bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status
Authors: Emmons C. , Stokes J. B.
Published: December 2017

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.
Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule
Authors: Poncet V. , Tauc M. , Bidet M. , Poujeol P.
Published: December 2017

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.
Trafficking of apical proteins into clathrin-coated vesicles isolated from rat renal cortex
Authors: Hammond T. G. , Verroust P. J.
Published: December 2017

The endosomal pathway of the rat renal cortex was labeled by intravenous infusion of fluorescent dextran small enough to cross the glomerular ultrafiltration barrier and be taken up by luminal endocytosis in the proximal tubule. Clathrin-coated vesicles (CCV) were isolated from the rat renal cortex utilizing discontinuous sucrose density gradients and negative lectin selection. More than 99 +/- 1% (n = 4) of the isolated vesicles contain entrapped fluorescein dextran when analyzed by small-particle flow cytometry techniques. Similarly, flow cytometry analysis demonstrates brisk H(+)-adenosinetriphosphatase activity in virtually all the vesicles. Western blot analysis of the vesicle proteins with a polyclonal anticlathrin antibody stains bands consistent with clathrin and adaptins. When the isolated vesicles are decoated by exposure to 0.5 M tris(hydroxymethyl)aminomethane, the proteins released match the molecular weights of the proteins identified on Western blot analysis. Flow cytometry demonstration of brush border enzymes in > 99% of the vesicles and Western blot identification of maltase suggests both that these vesicles are of apical origin and that apical enzymes traffic into endosomal elements. Additionally, two glycoproteins detectable in this fraction on Western blot analysis and flow cytometry immunocytochemistry are derived from intermicrovillar clefts traffic into the endosomal pathway. Hence, apical proteins traffic into a population of CCV isolated from the rat renal cortex.
Effects of endothelin on rat renal proximal tubule Na(+)-Pi cotransport and Na+/H+ exchange
Authors: Guntupalli J. , DuBose T. D.
Published: December 2017

Endothelin (ET), a powerful vasoconstrictive peptide, is distributed ubiquitously in various organs, including the vascular endothelium and tubules of the kidney. Although localized more abundantly to the glomerulus and inner medullary collecting duct, ET receptors have been identified in the proximal tubule. The possible effects of ET on proximal tubule transport and the potential role of second messengers in this process have not been described fully. To define the role of ET in proximal tubule transport, renal cortical slices were incubated for 3 min in the presence of various concentrations of ET. Incubation with low concentrations of ET-1 (1 x 10(-9) to 1 x 10(-11) M) within the physiological range stimulated both Na(+)-Pi cotransport and Na+/H+ exchange. Pretreatment with staurosporine (0.6 microM) for 25 min abolished completely the ET-induced effects on Na(+)-Pi cotransport and Na+/H+ exchange. Similarly, preincubation with phorbol ester 12-O-tetradecanoylphorbol-13-acetate (200 nM) also abolished the effects of ET on these transporters. Incubation with ET decreased significantly intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Intravenous administration of pertussis toxin for 2 days prevented the ET-induced decrease in cAMP and abolished the stimulatory effects of ET on Na(+)-Pi cotransport and Na+/H+ exchange. These findings provide indirect evidence that ET participates in the regulation of proximal tubular Pi and bicarbonate homeostasis. These effects of ET are mediated by activation of protein kinase C and cAMP-dependent protein kinase A.
Interleukin-1 alpha stimulates KC synthesis in rat mesangial cells: glucocorticoids inhibit KC induction by IL-1
Authors: Feng L. , Xia Y. , Kreisberg J. I. , Wilson C. B.
Published: December 2017

To assess the possible role of the production of chemokines by intrinsic glomerular cells in the generation of inflammation in glomerulonephritis, the chemokine, KC, was cloned from a rat macrophage cDNA library. Transfection of rat KC into COS-7 cells resulted in increased neutrophil chemotactic activity. The KC cDNA was expressed as a fusion protein in Escherichia coli for generation of an antibody. By using a riboprobe derived from the cDNA and the antibody, interleukin-1 (IL-1) was found to induce the expression of KC in rat mesangial cells. The induction of KC by IL-1 could be inhibited by dexamethasone (DEX). The protein synthesis inhibitor cycloheximide reversed the DEX-mediated inhibition, which suggested that new protein synthesis was necessary for the inhibitory effect. A nuclear runoff analysis indicated that DEX inhibited the transcription of KC induced by IL-1. The stability of KC mRNA was not decreased in the presence of DEX. Furthermore, immunoblots showed that DEX also inhibited KC expression at the level of translation. Together the inhibition of transcription and translation of the KC gene by DEX contribute to decreased KC expression in mesangial cells. The finding that mesangial cells express KC in response to proinflammatory cytokines, such as IL-1, points to a central role for the mesangial cell as a chemotactic source in glomerular inflammation.
Endogenous renal dopamine production regulates phosphate excretion
Authors: Debska-Slizien A. , Ho P. , Drangova R. , Baines A. D.
Published: December 2017

