bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal adaptation to changes in dietary phosphate during development
Authors: Mulroney S. E. , Haramati A.
Published: December 2017

The present study tested the hypothesis that the influence of dietary phosphate (Pi) on the renal handling of Pi changes during development. We evaluated whether variations in the dietary Pi content would alter the tubular capacity of Pi reabsorption [Max RPi/glomerular filtration rate (GFR)] in immature rats, which have a relatively greater Max RPi/GFR compared with adult rats. Then we examined the response of immature and adult Pi-deprived rats to dietary Pi replenishment. Studies were performed in acutely thyroparathyroidectomized Wistar rats at three different stages of development: immature (3- to 4-wk old), young (6- to 7-wk old), and adult (12- to 13-wk old). Animals were fed either low (0.07%)-, normal (0.7%), or high (1.8%)-phosphate diet (LPD, NPD, and HPD, respectively) for 4 days and were then prepared for renal clearance experiments to determine the Max RPi/GFR. On all dietary regimens, the Max RPi/GFR was highest in immature rats and decreased progressively with age. When fed LPD, immature rats, with an already elevated rate of phosphate transport, displayed a remarkable 68 +/- 13% increase in the Max RPi/GFR (from 5.58 +/- 0.29 to 9.47 +/- 0.76 mumol/ml, P less than 0.01). This was significantly greater than the 38 +/- 3% increase observed in adult rats (from 3.50 +/- 0.18 to 4.81 +/- 0.09 mumol/ml). Conversely, in response to HPD, the decrease in the Max RPi/GFR was smallest in immature rats (-42 +/- 2%) compared with the decrement in either young (-54 +/- 3%) or adult (-61 +/- 6%) rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Bulk isolation of renal PCT and PST. I. Glucose-dependent metabolic differences
Authors: Ruegg C. E. , Mandel L. J.
Published: December 2017

A new procedure for separately isolating milligram quantities of rabbit renal proximal straight (PST) or convoluted (PCT) tubules is described, and the differential abilities of these segments to utilize glucose as a metabolic substrate are investigated. Separate dissection of the cortical cortices and the outer medullary stripe, followed by collagenase digestion and discontinuous Percoll centrifugation, provide enriched populations (greater than 98% pure) of PCT (37 mg) and PST (14 mg), respectively, per rabbit. The purity of PCT and PST fractions was quantitated morphologically and by comparing the enriched activity of the proximal tubular marker leucine aminopeptidase and deenriched activity of the distal marker hexokinase to previously published values reported from microdissection studies. To investigate glucose-dependent metabolic differences, PCT and PST suspensions (1 mg/ml) were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being incubated for 30 min in buffer with or without glucose as the only available metabolic substrate. In glucose-containing buffer, PST segments maintained their oxygen consumption and ATP contents at levels significantly higher than PCT segments. These differential responses between PST and PCT were glucose-dependent because they were abolished when segments were incubated under glucose-free conditions. Because responses in PCT were glucose-independent, these results suggest that PCT cannot utilize glucose to support oxidative metabolism, whereas PST segments can oxidatively metabolize this substrate. These differences in glucose utilization do not correlate with the distribution of glycolytic enzyme activities, suggesting that differential metabolic regulation of these enzymes may determine the ability of each segment to utilize glucose.
Three peptides from the ANF prohormone NH2- terminus are natriuretic and/or kaliuretic
Authors: Martin Daniel R. , Pevahouse Joe B. , Trigg David J. , Vesely David L. , Buerkert John E.
Published: December 2017

Pages 1401-1408: Daniel R. Martin, Joe B. Pevahouse, David J. Trigg, David L. Vesely, and John E. Buerkert. “Three peptides from the ANF prohormone NH2- terminus are natriuretic and/or kaliuretic.” Pages 1404 and 1406, Tables 2 and 3, respectively: in the boxhead, the units for UNaV should be μeq·min-1·g kidney wt-1.
Aldosterone induces apical vesicles in rat distal colon
Authors: Perrone R. D. , Nye J. P. , McBride D. E. , Zahniser D. J. , Geyer J. J. , Ucci A. A.
Published: December 2017

Mineralocorticoid steroids markedly alter ion transport in responsive epithelia. Increases in absorption of Na+ and secretion of K+ and H+ are accompanied by increases in surface area of the basolateral membrane. The basolateral membrane changes are associated with increased Na(+)-K(+)-ATPase activity and increased numbers of Na(+)-K(+)-ATPase pump sites. It is thought that H+ secretion is mediated by H+ pumps contained in apical vesicles that are added to the luminal membrane in response to acidifying stimuli. Whether there are changes in the number or volume of apical vesicles in response to aldosterone has not been evaluated. To this purpose, we evaluated apical membrane morphology in rat distal colon, a mineralocorticoid-responsive epithelium. We found that aldosterone infused for 4-7 days by osmotic minipump significantly increased the number, surface density, and total volume of apical vesicles. Exposure of tissues to 5% CO2 for 15 min before fixation resulted in significant decreases in vesicle number, surface density, and volume in aldosterone-stimulated tissues. After CO2, apical vesicles in aldosterone-stimulated tissues tended to be closer to the luminal membrane; apical membrane surface density was increased but not to a significant degree. Fluorescence microscopy demonstrated acridine orange accumulation in discrete points under the lumen, suggesting the presence of acidic vesicles in this location. We propose that aldosterone increases the activity of a membrane shuttle system that is regulated by CO2 as found in other H(+)-secreting epithelia. This system may mediate aldosterone-induced changes in colonic H+ transport.
PGE2 inhibits AVP-induced water flow in cortical collecting ducts by protein kinase C activation
Authors: Hebert R. L. , Jacobson H. R. , Breyer M. D.
Published: December 2017

