bluuebunny/crossref_metadata_2025 huggingface dataset:
Rubidium absorption and proton secretion by rabbit outer medullary collecting duct via H-K-ATPase
Authors: Wingo C. S. , Armitage F. E.
Published: December 2017

Multiple lines of evidence support the hypothesis that the outer medullary collecting duct from the inner stripe (OMCDi) possesses a functional proton-potassium-activated adenosinetriphosphatase (H-K-ATPase). To examine the effect of inhibition of H-K-ATPase on Rb efflux, we measured the Rb tracer rate efflux coefficient (KRb) across the OMCDi of animals adapted to a K-restricted diet using the selective K-competitive H-K-ATPase inhibitor, Sch 28080. Sch 28080 (10 microM) did not significantly alter transepithelial voltage (VT) but significantly decreased KRb by 41%. We further examined the effect of 10% peritubular CO2 on KRb and the subsequent effect of Sch 28080 (10 microM) on KRb. After exposure to 10% CO2 for 120 min, vehicle-treated tubules exhibited a small but significant increase in KRb without a significant change in VT. In contrast, 10 microM Sch 28080 significantly decreased KRb by 44% without affecting VT. The lack of an effect of H-K-ATPase inhibition on VT in the presence of either 5% or 10% CO2 was in marked contrast to the effect of carbonic anhydrase inhibition (CAI). CAI consistently and significantly decreased VT either in the presence of 5% or 10% CO2. To address whether H-K-ATPase also participates in proton secretion we examined the effect of Sch 28080 (10 microM) on net bicarbonate absorption by the OMCDi of rabbits fed a normal rabbit ration and rabbits adapted to a K-restricted diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Interaction of Cl- and other halogens with Cl- transport systems in rabbit cortical collecting duct
Authors: Muto S. , Imai M. , Asano Y.
Published: December 2017

We have reported that in the rabbit cortical collecting duct (CCD) we can identify electrophysiologically three distinct cell types; the collecting duct (CD) cell and the alpha- and beta-intercalated (IC) cell. To further characterize the Cl- transport properties of each cell type, we examined the interaction between Cl- and other halogens or SCN- in the isolated and perfused CCD by intracellular microelectrode impalement. The rapid depolarization of the basolateral membrane potential (VB) caused by replacement of bath Cl- with each anion revealed that the sequences of apparent halogen selectivity for the basolateral Cl- conductance were similar in all three cell types. The ranking of Cl- > Br- > F- > I- corresponds to the sequence 5 of Eisenman's series, indicating “strong” interaction of the anions with the selectivity site. The basolateral Cl- conductance of these three cell types may share common characteristics, although I- permeability is less in IC cells than in CD cells. Hyperpolarization of the basolateral membrane of the beta-IC cell upon reduction of luminal Cl- reflects alterations in either Cl- entry across the apical membrane, or Cl- exit across the basolateral membrane, or both. Luminal Cl- replacement with each anion showed that the sequence of the hyperpolarization of the basolateral membrane was I- >> cyclamate = SCN- > F- > Br-, suggesting that I-inhibits either apical Cl- entry or basolateral Cl- exit. On the other hand, in the CD cell reduction of the perfusate Cl- by replacement with each anion caused the basolateral membrane to hyperpolarize with a different ranking: cyclamate = F- > I- = SCN- > Br-.(ABSTRACT TRUNCATED AT 250 WORDS)
Estimation of erythropoietin secretion rate in normal and uremic subjects
Authors: Coles G. A. , Liberek T. , Davies M. E. , Robinson M. , Jones J. , Thomas G. , Davies M. , MacDougall I. C. , Williams J. D.
Published: December 2017

We have developed a method for directly estimating the secretion rate of endogenous erythropoietin (EPO) in human subjects. Carrier-free recombinant human EPO was labeled with 125I by the chloramine T method. Human serum albumin was then added prior to removal of free iodide. Seven normal subjects and nine patients with chronic uremia and anemia were studied. Each subject received a bolus of 125I-EPO according to body size and then a constant infusion for 5 h. Secretion rate was calculated as the rate of infusion divided by serum EPO specific activity given by the protein-bound counts divided by the EPO concentration. Serum EPO concentration was significantly higher in the patients than the controls (mean 30.9 pg/ml vs. 11.1 pg/ml, P < 0.05), although all values were within the normal range. Mean EPO secretion rate was 255 in the patients (range 68–1,101) and 190 pg.kg-1 x h-1 in the normal group (range 52-382). This difference was not significant. As the subjects were in a steady state, EPO disappearance/degradation was the same in patients compared with controls despite a relatively hypoplastic marrow.
Age-related changes in alpha 1- and alpha 2-chain type IV collagen mRNAs in adult mouse glomeruli: competitive PCR
Authors: Peten E. P. , Garcia-Perez A. , Terada Y. , Woodrow D. , Martin B. M. , Striker G. E. , Striker L. J.
Published: December 2017