We examined the effect of endogenous dopamine production on Pi and citrate excretion by Wistar rats. Carbidopa (20-40 mumol/kg ip) decreased dopamine, Pi, and citrate excretion within 20 min (86%, 47%, and 38%, respectively); Pi reabsorption increased 11 +/- 4% (P = 0.03). The decreases were sustained for at least 18 h. 3-Hydroxybenzylhydrazine (45 mumol/kg ip) reduced Pi excretion 24%. Benserazide (40 mumol/kg ip and 0.1 mumol/min iv) reduced dopamine excretion (94%) and blocked the effect of carbidopa on Pi and citrate excretion. In isolated perfused kidneys benserazide, carbidopa, and 3-hydroxybenzylhydrazine all decreased Pi excretion. Dopamine (1 mumol/l) added to cortical minceates reduced brush-border membrane vesicle (BBMV) 32P uptake by 8% (P < 0.02) and amiloride-inhibitable 22Na uptake by 19%. Carbidopa added to minceates increased 32P uptake by 12%. Carbidopa pretreatment increased (75%) amiloride-sensitive 22Na uptake into BBMV of rats fed a high-salt diet. Uptake was not increased into BBMV from rats fed a low-salt diet. Carbidopa increased (17%) basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) gradually over 4 h. Na(+)-K(+)-ATPase did not increase in rats fed a low-phosphorous diet, but did increase when dopa was added to the diet. Thus endogenous dopamine appears to directly control Na(+)-Pi and Na+/H+ transport and secondarily alter basolateral membrane Na(+)-K(+)-ATPase.
In vivo modulation of rat distal tubule net HCO3 flux by VIP, isoproterenol, angiotensin II, and ADH
Authors: Levine D. Z. , Iacovitti M. , Buckman S. , Harrison V.
Published: December 2017

To examine the in vivo effects of agonists reported to influence bicarbonate flux (JtCO2), microperfusion experiments were carried out on distal tubules of normally fed or overnight-fasted rats. As we previously reported, distal tubules from fed rats reabsorbed no bicarbonate, whereas overnight-fasted rats consistently reabsorbed bicarbonate (JtCO2 10 +/- 3 pmol.min-1.mm-1; P < 0.01). Vasoactive intestinal peptide and isoproterenol infused intravenously (7.3 and 4.0 micrograms.kg-1.h-1, respectively) in fasted rats suppressed JtCO2 and, in the case of vasoactive intestinal peptide, elicited net bicarbonate secretion (JtCO2 -10 +/- 2 and -4 +/- 4 pmol.min-1.mm-1, respectively). In fed rats, angiotensin II infused at a rate of 1.2 micrograms.kg-1.h-1 stimulated bicarbonate reabsorption (JtCO2 16 +/- 3 pmol.min-1.mm-1), while antidiuretic hormone infused at 0.024 micrograms.kg-1.h-1 elicited a similar response (17 +/- 4 pmol.min-1.mm-1), both values being significantly different from control. These results, therefore, demonstrate for the first time that these agonists can modulate JtCO2 at the distal tubule site in vivo and therefore may be potential regulators of systemic acid-base balance.
Inhibition of bicarbonate transport in peanut lectin-positive intercalated cells by a monoclonal antibody
Authors: Fejes-Toth G. , Naray-Fejes-Toth A. , Satlin L. M. , Mehrgut F. M. , Schwartz G. J.
Published: December 2017

Intercalated cells (ICC) of the collecting duct are believed to secrete acid (alpha-type) or HCO3 (beta-type). Although these two types of ICC are functionally mirror images of each other, several components in their cell membranes are clearly unique. As a first step in defining the molecular composition of beta-ICC membranes, we raised cell-specific monoclonal antibodies (MAb) against surface antigens. One of these MAb, designated B63, reacts with the apical membrane of peanut lectin agglutinin (PNA)-positive cells of the kidney cortex. B63-positive cells do not react with antibodies against band 3 (the basolateral C1/HCO3 exchanger) or ST.48, markers for alpha-ICC and principal cells, respectively. Despite a significant positive correlation between PNA and B63 staining intensities, determined by flow cytometry, these markers react with separate antigens, as indicated by competition studies and the different distribution of the two antigens. In addition to renal beta-ICC, B63 antigen is present in tissues that are involved in HCO3 secretion, such as the pancreas, salivary glands, and the small intestine, suggesting that it might play a role in HCO3 secretion. To test this hypothesis more directly, we tested the effect of MAb B63 on HCO3 secretion and on changes in intracellular pH (pHi) in isolated perfused cortical collecting ducts. Luminal Cl removal in the presence of luminal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid resulted in a reversible increase in pHi. Luminal application of MAb B63 prevented this change in pHi. MAb B63 also significantly inhibited (by 37.7 +/- 7.3%) HCO3 secretion by isolated perfused tubules, whereas another MAb (MAb 601), which reacts with a separate antigen on beta-ICC, did not alter HCO3 secretion or pHi. These results indicate that B63 antigen plays an important role in HCO3 secretion: it might be either the apical anion exchanger of beta-ICC or an associated regulatory protein.
Angiotensin II type 1 receptor: role in renal growth and gene expression during normal development
Authors: Tufro-McReddie A. , Johns D. W. , Geary K. M. , Dagli H. , Everett A. D. , Chevalier R. L. , Carey R. M. , Gomez R. A.
Published: December 2017

