bluuebunny/crossref_metadata_2025 huggingface dataset:
Potassium conductance regulation by pH during volume regulation in rabbit proximal convoluted tubules
Authors: Beck J. S. , Breton S. , Giebisch G. , Laprade R.
Published: December 2017

When rabbit proximal convoluted tubules were microperfused in the presence of bicarbonate, a 90 mosmol hypotonic shock hyperpolarized the basolateral membrane by 5.5 +/- 1.4 mV, increased basolateral potassium selectivity (tK) from 0.30 +/- 0.02 to 0.45 +/- 0.02, and reduced the basolateral membrane resistance from 4,887 +/- 821 to 2,836 +/- 602 omega.cm. These data show that the hypotonic shock increased absolute basolateral potassium conductance. The same hypotonic shock elevated intracellular pH from 7.18 +/- 0.04 to 7.31 +/- 0.04. When bath pH was increased by 0.2 pH units (by reduction of CO2), intracellular pH rose by 0.13 +/- 0.01. In separate experiments this maneuver hyperpolarized the basolateral membrane by 5.0 +/- 0.8 mV and augmented basolateral tK from 0.58 +/- 0.06 to 0.68 +/- 0.04, suggesting that the basolateral potassium conductance is sensitive to pH changes of a magnitude similar to that evoked by a hypotonic shock. In the nominal absence of bicarbonate or presence of 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) in the bath, the hypotonic shock caused a transient intracellular acidification, suggesting involvement of basolateral bicarbonate transport in the hypotonic shock-induced alkalinization. In the absence of bicarbonate, the hypotonic shock did not increase basolateral tK or induce hyperpolarization of the basolateral membrane. We conclude that the increase in potassium conductance observed during hypotonic shock is at least partly mediated by a bicarbonate-dependent, SITS-sensitive intracellular alkalinization.
Induction of water diuresis by endothelin in rats
Authors: Schnermann J. , Lorenz J. N. , Briggs J. P. , Keiser J. A.
Published: December 2017

Experiments were performed in anesthetized rats to examine the possibility that endothelin (ET) modifies renal epithelial function in addition to its well-established hemodynamic actions. Infusion of ET-3 at rates between 34 and 178 ng.kg-1.min-1 was in many cases followed by a rise in urine flow and a persistent decrease in urine osmolality, whereas glomerular filtration rate (GFR) did not significantly change. The extent of ET-induced diuresis was dependent on the response of GFR: in rats in which ET-3 infusion caused a marked reduction of GFR (greater than 70%) ET-induced diuresis was not seen, even though urine osmolality still fell significantly. From animal to animal, ET-induced changes of urine flow or GFR did not correlate significantly with the rate of ET-3 infusion. ET-1, another ET isopeptide, also produced water diuresis when administered in GFR-neutral doses. Urinary excretion of total solutes and of sodium was not significantly altered by ET-3. Infusion of vasopressin blunted the diuretic effect of ET-3, whereas ET-3-induced water diuresis was not measurably altered by chronic or acute treatment with a converting enzyme inhibitor or by acute inhibition of prostaglandin synthesis. Induction of water diuresis was not secondary to an inhibition of vasopressin secretion since it could be demonstrated in homozygous Brattleboro rats in which antidiuresis was produced by the infusion of vasopressin at a rate of 200 microU.kg-1.min-1. These data suggest that ET may be an inhibitory modulator of the hydrosmotic action of vasopressin at the level of the renal collecting duct.
Enhanced intrarenal angiotensin II generation in response to obstruction of the pig ureter
Authors: Frokiaer J. , Knudsen L. , Nielsen A. S. , Pedersen E. B. , Djurhuus J. C.
Published: December 2017