Studies of age-related changes in glomerular extracellular matrix (ECM) synthesis in normal mice have been hampered by the difficulty of isolating sufficient numbers of intact glomeruli and by the inability to quantify different mRNA species. The purpose of this study was to identify and quantitate the individual mRNAs coding for alpha 1- and alpha 2-chains of type IV collagen in isolated, single glomeruli of normal mice at different ages. These data on normal ECM synthesis were necessary for the understanding of glomerulosclerosis, a condition characterized by excess deposition of collagen. Pools of freshly microdissected adult mouse glomeruli were reverse transcribed in situ, and alpha 1-IV and alpha 2-IV collagen mRNAs were individually amplified by means of specific primers and the polymerase chain reaction (PCR), according to a previously published method. A competitive PCR assay, based on utilization of mutated cDNAs, allowed the reproducible, quantitative, and separate determination of the absolute amounts of both alpha 1-IV and alpha 2-IV mRNAs measured, as their respective cDNAs, in one-tenth of one glomerulus. The levels of alpha 1-IV and alpha 2-IV collagen mRNA were 208 +/- 36.0 x 10(-4) and 161.2 +/- 18.6 x 10(-4) amol/glomerulus in 5-wk-old mice. There were no significant age-related differences at 8, 12, and 24 wk. The mean levels over this period were 60.2 +/- 4.9 x 10(-4) for alpha 1-IV collagen mRNA and 63.9 +/- 5.8 x 10(-4) amol/glomerulus for alpha 2-IV collagen mRNA. Two of three 24-wk-old mice had mild glomerulosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Vasomotor effects of purinergic agonists in isolated rabbit afferent arterioles
Authors: Weihprecht H. , Lorenz J. N. , Briggs J. P. , Schnermann J.
Published: December 2017

To examine the vasomotor effects of purinergic agonists, experiments were performed in isolated afferent arterioles of rabbit kidneys dissected together with their glomerulus and perfused with a pressure head of 120 cmH2O. Changes in vascular diameter were taken as measure of changes in vasomotor tone. Adenosine caused a dose-dependent and persistent decrease in vascular diameter along the entire afferent arteriole with significant changes being detectable at 10(-8) M. Constrictor effects were more pronounced in the glomerular entrance segment of the arteriole where adenosine caused a progressive diameter reduction with maximum contraction at 10(-4) M. Similar monophasic diameter reductions of the distal afferent arteriole were seen with increasing bath concentrations of 2-chloroadenosine (2-ClAdo), cyclohexyladenosine (CHA), and 5'-(N-ethylcarboxamido)adenosine (NECA). Concentrations to achieve half-maximum responses were 92.5 nM for 2-ClAdo, 39 nM for CHA, and 107 nM for NECA. The A2-receptor agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine had no effect on vessel diameter. Increasing bath concentrations of ATP caused significant diameter reductions in both the proximal and distal parts of the afferent arterioles. Addition of the A1-receptor blocker, 8-cyclopentyl-1,3-dipropylxanthine, eliminated the effect of ATP in the proximal region of the arteriole, but a significant diameter reduction was still seen in the glomerular entrance segment. The ATP analogue, beta gamma-methylene-ATP, caused a significant diameter reduction in this segment. These results are consistent with an essentially exclusive presence of A1 receptors in the glomerular entrance segment of the afferent arteriole, whereas in more proximal regions A2 receptors appear to also be expressed in low density. These studies also provide functional evidence for the presence of P2x receptors in renal afferent arterioles.
DOCA-enhanced sites of vasopressin-stimulated cAMP formation in rat cortical collecting tubule
Authors: McArdle S. , Fallet R. , Jeffries W. B. , Pettinger W. A.
Published: December 2017

In deoxycorticosterone acetate (DOCA)-NaCl hypertension, the effects of vasopressin (VP) in the cortical collecting tubule (CCT) are exaggerated. These include both the biochemical effect of VP-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in the CCT and physiological effects of VP-mediated sodium and water retention. In this study, we examined the mechanism of enhanced VP-stimulated cAMP formation in the CCT. We compared cAMP formation in response to activators (following in parentheses) of the VP receptor (VP), of the stimulatory guanine nucleotide binding (Gs) protein [guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S); F-], and of the catalytic subunit of adenylyl cyclase (forskolin, Mn2+) between control and DOCA-NaCl-treated rats. The effects of VP and forskolin were enhanced in CCT of DOCA-NaCl-treated animals by 201 and 139%, respectively, compared with control animals. Other activators, Mn2+ (150%), F- (142%), and GTP gamma S (156%), also caused augmented cAMP formation in the CCT of DOCA-NaCl-treated rats. The DOCA-NaCl-induced increment in cAMP response to VP remained after pretreatment of the rats with pertussis toxin (171 and 169% increase in response in DOCA-NaCl and control rats, respectively), suggesting that altered inhibitory guanine nucleotide binding (Gi) protein function is not the mechanism for the altered response to VP in the CCT. Further evidence that Gi function is intact in DOCA-NaCl animals is that epinephrine (via alpha 2-adrenoceptor stimulation) inhibited VP-stimulated cAMP accumulation to a similar degree in DOCA-NaCl and control rats (86 and 76%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
Cellular morphology in outer medullary collecting duct: effect of 75% nephrectomy and K+ depletion
Authors: Zalups R. K. , Henderson D. A.
Published: December 2017

The present study was designed to determine, in rats, whether 75% nephrectomy and potassium depletion affect the principal and intercalated cells in the outer medullary collecting duct in the same manner as they affect the principal and intercalated cells in the cortical collecting duct. Ten days after a 75% reduction of renal mass, whole animal glomerular filtration rate decreased and the fractional excretion of potassium increased in rats. However, no morphological changes occurred in either the principal or intercalated cells of the outer medullary collecting duct after the reduction of renal mass. When 75% nephrectomized rats were placed on a diet deficient in potassium, the concentration of potassium in plasma and the absolute and fractional excretion of potassium decreased significantly. In addition, marked hypertrophy occurred in both the principal and intercalated cells in the outer medullary collecting duct. Previous findings from the same animals used in the present study show that 75% nephrectomy caused hypertrophic changes in principal cells of the cortical collecting duct, which could be inhibited by potassium depletion induced by the dietary restriction of potassium. The findings also show that the intercalated cells of the cortical collecting duct in 75% nephrectomized rats were unaffected by potassium depletion. On the basis of our findings, it appears there is an absence of hypertrophy in either the principal or intercalated cells in the outer medullary collecting duct of the rat after renal mass in the animal is reduced significantly.(ABSTRACT TRUNCATED AT 250 WORDS)
Integrin receptors in renal tubular epithelium: new insights into pathophysiology of acute renal failure
Authors: Goligorsky M. S. , Lieberthal W. , Racusen L. , Simon E. E.
Published: December 2017