It is well known that prostaglandin E2 (PGE2) both inhibits arginine vasopressin (AVP)-stimulated water permeability (hydraulic conductivity, Lp) in the cortical collecting duct (CCD) or, if administered alone, modestly increases Lp in the CCD. These bifunctional effects on Lp correspond to PGE2's capacity to inhibit AVP-stimulated adenylate cyclase (AC) activity, or to singularly stimulate AC activity in the collecting duct. The present studies suggest that the inhibitory effect of PGE2 on Lp may also be mediated by phosphatidylinositol (PI) hydrolysis. Using in vitro microperfused rabbit CCDs, we show that PGE2 releases Ca from intracellular stores. We also demonstrate that the inhibitory effect of PGE2 on AVP-stimulated Lp in the CCD is significantly reversed by the protein kinase C (PKC) inhibitor, staurosporine (SSP). Although PGE2 does not reduce an established water flow response to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPTcAMP), when the sequence of addition is reversed and PGE2 is added first, marked inhibition of 8-CPTcAMP-induced Lp is observed. This provides independent evidence that PGE2 can act through a mechanism separate from modulating AC activity. PGE2 inhibition of 8-CPTcAMP-induced Lp is reversed by SSP pretreatment. Finally, SSP pretreatment also markedly potentiates the capacity of PGE2 itself to increase Lp. We conclude that PGE2 releases Ca from intracellular stores and, by activating PKC, inhibits AVP-induced osmotic water flow. This suggests an important role for PI hydrolysis in mediating PGE2's effects on the CCD.
PCO2 in renal cortex
Authors: De Mello Aires M. , Lopes M. J. , Malnic G.
Published: December 2017

In a number of recent investigations a renal cortical PCO2 higher than that of systemic blood was reported. We have studied this problem with the use of micro-Severinghaus electrodes based on antimony, H+ liquid ion exchange, and glass pH electrodes with an inner buffer solution containing 0.5 mg/ml carbonic anhydrase (CA). Measurements in renal cortical structures (renal tubules, star vessels, capillaries, and glomeruli in Munich-Wistar rats) were compared with determinations in renal vein or artery performed with the same electrode in sequence. No significant differences in PCO2 were found between cortical structures and renal vein in control rats, in metabolic alkalosis, respiratory acidosis and alkalosis, and after CA inhibition. Nevertheless, absolute PCO2 levels, which followed the PCO2 of systemic blood, were markedly different in these groups. Measurements of pH and PCO2 at the same tubule site were compatible with HCO3- determinations in tubule fluid in vitro (made with use of the Henderson-Hasselbalch equation) in control rats. When proximal tubules were pump-perfused in vivo with a solution containing 30 mM NaHCO3, measured PCO2 approached that of the perfusing solution at high pump rates, and approached the free-flow value as rates were reduced to zero, indicating that the CO2 generated in the lumen equilibrated rapidly across the epithelium. Reducing renal blood flow by aortic clamping reduced renal cortical PCO2. In conclusion, in a large number of experimental conditions renal cortical PCO2 was never higher than that measured in systemic blood.
Recruitment of renin gene-expressing cells in adult rat kidneys
Authors: Gomez R. A. , Chevalier R. L. , Everett A. D. , Elwood J. P. , Peach M. J. , Lynch K. R. , Carey R. M.
Published: December 2017

To define whether angiotensin I-converting enzyme (ACE) inhibition affects the distribution of renin gene-expressing cells within the kidney, a control group of adult male Wistar-Kyoto rats (C, n = 7) was compared with a group of rats treated with enalapril (E, n = 6) for 5 days. Renin mRNA distribution was assessed using in situ hybridization to a 35S-labeled 28 mer oligonucleotide complementary to rat renin mRNA. Whereas in control rats renin mRNA was confined to a juxtaglomerular location, in enalapril-treated rats, renin mRNA extended proximally along the length of the afferent arteriole. The percent of visible afferent arteriolar length containing renin mRNA was higher in enalapril-treated (71.7 +/- 2.8%) than in control (49.6 +/- 2.1%) rats (P less than 0.0001). These findings were accompanied by an increase in the percent of juxtaglomerular apparatuses (JGAs) containing renin mRNA (71 +/- 2.2 vs. 49 +/- 2.9%; E vs. C, P less than 0.0001). Also, the intensity of the JGA hybridization signals was higher in enalapril-treated (757 +/- 59 grains/JGA) than in control (167 +/- 11 grains/JGA) rats (P less than 0.00001). We conclude that the increased kidney renin gene expression elicited by ACE inhibition is the result of an increase in renin mRNA content per JGA, an increase in the number of JGAs expressing the renin gene, and a recruitment of renin gene-expressing cells along the afferent arteriole.
Glucocorticoid receptors mediate mineralocorticoid-like effects in cultured collecting duct cells
Authors: Naray-Fejes-Toth A. , Fejes-Toth G.
Published: December 2017