The effect of acute unilateral complete ureteral obstruction (UUO) on renal hemodynamics has been examined in a pig model. Pigs were operated on under general anesthesia, electromagnetic flow probes were inserted around renal arteries, and catheters were implanted in renal veins and aorta for blood sampling. The influence of 15 h of obstruction on the physiological variation in immunoreactive angiotensin II (ANG II) was measured together with detailed analysis of the renal handling of ANG II in anesthetized animals. Pelvic pressure rose significantly within 2 h to a maximum of 59.9 +/- 2.9 cmH2O (n = 10). The ipsilateral renal blood flow (RBF) was reduced by 30% from 319 +/- 35 to 241 +/- 42 ml/min (P = 0.001), whereas contralateral RBF did not change. A temporary significant increase in mean aortic blood pressure was seen during the first 2 h after UUO. Renal vascular resistance increased by 44% in the obstructed kidney from 37 +/- 3 to 55 +/- 9 mmHg.ml-1.min.g during the course of obstruction. Concomitantly, the plasma concentration of immunoreactive ANG II increased from all three sample sites by 8-15 times. This increase was highly significant compared with the sham-operated group. The results show that the pig kidney responds to UUO by a net secretion of ANG II from the ipsilateral kidney. Our observations on the ANG II renal secretion rate suggest that part of the immunoreactive ANG II in the renal venous effluent originated de novo from intrarenal generation.
Intrarenal handling of proteins in rats using fractional micropuncture technique
Authors: Tojo A. , Endou H.
Published: December 2017

Previous micropuncture studies on protein handling along the nephron could not exclude the possibility of contamination by extratubular proteins. Thus we developed a fractional micropuncture method. Renal tubules were punctured with an outer puncture pipette, into which an inner collection pipette was inserted repeatedly to collect tubular fluid, usually up to four fractions. The albumin concentration of tubular fluid was highest in the first fraction and gradually decreased to a constant level, indicating physiological albumin concentrations. On the other hand, low-molecular-weight protein (LMWP) concentrations showed no significant difference among the four fractions. By plotting the protein delivery in the fourth fraction along the nephron, glomerular filtrated protein concentrations were estimated by extrapolating the tubular fluid-to-plasma inulin concentration ratio into one. The glomerular filtrated albumin was 22.9 micrograms/ml (0.00062 in filtration coefficient), and that of LMWP was 72.1 (0.988). Albumin was almost evenly reabsorbed in early (37%) and late (34%) proximal convoluted tubules and the straight tubules (23%). On the other hand, LMWP was more strongly reabsorbed in the early proximal convoluted tubules (54%) than in the late ones (28%) or the straight portion (5%). The fractional micropuncture procedure provides direct evidence of protein handling along the nephron without extratubular protein contamination.
Inhibition of renin secretion from rat renal cortical slices by (R)-12-HETE
Authors: Henrich W. L. , Falck J. R. , Campbell W. B.
Published: December 2017

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) inhibits renin secretion both in vivo and in vitro, but the enzymatic origin of the 12-HETE responsible for renin inhibition is unknown. These studies examined the effect of the 12-HETE stereoisomers (R)-12-HETE (a product of the cytochrome P-450 monooxygenase enzyme system) and (S)-12-HETE (a product of the lipoxygenase enzyme system) on basal and stimulated renin secretion from superficial cortical slices in the rat. First, rat cortex was shown to produce 12-HETE; chiral-phase high-performance liquid chromatography revealed that cortex produced 81% (S)-12-HETE and 19% (R)-12-HETE. (R)-12-HETE reduced basal renin release by 28 +/- 4% to 49 +/- 5% at concentrations of 10(-9) to 10(-7) M (P < 0.05 to 0.01). (S)-12-HETE did not significantly affect renin release. (R)-12-HETE also blunted isoproterenol-stimulated renin secretion (P < 0.05) at a concentration of 10(-6) M. 20-HETE, another cytochrome P-450 product, did not exert a significant effect on renin release. In summary, both (R)-12-HETE and (S)-12-HETE are synthesized by renal cortical tissue. Only (R)-12-HETE directly inhibits in vitro renin release. These findings suggest that the renal cytochrome P-450 enzyme system is capable of directly influencing local renin secretion.
Vasopressin resistance in potassium depletion: role of Na-K pump
Authors: Mujais S. K. , Chen Y. , Nora N. A.
Published: December 2017