This review summarizes the existing evidence implicating disordered adhesion of renal tubular epithelial cells to the basement membrane in the pathophysiology of acute renal failure. The following three major lines of investigation are discussed: 1) exfoliation of renal tubular epithelial cells as a potential mechanism of tubular obstruction, 2) normal distribution of integrin receptors along the tubular apparatus, and 3) redistribution of integrin receptors and remodeling of the cytoskeleton following acute injury to renal tubular epithelium. We advance the hypothesis that the loss of the basolateral expression of integrin receptors is responsible for the exfoliation of viable proximal epithelial cells and that the redistribution of integrin receptors from the basolateral to the apical surface of epithelial cells facilitates adhesion of detached cells to the in situ cells. These two processes culminate in tubular obstruction.
Immunocytochemical characterization of the high-affinity thiazide diuretic receptor in rabbit renal cortex
Authors: Ellison D. H. , Biemesderfer D. , Morrisey J. , Lauring J. , Desir G. V.
Published: December 2017

Thiazide diuretics increase urinary NaCl excretion primarily by inhibiting Na and Cl transport across the apical membrane of cells in the renal distal tubule. Although these diuretics bind to a membrane protein that couples transport of Na and Cl directly, the molecular nature of this transporter and its localization in the mammalian kidney remain controversial. The present experiments were designed to develop monoclonal antibodies to the high-affinity thiazide diuretic receptor to investigate its molecular characteristics and its cellular and subcellular localization in rabbit kidney. Mice were immunized with high-affinity thiazide diuretic receptors that had been partially purified from rabbit kidney cortex. Resulting hybridomas were screened for the ability to immunoprecipitate thiazide diuretic receptors that were labeled with the thiazide-like diuretic [3H]metolazone. A single hybridoma (MAb JM5) produced antibodies capable of immunoprecipitating up to 80% of the labeled thiazide receptors from solubilized renal cortical membranes. MAb JM5 reacted with a 125-kDa protein on Western blots of solubilized renal cortical apical membranes. It stained the apical membrane of cells in the distal convoluted and connecting tubule but did not stain proximal tubules, glomeruli, or interstitial structures. Less intense staining of apical membranes of principal cells in the collecting tubule and a subpopulation of cells in the thick ascending limb were also present. These results indicate that the high-affinity thiazide diuretic receptor comprises a 125-kDa protein that localizes to the apical membrane of cells in the renal distal tubule.
Evidence against a proton pump in rabbit proximal convoluted tubule
Authors: Beck J. , Laprade R.
Published: December 2017

H+/OH- transport in the absence of bicarbonate was studied in the rabbit proximal convoluted tubule (PCT) perfused in vitro using measurements of membrane potential and intracellular pH (pHi). Blockade of apical Na/H exchange led to a cell acidification of 0.64 +/- 0.1 pH units from a control pHi of 7.27 +/- 0.04. A bafilomycin-insensitive recovery of pHi of 0.05 +/- 0.02 pH units occurred, but pHi did not exceed electrochemical equilibrium. A larger, sustained acidification of 0.87 +/- 0.07 from an initial control pHi of 7.25 +/- 0.05 induced by bilateral Na removal left pHi substantially below electrochemical equilibrium. These results suggest the absence of Na-independent active proton extrusion. We also examined the possibility that a passive electrogenic proton leak may exist. The removal of luminal glucose and alanine led to a basolateral membrane hyperpolarization of 31.3 +/- 0.5 mV, which was associated with a cell acidification of 0.15 +/- 0.02 pH units. These responses were reversed by addition of luminal glucose and alanine but not by depolarization by basolateral barium, suggesting that luminal glucose and alanine effects on pHi were due to changes other than cell potential. We conclude that, in the absence of bicarbonate, all active proton extrusion in the rabbit PCT is dependent on active Na transport and that a proton leak is negligible.
Effect of captopril on fluctuations of blood pressure and renal blood flow in rats
Authors: He J. , Marsh D. J.
Published: December 2017

Arterial blood pressure and renal blood flow (RBF) fluctuations in rats were studied by autoregressive (AR) and autoregressive-moving average (ARMA) modeling. These estimation procedures provided greater sensitivity and frequency resolution than classic fast Fourier transform (FFT)-based methods and also require shorter observation periods. We estimated the transfer function of RBF autoregulation to test whether inhibition of angiotensin-converting enzyme impairs whole kidney dynamic autoregulation. The transfer function in control animals measured with the ARMA method was similar to transfer functions obtained previously, using FFT methods. Because of better frequency resolution, we also detected an oscillation in RBF at 50 mHz, the same frequency as an oscillation in tubular pressure and glomerular filtration rate that had been attributed to tubuloglomerular feedback (TGF), but that FFT methods had not previously found in whole kidney blood flow. Captopril increased the amplitude of RBF fluctuations and increased the gain of the transfer function at frequencies below 100 mHz, a frequency bandwidth previously associated with TGF. The increased gain indicates that TGF operates less effectively to mediate dynamic autoregulation when angiotensin conversion is inhibited. Gain at frequencies greater than 100 mHz, previously ascribed to the myogenic mechanism, was not affected by captopril. These results show that angiotensin, by modulating TGF, reduces fluctuations of RBF about the mean value.
The Os/+ mouse: a genetic animal model of reduced renal mass
Authors: Zalups R. K.
Published: December 2017