To investigate the direct epithelial effects of corticosteroids on renal ion transport, we studied the influence of the pure glucocorticoid agonist RU 28362 and aldosterone on Na+ and K+ transport in primary cultures of immunodissected rabbit cortical collecting duct (CCD) cells. When grown on permeable supports in a steroid-free medium, CCD monolayers exhibited a lumen-negative transepithelial potential difference (PD) of 5.2 +/- 1.07 mV and a short-circuit current (SCC) of 8.54 +/- 2.2 microA/cm2. Transepithelial resistance averaged 660 +/- 49 omega/cm2. The cultures actively reabsorbed Na+ and secreted K+. Both aldosterone and RU 28362 significantly increased PD and SCC; the effects were time and dose dependent. The effect of RU 28362 was completely prevented by the glucocorticoid receptor antagonist RU 486, whereas ZK 91587, a specific mineralocorticoid receptor antagonist, did not block its effect. Both aldosterone and RU 28362 increased the bath-to-lumen concentration ratio of Na+ while lowering that of K+, indicating an increased Na+ reabsorption and K+ secretion. The number of Na(+)-K(+)-ATPase units was significantly enhanced (approximately 2-fold) by both RU 28362 and aldosterone. These results demonstrate that, in cultured CCD cells, not only aldosterone but also a pure glucocorticoid is able to exert mineralocorticoid-like effects, and this latter effect is mediated by glucocorticoid receptors. Because all parameters studied responded similarly to aldosterone and RU 28362, we speculate that in CCD cells glucocorticoids and mineralocorticoids might act by regulating the same gene(s).
Regulation of eicosanoid biosynthesis by phorbol ester in Madin Darby canine kidney cells
Authors: Coyne D. W. , Mordhorst M. , Morrison A. R.
Published: December 2017

We assessed the effects of the peptide agonist, bradykinin (BK), and phorbol myristate acetate (PMA) on prostaglandin E2 (PGE2) production, cyclooxygenase (COX) activity and mass, and arachidonic acid (AA) release in Madin Darby canine kidney (MDCK) cells. PMA stimulated PGE2 production by increasing both AA release and the activity of COX. Using [35S]methionine labeling and immunoprecipitation, we demonstrated that the increased COX activity is due to new COX synthesis. Actinomycin D and cycloheximide blocked the PMA-stimulated COX activity but not AA release. Both PMA-stimulated AA release and COX activity were reduced by the protein kinase C inhibitor staurosporine (STP). Glucocorticoids failed to alter PMA- or BK-stimulated PGE2 production was reduced by STP, indicating BK acts in part through protein kinase C activation. BK increased PGE2 production in PMA-treated cells, suggesting a protein kinase C-independent mechanism of action as well. BK did not stimulate any change in COX activity. We conclude that in MDCK cells PMA, but not BK, can stimulate both AA release and COX synthesis. Stimulation of COX synthesis requires either prolonged activation of protein kinase C and/or an additional nonprotein kinase C-mediated effect of PMA.
Three peptides from the ANF prohormone NH2- terminus are natriuretic and/or kaliuretic
Authors: Martin Daniel R. , Pevahouse Joe B. , Trigg David J. , Vesely David L. , Buerkert John E.
Published: December 2017

Pages 1401–1408: Daniel R. Martin, Joe B. Pevahouse, David J. Trigg, David L. Vesely, and John E. Buerkert. “Three peptides from the ANF prohormone NH2- terminus are natriuretic and/or kaliuretic.” Pages 1404 and 1406, Tables 2 and 3, respectively: in the boxhead, the units for UNaV should be μeg·min-1·g kidney wt-1.
Phosphate-dependent glutaminase activity in rat renal cortical and medullary tubule segments
Authors: Wright P. A. , Knepper M. A.
Published: December 2017

To determine whether local production of ammonium by medullary renal tubule segments may contribute to medullary ammonium accumulation, we measured activities of phosphate-dependent glutaminase (PDG) in microdissected tubule segments from rat medulla and cortex. PDG activities were very low in medullary loop of Henle segments but surprisingly high in inner medullary collecting duct (IMCD). In cortex, PDG levels were highest in distal convoluted tubule and cortical thick ascending limb, but substantial levels were also found in proximal segments, as reported previously. To determine effects of acid loading and alkali loading on PDG activity, 0.28 M NH4Cl (acid) or 0.28 M NaHCO3 (alkali) was added to rats' drinking water for 7 days. PDG activities in medullary segments were not affected by acid or alkali intake. Acid intake by rats increased PDG activity in S1 and S2 proximal convoluted tubules severalfold but did not affect the other cortical segments. We conclude that medullary loop of Henle segments probably contribute relatively little to medullary ammonium accumulation because of their low activities. The high PDG activity in IMCD suggests that ammonium could be produced and secreted by this segment. However, because total tubule length of IMCD is very low compared with proximal tubules, it appears unlikely that IMCD contributes substantially to overall renal ammonium production. PDG activity is regulated only in S1 and S2 proximal tubules, consistent with the view that the proximal tubule is the major site of regulation of renal ammonium production.
Effects of extra- and intracellular pH on vascular action of arginine vasopressin
Authors: Okada K. , Tsai P. , Briner V. A. , Caramelo C. , Schrier R. W.
Published: December 2017

Compared with control studies performed at an extra-cellular pH (pHe) of 7.4, when vascular smooth muscle cells (VSMC) were preincubated in an acid extracellular medium (pHe 7.0) for 60 min the maximal arginine vasopressin (AVP)-induced sustained cellular contraction was reduced, whereas preincubation in an alkaline extracellular medium (pHe 7.8) enhanced the vascular action of AVP. These changes in VSMC contraction were accompanied by a parallel decrease in AVP receptor affinity and cytosolic free calcium ([Ca2+]i) mobilization in the acid medium, increased AVP receptor affinity, and [Ca2+]i mobilization in alkaline medium. Because the changes in pHe were parallelled by changes in pHi, studies were performed in which only pHi was altered by administering the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10(-6) M for 10 min). CCCP inhibited VSMC contraction in the absence of changes in AVP receptor binding and AVP-induced [Ca2+]i mobilization but abolished the AVP-induced cellular alkalinization. The results therefore indicate that in a nonbicarbonate buffer alterations in pHe may modulate the vascular responses to AVP by altering hormone receptor affinity, [Ca2+]i mobilization, and pHi.
Role of renal nerves on pressure natriuresis in spontaneously hypertensive rats
Authors: Yoshida M. , Satoh S.
Published: December 2017