To determine whether angiotensin II (ANG II) modulates renal growth and renin and angiotensin type 1 (AT1) gene expression via AT1 during development, weanling rats were given ANG II antagonist losartan (DuP 753) for 3 wk. Body weight (g), kidney weight (g), and kidney weight-to-body weight ratio were lower in losartan-treated rats (162 +/- 7, 1.6 +/- 0.06, and 9.5 +/- 0.1 x 10(-3)) than in control rats (184 +/- 5, 1.8 +/- 0.07, and 10.1 +/- 0.1 x 10(-3); P < 0.05). Renal DNA content (mg/kidney) was lower in losartan-treated (2.4 +/- 0.17) than in control rats (3.3 +/- 0.31; P < 0.05), whereas protein-to-DNA and RNA-to-DNA ratios were similar in losartan-treated and control rats. Renin mRNA levels were sevenfold higher in losartan-treated than in control rats, as determined by quantitative standardized dot blot analysis. In addition, blockade of AT1 with losartan induced recruitment of renin-synthesizing and renin-containing cells in the renal vasculature, as determined by immunocytochemistry and in situ hybridization. To establish whether AT1 blockade has a direct effect on renin gene expression, freshly isolated renin-producing cells were exposed in vitro to losartan (10(-6) M) or culture media (control). Losartan induced a twofold increase in steady-state renin mRNA levels above control (P < 0.05). Intrarenal AT1 mRNA levels were not altered by losartan given either in vivo or in vitro to freshly dispersed cells. To define whether immature renin-secreting cells are responsive to ANG II, renin release was determined by reverse hemolytic plaque assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Distal tubule unidirectional HCO3 reabsorption in vivo during acute and chronic metabolic alkalosis in the rat
Authors: Levine D. Z. , Iacovitti M. , Buckman S. , Vandorpe D. , Harrison V. , Nadler S. P.
Published: December 2017

During metabolic alkalosis (MA) associated with 2 days of dietary chloride restriction, there is net bicarbonate secretion by rat distal tubules in vivo, whereas after 5 wk of chloride depletion alkalosis there is net bicarbonate reabsorption. To examine unidirectional components of net bicarbonate reabsorption during chronic MA, we measured distal tubule unidirectional bicarbonate secretion (Jsec) and reabsorption (Jreab), as well as the inhibitor sensitivity of Jreab. In control, 2-day, and 7-day alkalosis, Jsec was similar. Jreab, however, was only present in 7-day MA (17 +/- 3 pmol.min-1.mm-1, P < 0.05). This Jreab was completely suppressed by perfusion with 10(-7) M bafilomycin A1, partially suppressed with 10(-5) M Schering (Sch)-28080 (4 +/- 2 pmol.min-1.mm-1, P < 0.1), and converted into a secretory flux by 3 mM amiloride. We conclude that adaptation to chloride depletion MA from the acute secretory phase to the chronic state, where plasma bicarbonate is sustained at elevated levels, does not involve suppression of distal tubule Jsec but rather enhanced Jreab, which is sensitive to bafilomycin, Sch-28080, and amiloride.
ATP-dependent transport of tetraethylammonium by endosomes isolated from rat renal cortex
Authors: Pritchard J. B. , Sykes D. B. , Walden R. , Miller D. S.
Published: December 2017

During renal organic cation secretion by some species, intracellular concentrations greatly exceed the 10- to 15-fold ratio predicted by the potential-driven mechanism thought to mediate their basolateral uptake. Free cytoplasmic organic cation concentrations within the tubular cells might be decreased through sequestration within intracellular organelles. The data reported here show that endosomal vesicles isolated from rat renal cortex take up tetraethylammonium (TEA) by an ATP-dependent mechanism. Addition of 0.2-5 mM ATP to the medium stimulated uptake 5- to 10-fold at 5 min and 20-fold at 60 min. More than 80% of the ATP-dependent uptake was associated with an osmotically active space. The nonhydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate), did not stimulate TEA uptake. Mg2+ and Cl- were required for stimulation. Uptake was inhibited by several organic cations, including TEA itself. Uptake was also inhibited by inhibitors of intravesicular acidification, e.g., monensin and N-ethylmaleimide. Furthermore, the ATP requirement could be bypassed by establishing a pH gradient (inside acidic). These data show that endosomal TEA accumulation is mediated by proton/TEA exchange and is driven by the pH gradient maintained by H(+)-adenosinetriphosphatase. This potent sequestration mechanism may play an important role in organic cation secretion.
Differentiation of intercalated cells in developing rat kidney: an immunohistochemical study
Authors: Kim J. , Tisher C. C. , Madsen K. M.
Published: December 2017

Intercalated cells are present in both the collecting duct, which is derived from the ureteric bud, and the connecting tubule (CNT), which is part of the nephron and thus is developed from the metanephric blastema. However, the embryologic origin of the intercalated cells has not been established. Two populations of intercalated cells, type A and type B, exist in the CNT and the cortical collecting duct (CCD). It is uncertain, however, whether these cells represent truly distinct cell types or whether one is derived from the other. In this study we have used specific antibodies to carbonic anhydrase II (CA II), H(+)-adenosinetriphosphatase (H(+)-ATPase), and band 3 protein to identify subpopulations of intercalated cells, to determine the site and time of their appearance, and to follow their differentiation in the developing rat kidney. Prenatal kidneys from 16-, 17-, 18-, and 20-day-old fetuses, and postnatal kidneys from 0-, 3-, 7-, 14-, and 21-day-old pups were preserved for immunohistochemical studies. Immunostaining for CA II and H(+)-ATPase appeared simultaneously in a subpopulation of cells in the CNT and the medullary collecting duct (MCD) of the 18-day-old fetus, suggesting that intercalated cells differentiate from separate foci, one in the nephron and one in the collecting duct. Cells with apical and cells with basolateral labeling for H(+)-ATPase appeared in the CNT and MCD at 18 days of gestation, indicating that type A and type B cells differentiate simultaneously during renal development. Band 3 immunostaining was very weak in the fetal kidney, but a striking increase in labeling was observed in the 3-day-old kidney, suggesting that there is an activation of acid-secreting cells shortly after birth. In the fetal kidney, immunostaining for CA II and H(+)-ATPase was observed in cells throughout the MCD and on the papillary surface. After birth, immunostaining gradually disappeared from both the papillary surface and the terminal inner MCD, and cells with basolateral labeling for H(+)-ATPase gradually disappeared from the outer MCD. The results of this study suggest that type A and type B intercalated cells represent distinct cell types that derive from undifferentiated cells at two separate foci, one in the nephron and one in the collecting duct. Our results also suggest that entire populations of intercalated cells are eliminated from the collecting duct during normal renal development.
Hydrogen and potassium regulation of (pro)renin processing and secretion
Authors: King J. A. , Fray J. C.
Published: December 2017