The ROP [Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)] mouse possessing the gene for oligosyndactylism (Os) was evaluated as a potential genetic animal model of reduced renal mass. Young male ROP mice that were heterozygotes with respect to the Os gene (Os/+) and their normal homozygote litter mates (+/+) were used in the present study. Approximately 50% fewer nephrons were present in the Os/+ mice than in the +/+ mice. Hypertrophic changes were detected in the epithelial cells in proximal convoluted and proximal straight tubules as well as in cortical collecting ducts in the kidneys of the Os/+ mice. Glomerular hypertrophy was also noted in the kidneys of these mice. After unilateral nephrectomy, further hypertrophic changes occurred in both Os/+ and +/+ mice, but the magnitude of compensatory growth was greater in the +/+ mice. From a functional standpoint, the Os/+ mice appeared to be capable of maintaining normal fluid and electrolyte homeostasis, even after unilateral nephrectomy. The findings from the present study indicate that the ROP Os/+ mouse can be a useful animal model to study the effects of reduced renal mass.
Renal effects of acute endothelial-derived relaxing factor blockade are not mediated by angiotensin II
Authors: Baylis C. , Engels K. , Samsell L. , Harton P.
Published: December 2017

The renal responses to acute blockade of the endothelial-derived relaxing factor (EDRF) resemble the renal actions of angiotensin II (ANG II), and the present studies were conducted to establish what role, if any, the endogenous renin-angiotensin system plays in mediating the renal response to acute EDRF blockade. These studies were conducted in the conscious chronically catheterized rat. In control experiments we observed that acute blockade of ANG II synthesis with converting-enzyme inhibition (CEI) led to a fall in blood pressure (BP) and a slight renal vasodilation but no significant change in glomerular filtration rate (GFR) or renal plasma flow (RPF). Urine flow and sodium excretion were unchanged by CEI. Use of the nonpeptide ANG II receptor antagonist losartan had no effect on BP, renal vascular resistance (RVR), GFR, or RPF; however, urine flow and sodium excretion did rise significantly. Because of the high specificity of losartan, this suggests that, in the normal conscious rat, endogenous ANG II does not control renal vascular tone but does enhance renal sodium reabsorption. ANG II blockade with either CEI or losartan had little effect on BP and no effect on the renal hemodynamic responses to acute EDRF blockade. The marked natriuretic and diuretic response to acute EDRF blockade persists during concomitant losartan but is abolished by CEI. These studies suggest that in the conscious rat the renal hemodynamic response to EDRF blockade is not mediated by endogenous ANG II.
Immunohistochemical mapping of cellular and subcellular distribution of 5-HT1A receptors in rat and human kidneys
Authors: Raymond J. R. , Kim J. , Beach R. E. , Tisher C. C.
Published: December 2017

Northern blotting studies have demonstrated mRNA for the serotonin 5-HT1A receptor in human neonatal kidney (B. K. Kobilka, T. Frielle, S. Collins, T. Yang-Feng, T. S. Kobilka, U. Francke, R. J. Lefkowitz, and M. G. Caron. Nature Lond. 329: 75-79, 1987). To confirm expression of receptor protein in kidney, we raised antibodies to two peptides derived from the third intracellular loop of the human 5-HT1A receptor. Specific immunoglobulin G (IgG) was purified sequentially on protein A-Sepharose and peptide-Affigel 10 columns. Each IgG was able to: 1) quantitatively immunoprecipitate [3H]8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene ([3H]8-OH-DPAT)-labeled human and rat receptors; 2) immunoblot a new protein in cells transfected with human 5-HT1A receptor DNA; and 3) immunoautoradiographically label areas of rat brain (frontal cortex, hippocampus, and lateral septum) in a highly characteristic pattern similar to that labeled by 125I-Bolton-Hunter-8-methoxy-2-(N-propyl-N-propylamino)Tetralin, a specific 5-HT1A receptor autoradiography ligand. By use of a light microscopic immunoperoxidase labeling technique, incubation of each IgG antibody with sections of rat and human kidney demonstrated an identical pattern of immunoreactivity. Specific labeling of basolateral plasma membranes was detected throughout medullary and cortical thick ascending limbs (TAL), in distal convoluted tubules (DCT), in connecting tubule cells of the connecting tubule, and in principal cells of the initial collecting tubule. There was no labeling in the inner medulla, glomeruli, or blood vessels. The labeling was blocked by preincubation with the corresponding peptide, but not with noncorresponding peptide or carrier protein. There was no labeling with preimmune IgG. Electron microscopic immunoperoxidase labeling confirmed the specific localization of the IgG antibody along the basolateral plasma membrane in all positively staining cells in rat kidney. Radioligand binding studies with the specific 5-HT1A receptor ligand [3H]8-OH-DPAT confirmed the presence of 5-HT1A receptor binding sites in bulk-isolated rat medullary TAL. These studies provide the first evidence that the 5-HT1A receptor is expressed on the basolateral surface of TAL and DCT cells of human and rat kidney. The specific localization to these cells suggests a possible role for the 5-HT1A receptor in the regulation of salt and water transport in mammalian kidney.
Glycine protection against hypoxic but not phospholipase A2-induced injury in rat proximal tubules
Authors: Wetzels J. F. , Wang X. , Gengaro P. E. , Nemenoff R. A. , Burke T. J. , Schrier R. W.
Published: December 2017