An abnormal rightward shift of the pressure-natriuresis curve is a well known feature of the renal function in hypertension. The participation of intrinsic neural factors in the kidney in this phenomenon was investigated in anesthetized young and adult spontaneously hypertensive rats (SHR). At 7-8 wk of age, the renal pressure-diuresis curve and pressure-natriuresis curve were shifted to the left in denervated SHR compared with innervated animals. Fractional excretion of sodium was higher, and plasma renin activity was lower in denervated SHR. Glomerular filtration rate was not affected by renal denervation. In 13- to 15-wk-old SHR, renal denervation did not affect the pressure-diuresis and -natriuresis curves, although other parameters were changed compared with the results at 7-8 wk. In Wistar-Kyoto rats, the pressure-diuresis curve was shifted to the left by renal denervation at both ages. These results suggest that the renal nerves have an important effect on the renal pressure-diuresis and -natriuresis curves. However, renal innervation cannot be thought to cause an abnormal rightward shift of the pressure-diuresis and -natriuresis curves in SHR, especially in the established stage of hypertension.
Stimulation of renin release by hyperkalemia in the nonfiltering kidney
Authors: Lin H. B. , Young D. B. , Smith M. J.
Published: December 2017

This study was designed to analyze the acute effects of hyperkalemia on renin release in the normal filtering kidney and the nonfiltering kidney. Plasma K was increased by acute intravenous KCl infusion. In the normal filtering kidney experiment plasma K was 5.7 vs. 3.5 meq/l. Hyperkalemia resulted in a 45% increase in renal blood flow (RBF) and a 35% increase in glomerular filtration rate (GFR) at the 120-mmHg pressure level. Renin release was significantly greater in the hyperkalemic group than in the control group (P less than 0.01) with the greatest effect over the lower pressure range. In the nonfiltering kidney experiment plasma K was 6.09 vs. 3.5 meq/l. RBF was 33% greater in the hyperkalemic than in the normokalemic group at the 130-mmHg pressure level. Renin release was also greater in the hyperkalemic group than in the normokalemic group (P less than 0.01). However, unlike the normal filtering kidney experiments, in the nonfiltering kidneys the difference in renin release was most prominent at the highest level of renal perfusion pressure. These experiments demonstrate that acute hyperkalemia can cause renal vasodilation and stimulate renin release in both filtering and nonfiltering kidney preparations and that potassium may affect renin release both through a direct effect on the juxtaglomerular cells and indirectly by affecting delivery of fluid and/or NaCl to the macula densa.
Permeabilities of rat collecting duct segments to NH3 and NH4+
Authors: Flessner M. F. , Wall S. M. , Knepper M. A.
Published: December 2017

We have measured NH3 and NH4+ permeability coefficients in collecting ducts from the cortex, outer medulla, and inner medulla of the rat kidney. Isolated collecting duct segments of the rat were perfused with bicarbonate-buffered solutions containing carbonic anhydrase to eliminate any pH disequilibrium in the tubule lumen. NH3 or NH4+ concentration gradients were set up between the bath and the lumen. By measuring the total CO2 and total ammonia concentrations in the bath, the perfusate, and collected fluid, the NH3 and NH4+ concentrations were determined. Then, using the flow rate in the tubule and the tubule dimensions, we calculated the apparent permeability in each collecting duct segment for NH3 and NH4+. The NH3 permeabilities were as follows: 0.002 cm/s in the inner medullary collecting duct, 0.012 cm/s in the outer medullary collecting duct, and 0.024 cm/s in the cortical collecting duct. The NH4+ permeabilities for all segments were on the order of 10(-5) cm/s or less. The relative permeability values for the two ammonium species are consistent with the view that the secretion observed in vivo in collecting ducts is due to passive diffusion of NH3 from the interstitium to the lumen of the duct, parallel with H+ secretion.
Reset tubuloglomerular feedback permits and sustains glomerular hyperfunction after extensive renal ablation
Authors: Salmond R. , Seney F. D.
Published: December 2017

To determine the contribution of the tubuloglomerular feedback (TGF) system to glomerular hyperfunction after extensive loss of renal mass, TGF was assessed in anesthetized Sprague-Dawley rats 7 days after 5/6 reduction of renal mass (2/3 infarction of 1 kidney plus contralateral nephrectomy) or sham surgery. Five-sixths renal ablation significantly increased single-nephron (SN) glomerular filtration rate (GFR) 57%, late proximal tubule fluid flow 58-63%, and maximal proximal tubule stop-flow pressure (PSF) 24%. Despite these increments, 5/6 ablation did not increase TGF activation, as judged by the difference between proximally and distally measured SNGFR values, nor did it affect maximal TGF responses, measured as change in PSF during forward microperfusion of the loop of Henle. However, 5/6 ablation increased the late proximal perfusion rate eliciting half-maximal PSF suppression (V1/2) from 27.3 +/- 1.6 to 45.7 +/- 2.4 nl/min (P less than 0.001), a change closely matching the increment in native late proximal tubule fluid flow from 26.6 +/- 2.2 to 43.8 +/- 2.1 nl/min (P less than 0.001). Unilateral nephrectomy increased V1/2 less dramatically than did 5/6 ablation, thus showing that the effects of renal ablation on TGF are proportional to the degree of ablation. We conclude that extensive renal ablation resets TGF operation in a way that permits and helps sustain glomerular hyperfunction.
Glomerular B2-kinin-binding sites in two-kidney, one-clip hypertensive rats
Authors: Emond C. , Bascands J. L. , Rakotoarivony J. , Praddaude F. , Bompart G. , Pecher C. , Ader J. L. , Girolami J. P.
Published: December 2017