H and K ions play central roles in prorenin processing and secretion, and prorenin is abnormally expressed in H and K disorders. At the surface membrane of juxtaglomerular (JG) cells, K is sensed and regulated by K channels (coupled to Cl channels and activated by excess Ca), Na-K-adenosinetriphosphatase, and a KCl/H exchange transporter (regulated by Ca). In JG cell granular membrane, K flux is regulated by K channels and a KCl/H exchange transporter (activated by Ca). H channels and a H pump reside in the granular membrane, which maintain H concentration in the granular matrix at least two orders of magnitude greater than in cytosol. The H pump may also be responsible for maintaining the acidic matrix required for maximal prorenin processing to renin by prohormone convertase for human renin (PCren), the prorenin convertase. These molecules form the core of a chemiosmotic system, which appears to regulate both prorenin processing and renin secretion. Renin secretion and prorenin processing appear to be of more than causal significance in clinical disorders characterized by chemiosmotic imbalance. A critical review of the literature supports the following general conclusions. First, hyperrenin state defines the initial phase in the pathogenesis of heart disease, diabetes mellitus, and hypertension. Second, low-renin syndrome defines the transition-to-establish phase in the pathogenesis of heart disease, diabetes mellitus, and hypertension in which the key feature is renin secretory hyporesponsivity. Third, renin disorders are usually associated with other endocrine disorders (polyendocrinopathies types I, II, and III), suggesting that renin may be an important molecule in the processing of chemiosmotic forces. The key chemiosmotic molecules (K and H) are also important in the processing and export of most (if not all) hormones. Thus, by regulating K and H homeostasis, renin may regulate the endocrine system.
Intracellular Ca2+ release and Ca2+ influx during regulatory volume decrease in IMCD cells
Authors: Tinel H. , Wehner F. , Sauer H.
Published: December 2017

Volume changes and cytosolic Ca2+ concentration ([Ca2+]i) of inner medullary collecting duct (IMCD) cells under hypotonic stress were monitored by means of confocal laser scanning microscopy and fura 2 fluorescence, respectively. Reduction of extracellular osmolality from 600 to 300 mosmol/kgH2O by omission of sucrose led to an increase in cell volume within 1 min to 135 +/- 3% (n = 9), followed by a partial regulatory volume decrease (RVD) to 109 +/- 2% (n = 9) within the ensuring 5 min. In parallel, [Ca2+]i rose from 145 +/- 9 to 433 +/- 16 nmol/l (n = 9) and thereafter reached a lower steady state of 259 +/- 9 nmol/l. Under low-Ca2+ conditions (10 nmol/l) RVD was not impeded and reduction of osmolality evoked only a transient increase of [Ca2+]i by 182 +/- 22 nmol/l (n = 6). Preincubation with 100 mumol/l 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8) or 20 mmol/l caffeine, both effective inhibitors of Ca2+ release from intracellular stores, in low Ca2+ as well as in high Ca2+, inhibited the Ca2+ response and abolished RVD. The temporal relationship between Ca2+ release from intracellular stores and Ca2+ entry was analyzed by determining fura 2 quenching, using Mn2+ as a substitute for external Ca2+. Intracellular Ca2+ release preceded Mn2+ influx by 17 +/- 3 s (n = 10). Mn2+ influx persisted during the whole period of exposure to hypotonicity, indicating that there is no time-dependent Ca2+ channel inactivation. Preincubation with TMB-8 or caffeine reduced Mn2+ influx to the control level, indicating that activation of Ca2+ channels in the plasma membrane occurs via intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)
Detection of interactions between myogenic and TGF mechanisms using nonlinear analysis
Authors: Chon K. H. , Chen Y. -M. , Marmarelis V. Z. , Marsh D. J. , Holstein-Rathlou N. -H.
Published: December 2017

Pages F160-F173: K. H. Chon, Y.-M. Chen, V. Z. Marmarelis, D. J. Marsh, and N.-H. Holstein-Rathlou. “Detection of interactions between myogenic and TGF mechanisms using nonlinear analysis.” Pages F168-F169: The original Figs. 8-11 do not give sufficient detail of the contour plots. New, higher resolution images are reproduced here with the original legends.
Cyclooxygenase inhibitors suppress inhibitory effect of PGE2 on Na-K-ATPase in MDCK cells
Authors: Cohen-Luria R. , Moran A. , Rimon G.
Published: December 2017

