 60 mmHg MAP and 4.7 +/- 1.8 at 50 mmHg MAP, with associated decreases in RBF (-33 and -60%, respectively) and GFR (-27 and -53%, respectively). During hypotension with Phe and NaCl, PRA and RBF were unchanged from baseline but GFR decreased. Urinary Na secretion decreased comparably in all three groups. In conclusion, during sympathetic blockade with epidural anesthesia, marked reductions in both renal perfusion pressure and distal nephron Na delivery were insufficient to increase renin secretion. beta-Adrenergic stimulation (e.g., Epi) was required to increase PRA. Epi decreased RBF suggesting an angiotensin II-mediated effect.
22-Oxacalcitriol: dissection of 1,25(OH)2D3 receptor-mediated and Ca2+ entry-stimulating pathways
Authors: Farach-Carson M. C. , Abe J. , Nishii Y. , Khoury R. , Wright G. C. , Norman A. W.
Published: December 2017

22-Oxa-1,25-dihydroxyvitamin D3 (oxacalcitriol, or OCT) is a bioactive analogue of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] with lower calcemic activity than the parent compound. We investigated the ability of OCT to stimulate 1) genomic pathways mediated by nuclear receptors for 1,25(OH)2D3 versus 2) nongenomic pathways mediated by voltage-sensitive Ca2+ channels in growth phase rat osteosarcoma cells (ROS 17/2.8) and in chick intestine. Effects on nuclear receptor-mediated pathways were evaluated by measuring the ability of OCT to compete with [3H]1,25(OH)2D3 for soluble receptors. We also measured the ability of OCT to increase mRNA encoding osteoblast marker proteins osteopontin (OPN) and osteocalcin (OCN), which are both increased by 1,25(OH)2D3. Effects on Ca2+ entry into osteoblasts were measured using 45Ca2+ influx assays. The rapid stimulation of calcium absorption (transcaltachia) in chick intestine treated with OCT also was measured. We found that OCT bound to the nuclear receptor with lower binding affinity [relative competitive index (RCI) = 48.1 for ROS 17/2.8; RCI = 14.8 for chick intestine] than 1,25(OH)2D3 (RCI = 100). Like 1,25(OH)2D3, OCT increased mRNA levels of OPN and OCN in ROS 17/2.8 cells over a 48-h period. In contrast, OCT had no effect on transmembrane influx of 45Ca2+ across ROS cell membranes, whereas uptake was stimulated within 1 min by 1 nM 1,25(OH)2D3. In transcaltachia assays in perfused duodenum, OCT stimulated absorption with a maximum response at 6.5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
NHE3: a Na+/H+ exchanger isoform of renal brush border
Authors: Biemesderfer D. , Pizzonia J. , Abu-Alfa A. , Exner M. , Reilly R. , Igarashi P. , Aronson P. S.
Published: December 2017

Na+/H+ exchangers in the brush-border (luminal, apical) membrane of renal proximal tubules are responsible for active, transcellular reabsorption of NaHCO3 and NaCl. Although well characterized kinetically, the protein that mediates Na+/H+ exchange in the renal brush border has not been identified. Several Na+/H+ exchanger genes, including NHE1, NHE2, NHE3, and NHE4, are expressed in the kidney. To identify the NHE3 gene product and to determine its cellular and subcellular localization in the rabbit kidney, an NHE3-isoform-specific antibody was prepared. Guinea pigs were immunized with purified fusion protein containing the carboxy-terminal 40 amino acids of NHE3 (fpNHE3-C40). After affinity purification, immune sera demonstrated specific reactivity to the NHE3 sequence within the fusion protein as well as to an 80-kDa polypeptide expressed in NHE3-transfected LAP1 cells. Western blot analysis showed that anti-fpNHE3-C40 specifically reacted with an 80-kDa protein that is relatively enriched in renal brush-border membrane compared with basolateral membrane. Immunocytochemical studies confirmed that the Na+/H+ exchanger isoform NHE3 is expressed along the microvillar membrane of the brush border of proximal tubule cells in the rabbit kidney.
Carbonic anhydrase II mRNA is induced in rabbit kidney cortex during chronic metabolic acidosis
Authors: Schwartz G. J. , Winkler C. A. , Zavilowitz B. J. , Bargiello T.
Published: December 2017