We studied the effects of glycine (2 mM) on hypoxia-induced changes in phospholipids and fatty acids in isolated rat proximal tubules. In this preparation, 25 min of hypoxia caused cell injury, as reflected by the release of lactate dehydrogenase (LDH) (13.1 +/- 0.8 vs. 43.5 +/- 3.2%; P < 0.01). Hypoxia caused increases in fatty acids and in lysophospholipids. Glycine prevented the hypoxia-induced cell injury (LDH 13.1 +/- 0.8 vs. 11 +/- 0.7%; not significant) but did not attenuate the increases in fatty acids or lysophospholipids. In additional experiments, the effects of glycine on phospholipid changes and cell injury induced by exogenous phospholipase A2 (PLA2) were studied. PLA2 caused dramatic increases in fatty acids and lysophospholipids and mild cell injury; these effects were not influenced by glycine. In contrast, glycine attenuated increases in LDH release induced by exposing the tubules to exogenous arachidonic acid. In conclusion, glycine does not prevent the phospholipid degradation induced by either exogenous PLA2 or hypoxia in isolated proximal tubules and yet affords protection against hypoxia and exogenous arachidonic acid.
Protein kinase C inhibits arginine vasopressin-stimulated cAMP accumulation via a Gi-dependent mechanism
Authors: Teitelbaum I.
Published: December 2017

Studies were performed to identify the site at which activation of protein kinase C (PKC) inhibits arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cultured rat inner medullary collecting tubule (RIMCT) cells. Neither endogenous stimulation of PKC by epidermal growth factor (EGF) nor the addition of exogenous 1,2-dioctanoyl-sn-glycerol (DOG) impaired forskolin-stimulated cAMP accumulation. Similarly, neither EGF nor DOG altered cAMP generation in response to cholera toxin. However, pretreatment of RIMCT cells with pertussis toxin resulted in loss of inhibition of AVP-stimulated cAMP accumulation by DOG. Likewise, the ability of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), to inhibit AVP-stimulated cAMP accumulation was eliminated by pretreatment with pertussis toxin. PMA also inhibited AVP-stimulated adenylyl cyclase activity in plasma membranes prepared from rat inner medullas. In contrast to its effects on AVP, activation of PKC did not impair cAMP accumulation in response to isoproterenol or prostaglandin E2. These studies demonstrate that PKC-mediated inhibition of AVP-stimulated cAMP accumulation in cultured RIMCT cells requires the intact inhibitory guanine nucleotide binding protein Gi.
Dietary calcium modulates blood pressure through alpha 1-adrenergic receptors
Authors: Hatton D. C. , Scrogin K. E. , Levine D. , Feller D. , McCarron D. A.
Published: December 2017

To investigate the role of adrenergic receptor activity in dietary calcium-induced alterations in blood pressure, weanling spontaneously hypertensive rats (SHR) were placed on either high-calcium (2.0%) or low-calcium (0.1%) diets for 1-2 wk. Baseline blood pressure was higher and pressor responses to exogenous norepinephrine (NE) were greater in the SHR on low-calcium diets than high-calcium diets. There was no difference between diet groups in circulating NE or in the pressor response to angiotensin II. The difference in basal blood pressure was eliminated by the alpha 1-adrenergic receptor blockers phentolamine and prazosin but was not altered by alpha 2-, beta 1-, or beta 2-adrenergic receptor blockade (idazoxan, metoprolol, and butoxamine, respectively). Furthermore, hypotension produced by either calcitonin gene-related peptide, captopril, or nitroprusside failed to eliminate the diet-induced difference in blood pressure. The results suggest the possibility that diet-induced differences in alpha 1-adrenergic activity may be responsible, in part, for variations in blood pressure on different calcium diets.
Kinetics of endocytosis in renal proximal tubule studied with ruthenium red as membrane marker
Authors: Birn H. , Christensen E. I. , Nielsen S.
Published: December 2017

This study was performed to determine the membrane area of the different compartments involved in endocytosis in rat kidney proximal tubule. This enables a direct estimation of the kinetics of the membrane turnover in the subprocesses of endocytosis and recycling of membrane constituents. To accomplish this, cross-sectioned invaginations have to be distinguished from endocytic vesicles. To obtain a continuous heavy staining of the apical plasma membrane, we have developed a sandwich-staining technique involving initial staining with ruthenium red, osmium, and thiocarbohydrazide functioning as a molecular bridge between osmium molecules. The distribution of invaginations, noncoated and coated endocytic vesicles, as well as the surface density of invaginations, small endocytic vesicles, large endocytic vacuoles, lysosomes, and dense apical tubules are determined in segment 1 (S1) and 2 (S2) of the proximal tubule. This has shown that invaginations constitute most [54% (S1 and 62% (S2)] of the membrane in small membrane-bounded structures in the apical cytoplasm. No morphological characteristics enabled direct differentiation between cross-sectioned invaginations and small endocytic vesicles, but a method for correct identification of 72% of all invaginations and 82% of all small endocytic vesicles is presented. Using the surface densities of various compartments together with previous data on the internalization of membrane markers, we developed a kinetic model enabling calculation of the velocity of membrane internalization and subsequent recycling. The velocity of internalization is 6.4 X 10(-3) microns2.micron-3.s-1, corresponding to internalization of a membrane area equivalent to the entire surface of invaginations within 78 s. The velocity of membrane transfer from the vacuolar compartment to dense apical tubules and of recycling from dense apical tubules to the luminal membrane is 6.2 x 10(-3) microns 2.microns-3.s-1. The entire membrane area equivalent to the surface of the vacuolar compartment and dense apical tubules is transferred in 43 and 92 s, respectively. The amount of membrane that surrounds lysosomes is transported from the vacuolar compartment within 23 min.
Role of Na-dependent Cl/HCO3 exchange in basolateral Cl transport of rabbit proximal tubules
Authors: Ishibashi K. , Rector F. C. , Berry C. A.
Published: December 2017