In a previous work, we showed that picomolar concentrations of prostaglandin E2 (PGE2) inhibit Na-K-ATPase activity and ouabain binding in a clone of Madin-Darby canine kidney (MDCK) cells. In the present study, we demonstrate that the inhibitory effects of PGE2 on Na-K-ATPase activity, ouabain-sensitive Rb+ uptake, and ouabain binding in MDCK cells were diminished by treatment of the cells with nonsteroidal anti-inflammatory drugs. These results suggested that products of arachidonic acid synthesized through the cyclooxygenase pathway are involved in the inhibitory mechanism of PGE2. Treatment of the cells with arachidonic acid resulted in inhibition of ouabain binding, and the inhibition was eliminated by cyclooxygenase inhibitors. These observations further support the involvement of cyclooxygenase products in the PGE2-induced inhibitory process. Finally, we demonstrated that dopamine inhibits Rb+ influx and ouabain binding in MDCK cells similarly to PGE2. Cyclooxygenase inhibitors suppressed the inhibition of ouabain binding by dopamine, thus also suggesting the involvement of cyclooxygenase products in the inhibitory effect of dopamine.
Tubuloglomerular feedback and tubular reabsorption during acute potassium loading in rats
Authors: Braam B. , Boer P. , Koomans H. A.
Published: December 2017

Acute hyperkalemia has been associated with changes in reabsorption, glomerular filtration rate (GFR), and autoregulation, which might represent altered tubuloglomerular feedback (TGF) responsiveness. Therefore, TGF responsiveness, segmental reabsorption of water, sodium and potassium, and single-nephron GFR were evaluated during acute potassium loading in male Sprague-Dawley rats. Rats receiving 300 mM KNO3, KHCO3, and KCl showed significantly increased plasma potassium levels and attenuation of stop-flow pressure responses 45-90 min after starting the potassium infusion compared with that observed in time controls and rats infused with 300 mM NaCl. Attenuation of TGF responsiveness could not be related to plasma and kidney angiotensin II levels. Segmental water and sodium handling and proximal to distal single-nephron GFR differences assessed in a time control group and a group receiving 300 mM KCl revealed no changes related to KCl infusion. However, late proximal and early distal potassium concentrations increased significantly from 4.7 +/- 0.2 to 6.3 +/- 0.3 mM (P < 0.01) and from 1.5 +/- 0.1 to 2.7 +/- 0.4 mM (P < 0.01), respectively. In summary, although attenuated TGF responsiveness was demonstrated at higher perfusion rates, this study does not support a significant role for either the TGF mechanism or changes in reabsorption upstream of the early distal tubule for the initiation of kaliuresis during acute potassium loading.
Ammonia transport in a mathematical model of rat proximal tubule
Authors: Weinstein A. M.
Published: December 2017

Pathways for ammonia transport have been incorporated within a model of rat proximal tubule [A. M. Weinstein. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F784-F798, 1992]. The luminal membrane includes a Na+/NH4+ exchanger, while at the peritubular membrane there is uptake of NH4+ on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase); both luminal and peritubular cell membranes contain conductive pathways for NH4+. The model equations have been expanded to include cellular ammoniagenesis. The principal focus of this study is the interplay of forces that can raise proximal tubule fluid total ammonia concentration 10-fold higher than in arterial plasma. Analysis of a cellular model reveals that luminal membrane Na+/NH4+ exchange, cellular production of ammonia, and peritubular membrane NH4+ uptake (via Na(+)-K(+)-ATPase or via K+ channel) all act in parallel to drive ammonia secretion. This derives from the cellular interconversion of NH4+ and NH3 and the free permeation of NH3 across cell membranes. It implies that inhibition of the luminal membrane transporter does not block the contribution of peritubular uptake to the overall active transport of ammonia. Conversely, when inhibition of the luminal membrane Na+/NH4+ entry (i.e., Na+/H+ inhibition) depresses transcellular Na+ flux, then the decrease of NH4+ flux through the peritubular Na+ pump enhances the apparent importance of the luminal membrane pathway. This analysis is confirmed in the numerical calculations and is a departure from the Ussing paradigm of series membrane Na+ transport. Although active secretion of ammonia by this tubule is substantial, the relative contribution of luminal Na+/NH4+ exchange and of peritubular uptake via the Na+ pump remains uncertain. The determination of peritubular capillary NH4+ concentration will be crucial to resolving this uncertainty, with lower concentration (i.e., closer to systemic arterial ammonia) obligating greater luminal membrane Na+/NH4+ exchange.
Juxtamedullary microvascular responses to arginine vasopressin in rat kidney
Authors: Harrison-Bernard L. M. , Carmines P. K.
Published: December 2017