To extend our recent observations of the possible downregulation of glomerular B2-kinin-binding sites, we investigated density (Bmax) of bradykinin (BK)-binding sites in glomerular membranes of both the clipped (C) and nonclipped (NC) kidneys of two-kidney, one-clip (2K-1C) Goldblatt hypertensive rats, in relation to tissue kallikrein activity and glomerular three-dimensional structure. Compared with the Bmax of sham-operated (SO) kidney (31.8 +/- 7 fmol/mg protein), a significant increase in Bmax was observed in glomeruli of both kidneys in hypertensive rats, the Bmax being higher in glomeruli of NC than in C kidneys (98 +/- 11 vs. 59 +/- 12 fmol/mg protein). NC kidney compensatory hypertrophy was expressed by an increase in glomerular diameter, surface area, and volume. When expressed per unit of area or volume, Bmax in NC kidneys remained significantly higher than in both C and SO kidneys. Increased Bmax in both kidneys of 2K-1C rats was associated with a decreased intrarenal level of kallikrein. We also examined prostaglandin (PG) E2 release by isolated glomeruli from SO, C, and NC kidneys as a possible biological effect induced by BK. Whereas C kidney released more PGE2 than NC kidney under basal conditions, addition of BK (10 nM) induced greater PGE2 production in NC kidney consistent with the difference in Bmax between C and NC kidneys. These results suggest a possible downregulation of glomerular B2-binding sites by bradykinin, which may explain the difference between SO and C kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)
Cl-HCO3 exchange and Na-HCO3 symport in rabbit outer medullary collecting duct cells
Authors: Kuwahara M. , Sasaki S. , Marumo F.
Published: December 2017

The mechanisms of basolateral HCO3- (or related base) transport were examined in the outer stripe of the outer medullary collecting duct (OMCDo). Rabbit OMCDo were perfused in vitro and cell pH (pHi) of principal cells (PC) and intercalated cells (IC) was monitored with 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) using fluorescence ratio imaging microscopy. Basolateral HCO3- exit was stimulated either by bath HCO3- reduction or by NH3/NH4(+)-induced cell alkalinization. Rates of pHi decrease were twice as high in IC than in PC. In both cell types HCO3- exit was dependent on Cl(-)-coupled and Na(+)-coupled mechanisms; the former was dominant in IC, whereas the latter was dominant in PC. pHi of PC and IC significantly increased by bath Cl- removal in the absence of Na+ and decreased by bath Na+ removal in the absence of Cl- but with amiloride. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited these pHi responses to a reduction of bath HCO3-, Cl-, or Na+, and an alkali loading. Our results suggest that 1) the capacity for basolateral HCO3- transport is twice as high in IC than in PC, 2) both PC and IC possess basolateral Na(+)-independent Cl(-)-HCO3- exchange and Na(+)-HCO3- cotransport, and 3) basolateral HCO3- transport is mediated mainly by Na(+)-HCO3- cotransport in PC and by Na(+)-independent Cl(-)-HCO3- exchange in IC.
Regulation of osmotic water permeability during differentiation of inner medullary collecting duct
Authors: Siga E. , Horster M. F.
Published: December 2017

Urinary osmotic concentration capacity during renal ontogeny is subject to changes of medullary cytoarchitecture and of segmental epithelial transport characteristics. Osmotic equilibrium between interstitial and tubular fluid of the terminal nephron segment in response to vasopressin is an absolute essential of maximal urinary osmotic concentration. The regulation of osmotic water permeability (Pf) in this terminal epithelial segment during ontogenetic differentiation has not been documented. The inner medullary collecting duct (IMCD), the terminal 40% of total segmental length, was dissected at two stages of postnatal ontogenetic differentiation from immature (days 7-15) and from mature (days 33-37) rat kidneys and perfused in vitro. Pf (micron/s) was measured (bath hyperosmotic) in the absence and presence of arginine vasopressin (AVP, 230 pM). Basal Pf was 32.3 +/- 4.03 (n = 26) in the immature IMCD (IMCDi) and 111.5 +/- 20.6 (n = 15) in the mature segment (IMCDm). AVP increased Pf in IMCDi from 46.4 +/- 10.5 to 102 +/- 25.7 micron/s, whereas in IMCDm the AVP-dependent change of Pf was from 104.2 +/- 41.2 to 693 +/- 176 micron/s. AVP (2,300 pM) did not further increase Pf in IMCDi. Forskolin (50 microM) changed Pf in IMCDi from 34.9 +/- 6.3 to 104.1 +/- 16 micron/s; the corresponding change in IMCDm was from 150 +/- 32 to 985.8 +/- 133 micron/s. An analogue of adenosine 3',5'-cyclic monophosphate (cAMP; 10(-3) M) increased Pf in IMCDi from 35.5 +/- 11.4 to 138.5 +/- 32.6 and in IMCDm from 79.6 +/- 32.3 to 702.2 +/- 283 micron/s.(ABSTRACT TRUNCATED AT 250 WORDS)
Tubuloglomerular feedback responses to acute contralateral nephrectomy
Authors: Blantz R. C. , Peterson O. W. , Thomson S. C.
Published: December 2017