Resistance to the hydrosmotic effects of vasopressin has been described in K depletion. It is not clear whether other effects of vasopressin, notably its effects on the Na-K pump in the collecting duct, are similarly affected. Adrenalectomized male Sprague-Dawley rats were allocated to either a normal K (NK) or low-K (LK) diet. Na-K pump activity (pmol.mm-1.h-1) in cortical collecting duct (CCD) and medullary collecting duct (MCD) was determined at 21 days after allocation to the dietary groups before and after exogenous vasopressin (0.1 U twice daily for 3 days). In animals on NK diet, vasopressin (AVP) led to a doubling of Na-K pump activity in the CCD from 502 +/- 47 to 1,144 +/- 41 pmol.mm-1.h-1 (P < 0.01). In K-depleted animals, which had a higher baseline Na-K pump activity, an increase was also observed from 1,056 +/- 97 to 1,239 +/- 65 pmol.mm-1.h-1 (P < 0.05), but this increase was quantitatively less, with the change being 183 vs. 642 pmol.mm-1.h-1 in K-replete rats. The findings in the MCD were similar; in rats on a NK diet, AVP led to a significant increase in Na-K pump activity from 498 +/- 29 to 830 +/- 28 pmol.mm-1.h-1 (P < 0.01). With K depletion, this directional change was preserved, increasing from 1,380 +/- 49 to 1,556 +/- 45 pmol.mm-1.h-1 (P < 0.05), but was quantitatively less than in K-replete rats, the change being 176 vs. 332 pmol.mm-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Adriamycin nephropathy: a model to study effects of pregnancy on renal disease in rats
Authors: Podjarny E. , Bernheim J. , Rathaus M. , Pomeranz A. , Tovbin D. , Shapira J. , Bernheim J.
Published: December 2017

The influence of pregnancy on the evolution of primary renal disease is still a matter of controversy. Hypertension and derangement of renal function may occur. The pathophysiology of these complications is poorly understood. In the present study, we assessed the influence of pregnancy on the evolution of adriamycin (Adr) nephropathy. Four groups of animals were studied: 1) control virgin rats (C), 2) normal pregnant rats (NP), 3) virgin rats with nephropathy (Adr), and 4) pregnant rats with nephropathy (Adr-P). Inulin clearance measured at the end of pregnancy in awake rats was similar in NP (1.68 +/- 0.20 ml/min) and C (1.39 +/- 0.03 ml/min). In Adr-P rats, it tended to decrease (1.22 +/- 0.7 vs. 1.93 +/- 0.44 ml/min in Adr rats). Mean arterial pressure was increased in Adr-P rats (137 +/- 2.5 vs. 95 +/- 3.2 mmHg in NP; P < 0.001). Urinary protein excretion was 216 +/- 61 mg/day in Adr-P compared with 28.7 +/- 18 mg/day in Adr (P < 0.001). A significant increase in the glomerular thromboxane B2-to-prostaglandin E2 ratio was found in Adr-P rats (1.15 +/- 0.26 vs. 0.52 +/- 0.12 in Adr rats; P < 0.03). In NP rats, no change was observed. Kidneys and placentas were normal on light and electron microscopy. Thus pregnant rats with adriamycin nephropathy developed a clinical picture with several features of preeclampsia. Changes in glomerular prostanoid synthesis might play a role in the development of this complication.
Intracellular pH regulation in cultured renal proximal tubule cells in different stages of maturation
Authors: Ekblad H. , Aperia A. , Larsson S. H.
Published: December 2017

This study examines the ontogeny of cellular pH regulation in renal proximal tubule cells (RPTC). RPTC from 8- to 40-day-old Sprague-Dawley rats (RPTC-8 to RPTC-40) were studied after 48 h of primary culture. Intracellular pH (pHi) was measured by quantitative fluorescence microscopy using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Recordings were made under basal conditions and after imposing a cytoplasmic alkalosis and acidosis using 15 mM NH4+ salt. The net recovery rate (dpHi/dt) from intracellular acidosis increases significantly between 10 and 12 days of age from 0.39 +/- 0.04 to 0.54 +/- 0.06 pH units/min (P < 0.05, n = 10 vs. 6). This increase can be completely accounted for by an increase in the rate of amiloride (100 microM)-inhibitable Na(+)-H+ exchange (0.29 +/- 0.04 vs. 0.42 +/- 0.05 pH units/min, P < 0.05, n = 6 vs. 6). The rate of Na(+)-H+ exchange increases similarly in RPTC-10 and RPTC-40 when the transmembrane Na+ gradient is increased by Na+ depleting the cells (48 and 49%, respectively). The amiloride-insensitive recovery is Na+ independent and insensitive to 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS, 500 microM) (range 0.08-0.14 pH units/min). The net recovery rate from intracellular alkalosis is significantly lower in RPTC-10 than in RPTC-40 (0.16 +/- 0.02 vs. 0.28 +/- 0.02 pH units/min, P < 0.01, n = 4 vs. 5). SITS (500 microM) inhibits the recovery by 27 +/- 8 and 26 +/- 9%, respectively, whereas amiloride has no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
Protective and specificity-conferring mechanisms of mineralocorticoid action
Authors: Morris D. J. , Souness G. W.
Published: December 2017