To analyze the rate of basolateral Cl exit and the magnitude and relative contributions of KCl cotransport and Na-dependent and -independent Cl/HCO3 exchange to Cl exit across the basolateral membrane (BLM) during transcellular Cl absorption, rabbit proximal convoluted tubules (PCT) were perfused with high-Cl, low-HCO3 plus formate solutions and bathed with plasma ultrafiltrate-like plus formate solutions. The initial rates of intracellular Cl activity (AiCl) reduction following bath Cl removal were compared when bath Cl was 0, when bath Na and Cl were 0, and when bath HCO3 and Cl were 0. The initial rate of AiCl reduction following bath Cl removal was 4.4 +/- 0.4 mM/s. After bath Na and Cl removal, this rate was reduced to 25.1 +/- 5.0%. After bath HCO3 and Cl removal, it was reduced to 18.0 +/- 4.8%. The difference between bath Na and Cl removal and bath HCO3 and Cl removal was not significant. Cl efflux following bath HCO3 and Cl removal may be due to a KCl symporter. The contribution of a KCl symporter was examined by raising bath K to 20 mM, thus eliminating the chemical driving force for KCl exit. After bath K increase, the initial rate of AiCl increase was 0.057 +/- 0.005 mM/s. These data suggest that Cl efflux at BLM of rabbit PCT is 1) large enough to explain transcellular Cl transport, 2) predominately due to an Na-dependent Cl/HCO3 exchanger, and 3) negligibly due to an Na-independent Cl/HCO3 exchanger or a KCl symporter.
cAMP-dependent control of Na+/H+ antiport by AVP, PTH, and PGE2 in rat medullary thick ascending limb cells
Authors: Borensztein P. , Juvin P. , Vernimmen C. , Poggioli J. , Paillard M. , Bichara M.
Published: December 2017

Antidiuretic hormone and parathyroid hormone (PTH) inhibit HCO3- absorption by the rat medullary thick ascending limb (MTAL). Studies were performed on rat MTAL tubule suspension to specify the H(+)-HCO3- membrane transporters affected by these hormones and the implicated intracellular second messengers. Arginine vasopressin (AVP) and PTH stimulated cell adenosine 3',5'-cyclic monophosphate (cAMP) production with a relative rank order potency of AVP > rat PTH-(1-34) > bovine PTH-(1-84). Significant cell acidification in HCO3- -CO2-free medium, monitored in 2'7'-bis(carboxyethyl)-5(6')-carboxyfluorescein-loaded cells, was caused by 0.1 nM AVP, 1 nM rat PTH-(1-34), but not by < 100 nM bovine PTH-(1-84), as well as by 10(-4) M 8-bromo-cAMP and 2 x 10(-5) M forskolin; 10 nM AVP or rat PTH-(1-34) did not alter the intracellular pH when Na+/H+ antiport was inhibited by 2 mM amiloride. Prostaglandin E2 (PGE2, 10(-6) M), which inhibited AVP-stimulated cell cAMP production, reduced by 35% the cell acidification response to 10 nM AVP. AVP and 8-bromo-cAMP inhibited Na+/H+ antiport-dependent cell pH recovery from intracellular acidification, which was explained by a decrease in the Vmax of the antiporter. AVP did not directly affect K(+)-HCO3- cotransport and plasma membrane H(+)-ATPase of rat MTAL cells. Cytosolic calcium ([Ca2+]i), monitored in fura-2-loaded cells, was unaffected by up to 1 nM AVP, 100 nM PTH, glucagon, calcitonin, and oxytocin, and 1 microM PGE2; however, 100 nM AVP, but not 1-desamino-8-D-AVP (dDAVP), caused a peak increase in [Ca2+]i, even in the absence of extracellular Ca2+, and stimulated cell accumulation of [3H]inositol phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)
Basolateral K+, Cl-, and HCO3- conductances and cell volume regulation in rabbit PCT
Authors: Macri P. , Breton S. , Beck J. S. , Cardinal J. , Laprade R.
Published: December 2017

The relationship between changes in cellular volume, intracellular pH (pHi), basolateral membrane potential (VBL), and membrane partial basolateral conductances to K+ (tK) and Cl- (tCl) and mediated by the Na-HCO3 cotransporter (tNaHCO3) was determined in the collapsed proximal convoluted tubule (PCT) submitted to a 125-mosmol/kg hypotonic shock. The shock that produces a rapid swelling followed by partial volume regulation was accompanied by a rapid and transient VBL hyperpolarization of 10.0 +/- 1.5 mV and a second gradual hyperpolarization of 5.0 +/- 0.7 mV with respect to a control value of -44.0 +/- 4.6 mV.tK was 0.12 +/- 0.03 in control, increased transiently to 0.15 +/- 0.03, and then gradually increased to reach 0.32 +/- 0.06 at the end of hypotonic shock. In contrast, tCl was 0.03 +/- 0.01 in control, increased rapidly to a maximum of 0.16 +/- 0.01, and then decreased slowly to 0.08 +/- 0.02. During the same period, tNaHCO3 decreased rapidly from 0.41 +/- 0.04 to a minimum of 0.11 +/- 0.02 and slowly reincreased to reach 0.16 +/- 0.01.pHi increased transiently from 7.09 +/- 0.03 in control to 7.24 +/- 0.05 to come back gradually to 7.15 +/- 0.05 at the end of the hypotonic period. The membrane absolute conductance mediated by the Na-HCO3 cotransporter was found to increase only slightly in hypotonic conditions, whereas that to K+ and Cl-, GK and GCl, increased by at least factors of 8 and 17, respectively, with the increase of GCl being much faster than that of GK. In addition, the temporal variations in GCl followed closely those of the cellular water efflux. We conclude that the hypotonic swelling leads to important increases in the conductive pathways for K+ and Cl- and that the Cl- conductance pathway appears to be the rate limiting step in triggering and supporting regulatory volume decrease.
Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells
Authors: Takeda M. , Homma T. , Breyer M. D. , Horiba N. , Hoover R. L. , Kawamoto S. , Ichikawa I. , Kon V.
Published: December 2017