After unilateral nephrectomy adaptive events must occur in the remaining kidney within the first 12-14 h in anticipation of an increase in glomerular filtration rate (GFR) and eventual renal hypertrophy. Utilizing micropuncture and microperfusion techniques in the rat, we have examined tubuloglomerular feedback (TGF) and single-nephron GFR (SNGFR) responses while the late proximal tubule was microperfused [late proximal tubule flow (VLP)] from 0 to 40 nl/min in 10 nl/min intervals at 2-4 and 12 h after contralateral nephrectomy. Urinary excretion increased, but SNGFR derived from distal collections was reduced, and early distal flow rate remained constant 2-4 h after nephrectomy. The operating point was shifted, suggesting activation of TGF. The turning point half-maximal activity (V1/2) and slope were not statistically different when all nephron data were submitted to a curve-fitting procedure, but group mean data suggested a quantitatively lower V1/2 and steeper slope of the TGF profile. Twelve to fourteen hours after contralateral nephrectomy, values for SNGFR at all microperfusion rates were increased, as were late proximal and early distal flow rates. The values for V1/2 and slope of TGF were not statistically different from control values. We conclude that TGF activity and sensitivity are not suppressed at 2 and 12 h after nephrectomy. Increased urinary excretion does not require TGF alterations. Changes in TGF may be adaptive to increases in SNGFR and may not be causal to the increase in filtration rate after nephrectomy.
Volume regulation and intracellular calcium in the rabbit proximal convoluted tubule
Authors: Beck J. S. , Breton S. , Laprade R. , Giebisch G.
Published: December 2017

The hypothesis that an increase of calcium leads to activation of calcium-activated ionic conductances during cell swelling was examined in the isolated perfused proximal convoluted tubule of the rabbit. Reduction of bath and luminal osmolality by 90 mosmol/kgH2O caused the cells to swell by 23.6 +/- 1.5% (n = 5) and intracellular calcium to rise from 227 +/- 35 to 347 +/- 60 nM (n = 6). Both these increases were transient, with volume decreasing to 5.5 +/- 1.2% above control and intracellular calcium concentration decreasing to 272 +/- 46 nM after 5-9 min. The addition of glucose and alanine to the tubule lumen to increase transcellular sodium transport caused a sustained increase in cell volume of 15.6 +/- 3.4% (n = 4). In parallel experiments, no significant increase in intracellular calcium concentration was observed. Addition of 1 microM of the calcium ionophore, ionomycin, reversibly increased intracellular calcium by 224 +/- 60 nM from a control value of 301 +/- 29 nM (n = 7) and reversibly depolarized the basolateral membrane by 3.6 +/- 0.9 mV (n = 5). However, there was no initial increase in the apparent transference number for potassium or chloride and no significant change in cell volume. We conclude from these observations that the sustained increase in basolateral potassium conductance observed when cells are swollen by hypotonicity or increased sodium transport (J. S. Beck and D. J. Potts. J. Physiol. Lond. 425: 369-378, 1990) is not due to a calcium-activated potassium conductance.
Adaptation of distal convoluted tubule of rats. II. Effects of chronic thiazide infusion
Authors: Morsing P. , Velazquez H. , Wright F. S. , Ellison D. H.
Published: December 2017

Mammalian distal tubules adapt structurally and functionally when NaCl concentration in tubule fluid is altered chronically. These experiments were designed to test the hypothesis that chronic administration of hydrochlorothiazide (HCTZ), a drug that blocks Na and Cl uptake across apical membranes of rat distal tubule cells, would reduce intrinsic transport capacity of distal tubules and reduce the number of thiazide-sensitive transporters. Osmotic pumps were implanted into rats to deliver 3.75 mg/day HCTZ or vehicle for 10-14 days. All animals were offered a solution containing 0.8% NaCl and 0.1% KCl as drinking fluid. Free-flow micropuncture after 10–14 days indicated that Na and Cl delivery to distal tubule was not significantly different in HCTZ- and vehicle-treated animals. Microperfusion in vivo with an artificial interstitial solution, with no thiazide, indicated that 10-14 days of HCTZ infusion did reduce Na transport capacity of distal tubules from 390 +/- 32 to 203 +/- 24 pmol/min (P less than 0.01). In contrast, the number of thiazide-sensitive NaCl transporters, determined as high-affinity receptors for [3H]metolazone in renal cortical membranes, was higher in HCTZ group than in controls (2.2 +/- 0.4 vs. 1.0 +/- 0.1 pmol/mg protein, P less than 0.01). These data support the hypothesis that chronic blockade of NaCl entry across apical membranes of distal tubule cells reduces NaCl transport capacity, an effect that occurs despite an increase in the number of thiazide receptors. They indicate that thiazide receptor binding studies should be interpreted in combination with direct functional measurements.
ADH-induced water permeability and particle aggregates: alteration by a synthetic estrogen
Authors: Calamita G. , Le Guevel Y. , Bourguet J.
Published: December 2017