Experiments were performed to determine the site of arginine vasopressin (AVP)-induced vascular resistance adjustments that result in decreases in papillary blood flow. Simultaneous measurements of luminal diameter and centerline erythrocyte velocity allowed estimation of single-vessel blood flow in in vitro blood-perfused juxtamedullary nephrons. AVP (0.1-1,000 pM) caused concentration-dependent reductions in outer medullary descending vasa recta (OMDVR) blood flow (10 pM) without altering OMDVR diameter. Afferent arteriolar diameter was significantly decreased by 1 pM AVP, whereas arcuate artery diameter was decreased by 100 pM AVP. Increasing the concentration of AVP to 1,000 pM resulted in significant reductions of vessel diameter in interlobular arteries, distal afferent arterioles, and efferent arterioles. The effects of AVP to decrease afferent arteriolar diameter and blood flow were blocked in the presence of V1 receptor antagonist. These data indicate that afferent arterioles exhibit the greatest sensitivity to the vasoconstrictor effects of AVP, whereas OMDVR appear insensitive. We conclude that the change in OMDVR blood flow produced by AVP is not due to a direct effect of the peptide on this vascular segment but to its effect on upstream vessels.
Insulin stimulates NaCl transport in isolated perfused MTAL of Henle's loop of rabbit kidney
Authors: Ito O. , Kondo Y. , Takahashi N. , Kudo K. , Igarashi Y. , Omata K. , Imai Y. , Abe K.
Published: December 2017

To elucidate the mechanism of Na+ retention by insulin in vivo, the direct tubular effect of insulin on NaCl transport in the in vitro microperfused medullary thick ascending limb of Henle (MTAL) was examined. Insulin at 10(-6) mol/l in the bath increased transepithelial voltage (Vte) from 3.1 +/- 0.3 to 5.7 +/- 0.3 mV (n = 12, P < 0.0001). The effect of insulin on Vte was dependent on its concentration, and the half-maximal effect of insulin was observed at 5 x 10(-9) mol/l. Insulin at 10(-6) mol/l also caused a significant decrease of luminal Cl- concentration from 85.4 +/- 5.0 to 62.8 +/- 3.0 mmol/l (n = 5, P < 0.002) when the lumen was microperfused constantly at less than 1 nl/min. Insulin at 10(-6) mol/l also increased net lumen-to-bath Cl- flux (JCl) from 143 +/- 15 to 292 +/- 37 pmol.mm-1.min-1 (n = 5, P < 0.004). When the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in the basolateral membrane was blocked by 10(-4) mol/l ouabain, the insulin-mediated increase in Vte was completely suppressed. When the Na(+)-K(+)-2Cl- cotransporter in the luminal membrane of the MTAL was blocked by 10(-4) mol/l furosemide, the insulin-mediated increase in Vte was also abolished. To test whether adenosine 3',5'-cyclic monophosphate (cAMP) contributes to the action of insulin, we examined the effect of cAMP analogue and cAMP-dependent protein kinase inhibitor on the action of insulin. A maximal concentration (5 x 10(-4) mol/l) of dibutyryl-cAMP (DBcAMP) increased Vte and JCl.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of losartan and enalapril on renal adaptation sodium restriction in rat
Authors: Jover B. , Saladini D. , Nafrialdi N. , Dupont M. , Mimran A.
Published: December 2017

The influence of losartan (10 or 30 mg.kg-1.day-1), enalapril (10 mg.kg-1.day-1), and combined treatment by losartan and enalapril on the renal adaptation to dietary sodium withdrawal was assessed in normal rats. Treatments were given by gavage for 3 days before and during the 6-day period of low-sodium (LS) diet. Cumulative sodium excretion during LS was similar in untreated and low-dose losartan groups (0.62 +/- 0.07 and 0.75 +/- 0.07 mmol/6 days), whereas it was significantly increased in groups treated by the high dose of losartan and enalapril alone or combined with both doses of losartan (1.38 +/- 0.16, 1.50 +/- 0.10, 1.37 +/- 0.16, and 1.12 +/- 0.03 mmol/6 days, respectively). A decrease in conscious systolic arterial pressure was observed in all treated groups in response to LS. At the end of LS, conscious renal blood flow (microsphere method) was similarly increased in all treated groups. Creatinine clearance decreased to a similar extent with both doses of losartan, whereas a further reduction was observed with enalapril given alone or combined with losartan. These results demonstrate that the enalapril-induced disturbance in the response of renal sodium excretion to LS is mainly related to angiotensin-mediated mechanisms. However, non-angiotensin-related actions of enalapril may contribute to the deterioration of renal function in sodium-restricted animals. In addition, a high dose of losartan is required to impair renal sodium conservation, thus suggesting that the tubular renin-angiotensin system may play a crucial role in the renal adaptation to dietary sodium withdrawal.
Distribution of kallikrein-binding protein mRNA in kidneys and difference between SHR and WKY rats
Authors: Yang T. , Terada Y. , Nonoguchi H. , Tsujino M. , Tomita K. , Marumo F.
Published: December 2017

We investigated kallikrein-binding protein (KBP) mRNA distribution in the kidney of Sprague-Dawley (SD) rats, spontaneously hypertensive rats (SHR), and Wistar-Kyoto strain (WKY) rats. Northern blot analysis revealed that KBP mRNA was located mainly in the medulla and with lower amounts in SHR than in WKY rats. KBP mRNA in microdissected nephron segments was detected by reverse transcription and polymerase chain reaction (RT-PCR) followed by Southern blot analysis. In SD rats, the most abundant signals were consistently found in inner medullary collecting duct (IMCD), with small amounts in outer medullary collecting duct, proximal convoluted tubule, and glomerulus. No signals were found in connecting tubule and cortical collecting duct. The nephron distribution of KBP mRNA was similar in WKY and SD rats. Only a small amount of signal was found, however, in IMCD of SHR. In conclusion, 1) KBP mRNA was predominantly distributed in the medullary segments of the distal nephron, downstream from the known kallikrein activity site in the collecting duct, and 2) KBP mRNA expression was significantly decreased in the kidney of SHR.
cAMP-dependent effects of vasopressin and calcitonin on cytosolic calcium in rat CCD
Authors: Siga E. , Champigneulle A. , Imbert-Teboul M.
Published: December 2017