Several experiments using both the in vivo rat bioassay and the isolated toad bladder preparation have been reported that lend support to the hypothesis that the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD), in mineralocorticoid (MC) target tissues, acts as a “protective” mechanism that prevents endogenous glucocorticoids (GCs) from causing MC receptor-mediated effects on Na+ and K+. Additional experiments have also indicated that a second distinct protective mechanism exists that prevents endogenous MCs from eliciting excessive MC receptor-mediated Na+ retention. This second protective mechanism may involve steroid-metabolizing enzymes other than 11 beta-OHSD. Since the specific GC agonist, RU28362, can elicit Na+ retention and K+ secretion, it is possible that a third protective mechanism exists whereby renal 11 beta-OHSD prevents endogenous GCs from eliciting GC receptor-mediated effects on Na+ and K+.
Induction and intracellular localization of HSP-72 after renal ischemia
Authors: Van Why S. K. , Hildebrandt F. , Ardito T. , Mann A. S. , Siegel N. J. , Kashgarian M.
Published: December 2017

To determine whether heat shock proteins (HSPs) might be active in cellular recovery following transient ischemia, we examined rat kidneys for 70-kDa HSP (HSP-70) mRNA expression, protein elaboration, and intracellular localization after 45 min of renal ischemia and reflow of 15 min, 2, 6, and 24 h. Inducible HSP-70 mRNA is present at 15 min of reperfusion, peaks between 2 and 6 h, and falls by 24 h. Inducible 72-kDa HSP (HSP-72) protein accumulates progressively through 24 h and is found in both soluble and microsomal fractions following ischemia. Within proximal tubules, immunofluorescent localization of HSP-72 is restricted to the apical domain at 15 min, is dispersed through the cytoplasm in a vesicular pattern at 2 and 6 h, and has migrated away from the apical domain at 24 h. A portion of the vesicular HSP-72 is associated with lysosomes; no intranuclear HSP-72 is detected. The course of mRNA induction, protein elaboration, and HSP-72 localization coincides with previously described changes in proximal tubule morphology and polarity following sublethal ischemic injury. HSP-72 may be instrumental in cellular remodeling and restitution of epithelial polarity during recovery from ischemic renal injury.
Epidermal growth factor accelerates renal tissue repair in a model of gentamicin nephrotoxicity in rats
Authors: Morin N. J. , Laurent G. , Nonclercq D. , Toubeau G. , Heuson-Stiennon J. A. , Bergeron M. G. , Beauchamp D.
Published: December 2017

Epidermal growth factor (EGF) is a potent mitogen for renal tubular cells that possess specific high-affinity binding sites for this polypeptide. However, actual function of EGF within the kidney remains to be elucidated. We evaluated the effect of exogenous EGF administration on the rate of tubular regeneration in an experimental model of gentamicin (GT) nephrotoxicity. Female Sprague-Dawley rats were anesthetized, and a miniosmotic pump filled with mouse EGF or saline was implanted subcutaneously. Twenty-four hours later, GT (40 mg.kg-1 x 12 h-1 ip) was given for 4 and 8 days. Groups of treated animals and controls were killed either the day after cessation of treatment (days 5 and 9) or 4 and 8 days after the end of 8-day GT administration (days 12 and 16). Cortical GT levels of groups killed at days 5, 9, 12, and 16 were similar in animals infused with saline or EGF. Serum creatinine levels were significantly higher in GT-treated animals infused with EGF or saline and killed at days 9 and 12 compared with saline-treated animals infused with EGF or saline alone (P < 0.01). Blood urea nitrogen (BUN) also increased as a result of GT administration. However, in animals receiving GT and EGF and killed at day 16, mean BUN level was significantly lower (P < 0.01) compared with rats dosed with GT alone. In treated rats, the extent of tubular regeneration, evaluated by the rate of [3H]thymidine incorporation into renal cortical DNA or by the frequency of S-phase cells (histoautoradiography), was increased in a dose- and time-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of angiotensin II on proximal tubular cells stably transfected with the c-mas oncogene
Authors: Wolf G. , Neilson E. G.
Published: December 2017