In the amphibian urinary bladder, the increase in water permeability induced by antidiuretic hormone (ADH) is accompanied by the appearance of apical intramembrane particle (IMP) aggregates that are believed to contain specific channels for water. In a previous work, we have shown that 3,3'-diallyldiethylstilbestrol (DADES), a synthetic estrogen which is a blocker of the glucose transporter, also inhibits the hydrosmotic response to ADH in the bladder. Our aim in the present study was to analyze the alterations of the membrane fine structure further and to correlate them with the water permeability changes. The results point to a selective inhibition of the ADH-induced net water flow, probably due to an interference with one of the last steps of the response to the hormone. This inhibition is associated with an increase in the density of the apical IMP aggregates, which are thus probably not operational. The resting net water flow is not inhibited and, surprisingly, typical IMP aggregates are frequently observed in the apical membrane after DADES treatment. The compound also induces the appearance of unusual loose IMP clusters that can only be seen on the apical membrane of the granular cells and that share several ultrastructural similarities with the ADH-induced aggregates. These results suggest that 1) apical DADES treatment stimulates the insertion of IMP aggregates in the apical membrane of the urinary bladder and 2) DADES inhibits the ADH-induced water flow by interfering with the aggregates and thus probably by blocking the specific water channels.
Changes in NMR-visible kidney cell phosphate with age and diet: relationship to phosphate transport
Authors: Barac-Nieto M. , Dowd T. L. , Gupta R. K. , Spitzer A.
Published: December 2017

To test the hypothesis that growth and dietary Pi affect the intracellular concentration of Pi ([Pi]i) as well as its renal reabsorption, we measured nuclear magnetic resonance (NMR)-visible [Pi]i in isolated perfused kidneys of less than 1- and greater than 4-wk-old guinea pigs fed various amounts of Pi. Changes in [Pi]i were correlated with those in fractional Pi reabsorption (FRPi) in vivo and in capacity (Vmax) for Na(+)-Pi cotransport in microvilli derived from animals of similar age and fed the same diets. In animals fed normal (0.76% Pi) diet, [Pi]i was lower (0.91 +/- 0.14 vs. 1.85 +/- 0.23 mM, P less than 0.05), whereas FRPi was higher (0.90 +/- 0.02 vs. 0.70 +/- 0.03, P less than 0.01) in less than 1- than in greater than 4-wk-old guinea pigs. Pi deprivation decreased [Pi]i in mature animals to 0.74 +/- 0.29 mM, P less than 0.05, and increased FRPi to 0.99 +/- 0.01. Excess dietary Pi increased [Pi]i in immature animals to 1.67 +/- 0.56 mM, P less than 0.05, and decreased FRPi to 0.55 +/- 0.03. Diet-induced changes in [Pi]i were associated with reciprocal changes in Vmax of similar absolute magnitude in immature and mature animals. However, diets that resulted in comparable [Pi]i at the two ages were associated with higher (P less than 0.05) Vmax in less than 1- than in greater than 4-wk-old animals. The reciprocal nature of the relationship between [Pi]i and renal Pi transport indicates that [Pi]i is primarily determined by Pi efflux from the cells or Pi organification rather than Pi influx through Na(+)-Pi cotransport. Findings indicate that changes in [Pi]i with growth or diet may be a cause but cannot be the consequence of changes in abundance or maximal mobility of Na(+)-Pi cotransporters. Data also indicate that factors in addition to low [Pi]i contribute to the high Na(+)-Pi cotransport capacity observed in renal microvilli of growing animals.
A Ca-activated K channel from rabbit renal brush-border membrane vesicles in planar lipid bilayers
Authors: Zweifach A. , Desir G. V. , Aronson P. S. , Giebisch G. H.
Published: December 2017

Rabbit renal brush-border membranes were fused to planar lipid bilayers to gain insight into the nature and properties of ion channels from the luminal membrane of the proximal tubule. Fusion was obtained using osmotic gradients. A large conductance channel was commonly observed. Measurements of reversal potentials indicated that the channel was selective for K over Rb, Na, and Cl. Channel open probability was increased by membrane depolarization and by increased Ca activity on the intracellular face of the channel. The channel was inhibited by charybdotoxin (CTX), a protein from leiurus venom, from the external side of the channel. The channel was also blocked by Ba and quinidine added to the intracellular bathing solution. Na added to the intracellular bathing solution reduced current amplitude in a voltage-dependent fashion. In addition, methylisobutyl amiloride, an analogue of the K-sparing diuretic amiloride, inhibited channel activity when added to the external solution. The possible physiological role of the channel is discussed. The usefulness to the study of renal ion channels of the technique of fusing membrane vesicles to planar lipid bilayers is evaluated.
Role of cytoskeleton in isotonic cell volume control of rabbit proximal tubules
Authors: Linshaw M. A. , Macalister T. J. , Welling L. W. , Bauman C. A. , Hebert G. Z. , Downey G. P. , Koo E. W. , Gotlieb A. I.
Published: December 2017

Stability of mammalian cell volume depends primarily on the sodium pump. When active cation transport of rabbit renal proximal tubules is blocked by ouabain, cells swell, but their size is limited by residual volume control mechanisms. This “ouabain-resistant” volume control is not an active process, as it operates in the presence of cyanide and dinitrophenol and in the absence of exogenous energy. Nevertheless, it remains incompletely explained by known transmembrane oncotic and hydrostatic forces. We tested the hypothesis that the cytoskeleton contributes to isotonic cell volume control. Isolated, collapsed rabbit proximal convoluted tubules (PCT) were crimped at both ends with micropipettes and had their volume assessed optically. PCT in ouabain (1 mM) swelled to 1.40 above control with protein, 1.62 without protein, and 1.89 with the cytoskeleton inhibitors vincristine (5 microM) and cytochalasin B (50 microM) and without protein. Tubulozole-C and cytochalasin D gave similar results. A hydrostatic pressure of 50 cmH2O increased tubule volume to 1.93 before the tubule basement membrane (TBM) prevented further volume increase. We conclude that volume of renal tubule cells in ouabain is limited partly by external protein, but primarily by the cytoskeleton. The TBM prevents massive swelling and tubule disaggregation.
Effects of maleic acid on renal phosphorus transport: role of dietary phosphorus
Authors: Guntupalli J. , Delaney V. , Weinman E. J. , Lyle D. , Allon M. , Bourke E.
Published: December 2017