Fura 2 fluorescence measurements were carried out on microperfused rat cortical collecting ducts (CCD) to investigate the effect of adenosine 3',5'-cyclic monophosphate (cAMP) and adenylate cyclase-stimulating hormones on free cytosolic calcium ([Ca2+]i). Forskolin, 3-isobutyl-1-methylxanthine, and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) all triggered marked and sustained [Ca2+]i variations. Maximal increases elicited by 100 microM CPT-cAMP amounted to 101 +/- 11 nM (mean +/- SE, n = 18). This effect was mostly dependent on the presence of basolateral calcium and totally independent of luminal calcium. It remained unchanged in CCD perfused with sodium-free luminal fluid (82 +/- 10 nM, n = 5), pretreated with 1 mM bath ouabain (113 +/- 20, n = 4), or superfused with sodium-free bath in the presence of ouabain (82 +/- 22, n = 5). The V2 agonist 1-desamino-8-D-arginine vasopressin (DDAVP, 10 nM) increased [Ca2+]i by 57 +/- 5 nM (n = 27), a value 40% lower than that achieved with 10 nM AVP (141 +/- 7, n = 34) but similar to that observed with AVP + a V1a antagonist (57 +/- 6, n = 6). Significant effects could also be obtained with 200 pM DDAVP (31 +/- 6, n = 8) and arginine vasopressin (AVP) (72 +/- 6, n = 16). Rat calcitonin also raised [Ca2+]i by 43 +/- 10 (n = 8) and 66 +/- 8 nM (n = 17) at 1 and 10 nM, respectively, and its effect was not additive to that of CPT-cAMP. Calcitonin and DDAVP effects, like those of CPT-cAMP and forskolin, were nearly abolished in Ca(2+)-free bath, but AVP action on intracellular release persisted. These results show that, in rat CCD, cAMP effects on [Ca2+]i mainly result from basolateral calcium entry. In contrast to rabbit CCD the mechanism is independent on Na reabsorption and basolateral Na+/Ca2+ exchange. Calcitonin and DDAVP effects on [Ca2+]i are probably secondary to increased cAMP production.
Effect of respiratory acidosis and respiratory alkalosis on renal transport enzymes
Authors: Eiam-ong S. , Laski M. E. , Kurtzman N. A. , Sabatini S.
Published: December 2017

We studied the effect of respiratory acidosis and respiratory alkalosis on acid-base composition and on microdissected renal adenosinetriphosphatase (ATPase) enzymes. Rats were subjected to hypercapnia or hypocapnia of 6, 24, and 72 h duration. After 6 h of hypercapnia, collecting tubule (CT) ATPases were not changed. At 24 h, plasma bicarbonate was 35 +/- 1 meq/l (P < 0.01) and CT H-ATPase and H-K-ATPase activities were 90% greater than controls (P < 0.01). By 72 h, plasma bicarbonate was 37 +/- 1 meq/l (P < 0.005 vs. control) and CT enzyme activity had increased even more, averaging approximately 130% of control (P < 0.05). Significant increases in enzyme activities were also observed in the proximal convoluted tubule and medullary thick ascending limb. Plasma aldosterone was three to four times that of control at all three time periods. In hormone-replete adrenalectomized rats, acid-base parameters and ATPase activities were the same as those seen in adrenal intact animals. After 6 h of hypocapnia, plasma bicarbonate was not significantly changed, but H-ATPase and Na-K-ATPase activities were decreased by 35% along the entire nephron (P < 0.05). H-K-ATPase activity in CT also decreased by 35%. At 24 h, plasma bicarbonate was 20.5 +/- 0.5 meq/l (P < 0.05 vs. control) and CT H-ATPase and H-K-ATPase activities were 60% less than control (P < 0.01). By 72 h, plasma bicarbonate was 18.5 +/- 0.5 meq/l (P < 0.05); however, only CT H-ATPase activity continued to fall, averaging 75% less than control (P < 0.005). Hypocapnia had no effect on plasma aldosterone or potassium. These results demonstrate that chronic, but not acute, respiratory acidosis stimulates activity of both renal proton ATPases. By contrast, both acute and chronic respiratory alkalosis decrease the two renal proton pumps. The stimulatory effect of hypercapnia and the inhibitory effect of hypocapnia on the renal ATPases appear to be potassium and aldosterone independent. Although the precise mechanisms for these results are not known, a direct effect of PCO2, pH, or changes in bicarbonate delivery may be involved.
Dissociation of ammoniagenic enzyme adaptation in rat S1 proximal tubules and ammonium excretion response
Authors: DiGiovanni S. R. , Madsen K. M. , Luther A. D. , Knepper M. A.
Published: December 2017