Carbonic anhydrase II (CA II), the predominant isoform of carbonic anhydrase in the kidney, is believed to be localized primarily in the cytoplasm of proximal tubule and collecting duct intercalated cells. Carbonic anhydrase facilitates H+ secretion by catalyzing the formation of HCO3- from OH- in the presence of CO2. We have shown that renal cortical CA II activity is stimulated during 4-6 days of chronic metabolic acidosis [L.P. Brion, B.J. Zavilowitz, O. Rosen, and G.J. Schwartz. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30): R1204-R1213, 1991]. The purpose of these studies was to examine under similar conditions the regulation of CA II mRNA. We obtained a major portion of the rabbit CA II cDNA by reverse transcription of total RNA from rabbit kidney followed by amplification using oligonucleotide primers prepared from conserved areas in the coding regions of human, mouse, and chick CA II cDNAs in a polymerase chain reaction (RT-PCR). The 696-bp RT-PCR product was sequenced and found to be 71-86% homologous to CA II cDNAs from the other three species. The deduced amino acid sequence agreed closely (> 97%) with a previous Edman analysis of rabbit erythrocyte CA II. Northern analysis showed expression of a approximately 1.4 kb RNA, with cortex > outer medulla > inner medulla. Steady-state mRNA expression from kidney cortex of acid-treated rabbits was about twice that from controls, when normalized to the expression of beta-actin or malate dehydrogenase. The stimulation of CA II mRNA was greater after 3 days than after 5-6 days of acid treatment. (ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of cell pH by K+/H+ antiport in renal epithelial cells
Authors: Graber M. , Pastoriza-Munoz E.
Published: December 2017

Acid-loaded opossum kidney (OK) cells secrete H+ by Na+/H+ exchange and by a Na(+)- and HCO3(-)-independent pathway that has not been fully characterized. We studied the Na(+)-independent component by measuring H+ flux using the pH-sensitive trapped indicator 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein. Two Na(+)-independent H(+)-transport systems were identified in acid-loaded cells perfused with HCO3(-)-free buffers. The minor component appears to be a conductive pathway for H+, over 90% inhibitable by 5 mM barium. The major component is stimulated by extracellular K+ and was fully active in the presence of barium, amiloride, ouabain, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and bumetanide and in the absence of Cl-. Ammonium inhibited the H+ flux by 72% at 50 mM, and the H+ flux could be accelerated two- to threefold by limited proteolysis of intact cells using kallikrein or papain. In cells pretreated with barium, the K(+)-induced H+ flux caused no change of bis-oxonol fluorescence, suggesting an electroneutral pathway. The H+ flux was a saturable function of extracellular K+ (Michaelis constant 55 mM), and flux reversed when the K+ gradient was reversed. Similarly, the H+ flux was a linear function of the H+ gradient and reversed when the H+ gradient reversed. Evidence for ongoing K(+)-induced H+ flux was also found in nonacidified cells. First, changing perfusate K+ from 5 to 50 mM alkalinized baseline cell pH, an effect not reproduced by barium despite an equivalent depolarizing effect. Second, increasing perfusate K+ from 5 to 50 mM completely eliminated the acidification produced by 1 mM amiloride. We conclude that the OK cell expresses two Na(+)-independent acid-base transport systems. One is a barium-sensitive electrogenic H+ conductance and the other functions as an electroneutral K+/H+ antiporter. The antiporter is capable of H+ extrusion from acid-loaded cells but in normal cells functions in the reverse direction, as an H+ loader. The K+/H+ antiporter appears to be one of the major systems regulation cell pH in these cells, balancing the H+ efflux mediated by Na+/H+ exchange.
Disparate effects of adenosine A1- and A2-receptor agonists on intrarenal blood flow
Authors: Agmon Y. , Dinour D. , Brezis M.
Published: December 2017