We investigated whether cell volume decrease per se can activate intracellular mechanisms leading to mesangial cell contraction. For this purpose, we applied hyperosmotic stress to cultured glomerular mesangial cells and examined the effects on phosphorylation of myosin light chain (MLCP). Compared with control cells, hyperosmotic stress (390 mosmol/kg) attained by either NaCl or raffinose significantly increased MLCP to 140.7 +/- 7.0% (n = 5) and 134.8 +/- 7.7% (n = 4), respectively, in parallel with a decrease in the cell volume. This increase was comparable to that achieved by the following agonists: arginine vasopressin (AVP, 100 nM; n = 5) and endothelin-1 (ET, 10 nM; n = 5). By using two-dimensional tryptic phosphopeptide mapping, contribution of myosin light-chain kinase (MLCK) and protein kinase C (PKC) to the observed phosphorylation was examined by identifying phosphorylation at serine-19 (by MLCK) and at serine-1 or serine-2 (by PKC). Under resting conditions, relative distribution of phosphorylation between MLCK and PKC sites was 60.1 +/- 8.4 and 39.9 +/- 8.4%. The relative contribution by these enzymes remained similar during hyperosmotic stress or agonist stimulation. Since cytosolic Ca2+ concentration ([Ca2+]i) is an important determinant of MLCP, we also examined [Ca2+]i in these settings. While AVP and ET-induced a characteristic transient spike in [Ca2+]i, hyperosmotic stress caused a gradual and modest increase in [Ca2+]i. These studies show that, in mesangial cells, reduction in cell volume induces MLCP through mechanisms distinct from those involved in agonist-induced events.
Mechanisms of temporal variation in single-nephron blood flow in rats
Authors: Yip K. P. , Holstein-Rathlou N. H. , Marsh D. J.
Published: December 2017

Modified laser-Doppler velocimetry was used to determine the number of different mechanisms regulating single-nephron blood flow. Two oscillations were identified in star vessel blood flow, one at 20-50 mHz and another at 100-200 mHz. Tubuloglomerular feedback (TGF) mediates the slower oscillation, and the faster one is probably myogenic in origin. Acute hypertension increased autospectral power in the 20-50 mHz and 100-200 mHz frequency bands to 282 +/- 50 and 248 +/- 64%, respectively, of control even though mean single-nephron blood flow was autoregulated. Mean blood flow increased 24.6 +/- 6.1% when TGF was inhibited by intratubular perfusion with furosemide, and it decreased 42.8 +/- 3.9% when TGF was saturated by tubular perfusion with artificial tubular fluid at high rates. Autospectral power in the low-frequency band decreased 50.5 +/- 9.6% during furosemide and decreased 74.9 +/- 5.9% during TGF saturation, consistent with a TGF origin of the slow oscillation. In contrast, autospectral power of the high-frequency oscillation increased 75.4 +/- 23.9% during TGF inhibition and decreased 35.8 +/- 11% when TGF was saturated, suggesting interactions between the two spontaneously oscillating components in efferent arteriole blood flow.
Modulation of tubuloglomerular feedback responsiveness by extracellular ATP
Authors: Mitchell K. D. , Navar L. G.
Published: December 2017

Experiments were performed in pentobarbital sodium-anesthetized rats to determine the effects of activation of P2 purinoceptors sensitive to ATP on glomerular capillary pressure, as estimated from proximal tubule stop-flow pressure (SFP) measurements, and on the magnitude of maximal tubuloglomerular feedback-mediated reductions in SFP. To selectively expose nephrons in vivo to ATP without influencing arterial blood pressure, we infused ATP directly into the surrounding peritubular capillaries. Peritubular capillary infusion, at a rate of 20 nl/min, of an isotonic saline solution containing 10(-3) M ATP elicited a transient decrease in resting SFP. However, subsequent infusion of 10(-3) M ATP together with the adenosine receptor (P1 purinoceptor) antagonist 1,3-dipropyl-8-p-sulfophenylxanthine (PSPX, 10(-3) M) into the same vascular sites elicited a sustained decrease in resting SFP. Peritubular infusion of the slowly metabolizable ATP analogue, beta,gamma-methylene-ATP (10(-3) M), at a rate of 20 nl/min, also elicited a transient decrease in SFP, but this was not converted to a sustained response by PSPX. The SFP feedback responses to a late proximal perfusion rate of 40 nl/min were markedly attenuated during peritubular infusion of either 10(-3) M ATP (8.7 +/- 1.2 vs. 1.8 +/- 0.9 mmHg; P < 0.01, n = 9) or 10(-3) M beta,gamma-methylene-ATP (8.2 +/- 1.3 vs. 2.2 +/- 1.2 mmHg; P < 0.01, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)
Functional evidence for an apical V1 receptor in rabbit cortical collecting duct
Authors: Ando Y. , Asano Y.
Published: December 2017