The effects of maleic acid on renal phosphate (Pi) transport were examined by clearance and brush-border membrane vesicle (BBMV) transport studies. In normal rats, maleic acid 50 mg.kg body wt-1.h-1 increased the phosphaturia (P less than 0.001). Intraperitoneal administration of a similar dose of maleic acid decreased the BBMV uptake of Pi but not glucose. In rats fed a low-phosphate diet (0.03%), the maleic acid-induced phosphaturia was blunted, but the inhibitory effect on the BBMV transport of Pi persisted. In chronic parathyroidectomized rats fed a low-phosphate diet, where the filtered load of Pi was higher than in the previous groups, the phosphaturia was abolished, but the inhibition of the BBMV transport of Pi was sustained. Both the in vitro incubation of BBMVV and in vivo administration of maleic acid were associated with a competitive inhibition of Pi transport. These studies indicate that the maleic acid-induced phosphaturia is expressed at the apical membrane entry step of Pi, and the enhanced distal tubular reabsorption accounts for the lack of phosphaturia in dietary Pi deprivation.
Polyclonal antibodies in study of ADH-induced water channels in frog urinary bladder
Authors: Valenti G. , Calamita G. , Svelto M.
Published: December 2017

It is now generally accepted that changes in water permeability in anti-diuretic hormone (ADH)-responsive target epithelial cells result from the insertion in the plasma apical membrane of new components that contain channels for water. The specificity of these channels suggests that they are formed by intrinsic proteins having access to both facies and spanning the whole membrane. We have previously shown that Triton X-100 apical extracts from ADH-stimulated frog urinary bladder contain some proteins inserted under hormonal stimulation. In the present study we have developed polyclonal antibodies using Triton X-100 extract as an immunogen. After considering the inhibitory effect exerted by the whole immune serum on the osmotic water flow, we used different adsorption steps to select, from the immune serum, antibodies to apical membrane proteins inserted in response to the hormone. Immunoblot analysis of these selected antibodies shows that they recognize seven to eight proteins, of which 55-, 35-, 26-, and 17-kDa proteins are always present. Antibodies to these four proteins, affinity purified on nitrocellulose sheets, inhibited ADH-induced osmotic water flow. Altogether these results strongly suggest that proteins of 55, 35, 26, and 17 kDa (or at least one of them) are likely to be involved in the mechanism of water transport.
Pathways of acute pH regulation of ammoniagenesis in LLC-PK1 cells: study with [15N]glutamine
Authors: Sahai A. , Nissim I. , Tannen R. L.
Published: December 2017

The present study utilized [15N]glutamine labeled at amide (5-N) and amino (2-N) groups to analyze the metabolic fate of glutamine nitrogen in basal and in acute pH regulation of ammoniagenesis. One-hour incubation of LLC-PK1 cultures in a media of pH 7.4, 7.0, or 7.6 containing either [5–15N]glutamine or [2–15N]glutamine resulted in parallel alterations in glutamine consumption in response to acute acid-base maneuvers. Incubation with [5–15N]glutamine resulted in substantial enrichment and production of ammonia at pH 7.4, which was unaffected by the changes in media pH, and in no significant enrichment of alanine, aspartate, and glutamate. In contrast, significant enrichment and production of 15N-labeled ammonia, alanine, aspartate, and glutamate were detected from cultures incubated with [2–15N]glutamine. Ammonia formation, from incubation with [2–15N]glutamine, was stimulated significantly by a low pH and inhibited by high pH. Alanine production was altered in a fashion similar to ammonia formation, whereas aspartate production was unaltered and glutamate formation significantly decreased by a low pH. Furthermore, a low pH significantly increased the production of alpha-ketoglutaramate in a fashion qualitatively similar to alanine production. In contrast to our prior conclusions based on total metabolite production, these studies indicate that although ammonia formation at pH 7.4 is predominantly generated from the mitochondrial phosphate-dependent glutaminase pathway, the increased ammonia formation in acute acidosis is a result of increased flux through glutamate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
Thromboxane stimulates synthesis of extracellular matrix proteins in vitro
Authors: Bruggeman L. A. , Horigan E. A. , Horikoshi S. , Ray P. E. , Klotman P. E.
Published: December 2017

The vasoconstrictor eicosanoid thromboxane plays an important role in the pathogenesis of several renal diseases. As an autacoid, its local release alters blood flow and induces platelet aggregation. We report a direct stimulatory effect of thromboxane on extracellular matrix protein production and gene expression in vitro. Treatment of two cell types, differentiated mouse teratocarcinoma cells (F9+) and human glomerular mesangial cells, with two different thromboxane analogues resulted in increased production of components of the extracellular matrix including fibronectin and the basement membrane proteins laminin and type IV collagen. These responses to thromboxane were not the result of a mitogenic effect of thromboxane nor the result of an increase in total cellular protein. The increased production of extracellular matrix proteins was, at least in part, due to an increase in the steady-state level of mRNA for these genes. Furthermore, the effect of thromboxane was markedly inhibited by cotreatment with a thromboxane-receptor antagonist. These results suggest a new potential role for thromboxane as a mediator of the sclerotic and fibrotic responses to injury.