Endogenous adenosine, secreted locally by the kidney during tissue hypoxia, induces heterogeneous renal hemodynamic responses. We investigated the cortical and outer medullary blood flow responses to intrarenal infusions of adenosine and adenosine A1- and A2-receptor agonists in anesthetized rats. These agents were infused into the renal interstitium through chronically implanted capsules, and blood flow was measured by laser-Doppler probes. Short (1 min, 0.05 ml) intrarenal infusions of adenosine (0.5 mumol) lowered cortical blood flow to 27 +/- 10% of baseline (n = 7, P < 0.0005). Medullary blood flow response was biphasic, i.e., a transient decrease in flow to 52 +/- 8% of baseline (n = 17, P < 0.0001) followed by a more-sustained increase in flow to 135 +/- 6% (n = 17, P < 0.0001). N6-cyclopentyladenosine, an adenosine receptor A1 agonist, reduced both cortical and medullary blood flow to 59 +/- 4% (n = 10, P < 0.0001) and 38 +/- 5% (n = 11, P < 0.0001) of baseline, respectively. By contrast, 2-[p- (carboxyethyl)phenethylamino]-5'-N-ethycarboxamidoadenosine (CGS-21680C), an adenosine receptor A2 agonist, increased dramatically the medullary blood flow to 184 +/- 15% of baseline (n = 12, P < 0.0005), without major changes in cortical flow. We conclude that intrarenal adenosine reduces cortical blood flow and predominantly increases medullary flow via A1 and A2 receptors, respectively. These hemodynamic responses could play a role in protection of the outer medulla from hypoxia.
Interactions of cAMP-mediated vasodilators with angiotensin II in rat kidney during hypertension
Authors: Chatziantoniou C. , Ruan X. , Arendshorst W. J.
Published: December 2017

In previous studies [C. Chatziantoniou and W.J. Arendshorst. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F573-F580, 1992], we reported that vasodilator prostaglandins (PGs) are defective in buffering the angiotensin II (ANG II)-induced vasoconstriction in the renal vasculature of spontaneously hypertensive rats (SHR). The purpose of the present study was to determine whether this defect in SHR kidneys is specific to PGs or generalized to the action of vasodilators and to gain insight into which intracellular signal(s) mediates this abnormality. Renal blood flow (RBF; electromagnetic flowmetry) was measured in 7 wk-old anesthetized, euvolemic SHR and normotensive Wistar-Kyoto (WKY) rats pretreated with indomethacin to avoid interactions with endogenous PGs. ANG II (2 ng) was injected into the renal artery before and during continuous intrarenal infusion of fenoldopam [DA1 receptor agonist and G protein-dependent stimulator of adenosine 3',5'-cyclic monophosphate (cAMP)], forskolin (G protein-independent stimulator of cAMP), dibutyryl-cAMP (soluble cAMP), and acetylcholine (cGMP stimulator). Each vasodilator was infused at a low dose that did not affect baseline arterial pressure or RBF. In the control period, ANG II reduced RBF by 50% in both strains. Infusion of fenoldopam significantly blunted the ANG II-induced vasoconstriction in WKY, but not in SHR. In contrast, forskolin, dibutyryl-cAMP, and acetylcholine effectively buffered the vasoconstriction due to ANG II in both SHR and WKY. These results suggest that renal vasodilators acting through receptor binding to stimulate the cAMP signaling pathway are ineffective in counteracting the ANG II-induced vasoconstriction in SHR kidneys. (ABSTRACT TRUNCATED AT 250 WORDS)
Insulin-like growth factor I produces renal hyperfiltration by a kinin-mediated mechanism
Authors: Jaffa A. A. , LeRoith D. , Roberts C. T. , Rust P. F. , Mayfield R. K.
Published: December 2017

Insulin-like growth factor-I (IGF-I) infusion into rats and humans reduces renal vascular resistance and raises glomerular filtration rate (GFR) and renal plasma flow (RPF). To investigate whether kinins mediate the renal vasodilatory effects of IGF-I, we infused rats with IGF-I alone or in the presence of a B2 kinin receptor antagonist. Left kidney GFR, RPF, and kinin excretion were measured during infusion of vehicle and subsequently during 60-min infusion of IGF-I or IGF-I plus kinin antagonist. IGF-I was given as a bolus (150 micrograms/kg body wt), followed by infusion at a rate of 8.3 micrograms.kg-1 x min-1 for 60 min. The kinin antagonist was infused at a dose of 1 microgram.kg-1 x min-1 for 60 min before the start of IGF-I infusion. GFR and RPF increased significantly after IGF-I infusion was begun, from baseline levels of 1.70 +/- 0.12 and 6.21 +/- 0.34 to 2.12 +/- 0.11 and 7.91 +/- 0.29 ml/min, respectively, at 20 min (P< 0.001). This effect was maintained throughout 60 min of infusion. The increase in GFR and RPF was associated with a marked rise in urinary kinin excretion, from a baseline of 8.51 +/- 6.7 to 24.7 +/- 6.7 pg/min at 20 min and 40.3 +/- 10.4 pg/min at 40 min (P< 0.001). Pretreatment with the kinin receptor antagonist blocked the rise in GFR and RPF in response to IGF-I. These data suggest that the renal vasodilatory effect of IGF-I is mediated by kinins.