In the rabbit cortical collecting duct (CCD) perfused in vitro, we recently found that luminal arginine vasopressin (AVP) hyperpolarizes the transepithelial voltage (Vt) and inhibits the hydrosmotic effect of basolateral AVP. The present study was undertaken to characterize the apical receptor of the CCD for AVP. In contrast to AVP, luminal application of 1-desamino-8-D-arginine vasopressin (DDAVP), a V2 agonist, did not significantly induce hyperpolarization. Luminal oxytocin (OXT) hyperpolarized Vt, interfering with the effect of superimposed luminal AVP, whereas [Thr4,Gly7]OXT, an OXT agonist, did not reproduce the effect of OXT. The effects of luminal AVP and OXT were abolished by [d(CH2)5,Tyr(Me)]-AVP, a V1 antagonist. Finally, luminal applications of AVP metabolite neuropeptides, pGlu-Asn-Cys(Cys)-Pro-Arg and pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH2, were without effect on Vt. These data suggest that luminal AVP induces hyperpolarization through an apical V1 receptor but not through a V2 receptor or an OXT receptor.
Regulation of basolateral K channels in proximal tubule studied during continuous microperfusion
Authors: Beck J. S. , Hurst A. M. , Lapointe J. Y. , Laprade R.
Published: December 2017

Potassium channel activity of the basolateral membrane of the collagenase-treated rabbit proximal convoluted tubule (PCT) was studied during continuous luminal microperfusion. In cell-attached patches (high-K pipette) an inwardly rectifying potassium channel was observed with an inward slope conductance of 60.8 +/- 3.3 pS (n = 12) and outward slope conductance of 17.1 +/- 2.7 pS (n = 6). Stimulation of transcellular sodium transport with luminal glucose and alanine increased channel activity [measured as single-channel open probability (NPo)] from 0.19 +/- 0.11 to 0.44 +/- 0.09 (n = 8). This increase in channel activity was not likely to be mediated by either cell depolarization or cell swelling, because channel activity was voltage insensitive over physiological potentials and because the channel was not activated by stretch. However, channel activity was pH sensitive; reducing luminal pH from 7.4 to 6.5 reduced NPo from 0.63 +/- 0.24 to 0.26 +/- 0.16 (n = 5). Our work demonstrates the feasibility of patch clamping the basolateral membrane of microperfused nephron segments. This has allowed us to follow the activity of this potassium channel during an increase in sodium transport and show that its activity does increase during this maneuver. We conclude that: 1) it is possible to patch clamp the basolateral membrane of microperfused nephron segments, and 2) basolateral membrane of the rabbit PCT contains an inwardly rectifying, pH-sensitive potassium channel. The behavior of this channel on stimulation of transcellular sodium transport could explain the macroscopic increase in basolateral potassium conductance observed under similar conditions.
Vasopressin increases density of apical low-conductance K+ channels in rat CCD
Authors: Cassola A. C. , Giebisch G. , Wang W.
Published: December 2017

The effect of arginine vasopressin (AVP) on the low-conductance K+ channel in the apical membrane of rat cortical collecting duct (CCD) principal cells from animals on a control and high-K+ diet was studied using patch-clamp techniques. AVP stimulated apical low-conductance K+ channel activity in both control and high-K+ animals: application of 110-220 pM AVP induced a significant increase in the density of low-conductance K+ channels. In the presence of phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), administration of 22 pM AVP also increased channel activity. The action of AVP on low-conductance K+ channel activity was mimicked by simultaneous application of forskolin and 3-isobutyl-1-methylxanthine. Exogenously applied N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP, 0.4-0.8 mM) also increased apical low-conductance K+ channel activity. Since channel open probability (Po) was almost saturated in the absence of AVP, the increase of channel activity induced by AVP, forskolin, and dibutyryl-cAMP resulted predominantly from stimulating previously silent K+ channels. We conclude that AVP induces an increase of low-conductance K+ channel activity of principal cells in rat CCD by the stimulation of cAMP-dependent protein kinase. The AVP-induced increase of low-conductance K+ channel activity can thus significantly contribute to the hormone-induced K+ secretion in the rat CCD.
Angiotensin II conditions the slow component of autoregulation of renal blood flow
Authors: Cupples W. A.
Published: December 2017

Release of a suprarenal aortic clamp results in angiotensin-dependent, arterial pressure-mediated renal vasoconstriction. The experiments reported here were designed to show whether this represents operation of autoregulation and whether the slow component of autoregulation is affected by angiotensin II (ANG II). They were performed using halothane-anesthetized Sprague-Dawley rats. In the 1st experiment renal perfusion pressure (RPP) was reduced in steps from spontaneous level to 45 mmHg and then returned in steps to the spontaneous level. The autoregulatory plateau was left-shifted some 20-30 mmHg, with the lower limit of autoregulation reduced from approximately 85 mmHg on the downward leg to approximately 60 mmHg on the upward leg. This resetting was blocked by captopril. Two experiments examined low pressure autoregulation in more detail. After RPP was reduced, three pairs of steps between 65 and 75 mmHg were performed. Significant renal vasodilatation was observed after downward pressure steps in both experiments. Time constants (tau) of resistance adjustment were recovered from most steps by curve fitting. In both experiments tau down = 0.07 +/- 0.01 Hz was faster than tau up = 0.04 +/- 0.01 Hz. Blockade of ANG II by enalaprilat or by the AT1-receptor blocker losartan potassium significantly inhibited regulatory vasodilatation and vasoconstriction at low RPP. Also, tau down = 0.04 +/- 0.01 Hz collapsed to the value of tau up = 0.04 +/- 0.01 Hz. These results demonstrate a significant role for ANG II in renal autoregulation. They show that ANG II is necessary for autoregulation to reset to operate at reduced arterial pressure and to defend a lower blood flow.(ABSTRACT TRUNCATED AT 250 WORDS)