We measured ammonium production rates, phosphate-dependent glutaminase (PDG) activity, and glutamate dehydrogenase (GDH) activity in microdissected S1 proximal tubules of rats to investigate the role of adaptations of PDG activity and GDH activity in response to a step increase in acid intake. In vivo ammonium excretion increased much more rapidly than did single-tubule ammonium production in vitro or ammoniagenic enzyme activities measured in microdissected tubules, manifesting an 85-fold increase in the first 24 h. In vitro ammonium production rates in microdissected tubules rose only twofold in the first 24 h, fourfold by day 2, and fivefold by day 4 of acid loading. The adaptation of PDG activity paralleled the increase in single-tubule ammoniagenic capacity measured in vitro. GDH activity, on the other hand, did not change significantly even after 4 days of acid loading. From these observations, we conclude that 1) the adaptation of in vitro ammoniagenic capacity in S1 proximal tubules is temporally associated with an adaptation in PDG activity and not GDH activity, and 2) a major portion of the increased ammonium excretion seen in the first 24 h is due to factors other than an adaptive increase in ammoniagenic enzyme activity.
Epidermal growth factor receptor activation in developing rat kidney
Authors: Cybulsky A. V. , Goodyer P. R. , McTavish A. J.
Published: December 2017

Epidermal growth factor (EGF) binding increases in late-gestational rat kidney and then falls toward basal adult levels postnatally during the 1st wk. We report that the increase in EGF binding is accompanied by an increase in EGF receptor (EGFR) protein and activation of EGFR tyrosine kinase. Multiple proteins were endogenously tyrosine phosphorylated in kidney membranes from fetal rats, and the phosphorylation pattern was similar in rats ranging from 16 to 21 days of gestation. Tyrosine phosphorylation was, however, almost undetectable in 12-wk adult rat kidneys (controls). Among the phosphoproteins in fetal kidney, a prominent 170-kDa protein was identified as EGFR. Endogenous tyrosine phosphorylation of EGFR (reflecting receptor activation) was 30-fold higher in fetal kidney membranes than in adult (3- to 7-fold higher when adjusted for differences in EGF binding or EGFR protein content). The EGFR substrate, phospholipase C-gamma 1, was tyrosine phosphorylated in fetal kidneys but not adult, and a greater proportion was membrane-associated in fetal kidneys, consistent with activation of phospholipase C-gamma 1. Thus EGFR tyrosine kinase activity is increased in late-gestational rat kidney. Induction and activation of EGFR may mediate perinatal renal cell growth and development.
Effects of ANG II, ETA, and TxA2 receptor antagonists on cyclosporin A renal vasoconstriction
Authors: Conger J. D. , Kim G. E. , Robinette J. B.
Published: December 2017

The renin-angiotensin system, endothelin (ET), and vasoconstrictor prostaglandins have been reported in separate studies to mediate the renal vasoconstrictor effect of cyclosporin A (CsA). However, direct comparison of the relative importance of these potential mediators has not been performed. In this study, the attenuating effects of comparable agonist-inhibiting doses of receptor antagonists for angiotensin II (ANG II), DuP-753 at 2.5 mg/kg, for ETA, BQ-123 at 0.5 mg/kg, and for thromboxane A2 (TxA2), SQ-29,548 at 1.6 mg.kg-1.h-1, or saline vehicle on acute CsA (20 mg/kg) renal vasoconstriction were compared in anesthetized Sprague-Dawley rats. All three receptor antagonists significantly limited the CsA-induced increase in renal vascular resistance; however, BQ-123 and SQ-29,548 were more effective than DuP-753. Because all three receptor antagonists demonstrated at least some attenuation of CsA-induced renal vasoconstriction, the potential role of acute CsA-related nitric oxide synthase (NOS) inhibition and nonspecific heterologous effects of specific receptor antagonists on other agonists were determined to exclude the possibilities that there was a general increased agonist sensitivity and that detection of a single or primary constrictor mediator was obscured by "crossover" receptor antagonist effects. CsA significantly reduced renal blood flow (39%) in the presence of the NOS inhibitor, N omega-nitro-L-arginine methyl ester, and there was negligible indication that receptor antagonists had nonspecific effects. It is concluded that CsA-induced renal vasoconstriction is complex and involves activation of multiple constrictor agonists independently or sequentially.
Modulation of thromboxane receptor activation in rat glomerular mesangial cells
Authors: Spurney R. F. , Middleton J. P. , Raymond J. R. , Coffman T. M.
Published: December 2017

Rat glomerular mesangial cells were used to investigate mechanisms of thromboxane A2 (TxA2) receptor regulation in the kidney. Exposure of mesangial cells to the TxA2 agonist U-46619 for 10 min reduced subsequent TxA2-induced increases in inositol phosphates and intracellular Ca2+ levels by approximately 70%. This loss of receptor responsiveness could be blocked by the TxA2 receptor antagonist SQ-29548 and was reversible after removal of agonist from the incubation medium. Radioligand binding studies using the TxA2 agonist [125I]BOP suggested that exposure of mesangial cells to U-46619 for 10 min reduced TxA2 receptor responsiveness without a loss of receptor sites from plasma membrane fractions of the cell, although the density of mesangial cell TxA2 receptors was decreased by approximately 60% after more prolonged exposure of mesangial cells to thromboxane agonists. Both desensitization to U-46619 and loss of TxA2 binding sites could be attenuated by the protein kinase C (PKC) inhibitors staurosporine, sphingosine, or H-7, and TxA2 receptor responsiveness was reduced in cells incubated with phorbol esters before stimulation with thromboxane agonists. We conclude that 1) agonist-specific decreases in TxA2 receptor responsiveness may involve initial uncoupling of the receptor from its effector systems, followed by a loss of TxA2 receptor sites from plasma membrane fractions of the cell, and 2) PKC may be involved in these processes.