Angiotensin II (ANG II) normally induces cellular hypertrophy in proximal tubular epithelium by engaging receptor systems that use a G-protein-signaling mechanism. The c-mas oncogene also encodes part of a superfamily of vasoactive peptide receptor-like moieties that couple to G proteins. To determine whether the stable expression of the c-mas gene might alter or modify the induction of cellular hypertrophy by ANG II in tubular epithelium, a rat c-mas cDNA was cloned into the pSV2 expression vector for use in cell transfection. Scatchard analysis of ANG II binding revealed no significant differences in ANG II receptor number or in the dissociation constant between pSV2mas-transfected or wild-type MCT cells, but rather an increase in the number of receptors not replaceable by known inhibitors. ANG II also induced proliferation in pSV2mas-transfected MCT cells that was not blocked by conventional inhibitors and increased intracellular levels of inositol trisphosphate. ANG II, furthermore, did not increase de novo protein synthesis in pSV2-transfected MCT cells and failed to lower their intracellular concentration of adenosine 3',5'-cyclic monophosphate, both expected parameters of cellular hypertrophy. Our findings demonstrate that expression of c-mas in tubular epithelium can modulate tubular cell phenotype toward proliferation rather than hypertrophy. This effect is likely mediated by a reshuffling of the heterogeneity of ANG II receptors on the cell surface, or perhaps by the emergence of a new ANG II receptor, followed by alterations in the process of signal transduction.
Organ perfusion by dynamic scintigraphy convection-diffusion tracer kinetics in a phantom
Authors: Kegeles L. S. , Stritzke P. , Kupfer S. , Vallabhajosula S. , Burrows L. , Schanzer H. , Goldsmith S. J.
Published: December 2017

Dynamic scintigraphy is used widely to evaluate qualitatively the perfusion of an organ. Attempts to quantify blood flow to an organ by means of scintigraphic imaging modalities have often employed assumptions that lead to oversimplifying the physiology of the tracer kinetics. We used a mathematical formalism described by W. Perl and F. P. Chinard (Circ. Res. 22: 273-298, 1968), the convection-diffusion tracer kinetics, model, for parameter evaluation of flow (F) and volume of distribution (V). This modeling methodology was evaluated using a circulatory phantom with absolute flow measured independently by flowmeter. In a series of 22 phantom experiments with F/V < 0.32 s-1, there was a strong correlation between F and flow probe measurement [r = 0.97; slope = 1.08 +/- 0.06 (SE)]. The theoretical analysis comparing this approach with classical tracer kinetics methods explains both the satisfactory results for F/V using mean transit time and the systematic overestimation of F/V using decay constant methods.
Renal expression of the gene for atrial natriuretic factor
Authors: Greenwald J. E. , Ritter D. , Tetens E. , Rotwein P. S.
Published: December 2017

To date, atrial natriuretic factor (ANF) mRNA has eluded detection in the mammalian kidney, although we and others have identified ANF protein in the kidney using immunohistochemical and immunoassay techniques. Furthermore, we have demonstrated the synthesis and secretion of the ANF prohormone in the distal cortical nephron of the intact rat kidney and from rat primary cultured renal distal cortical tubular epithelial cells. In the present study, we show that the ANF gene is expressed in the kidney. Amplification of RNA isolated from rat distal cortical tubular epithelial cultures using ANF specific primers produced a 213-bp fragment that specifically hybridized to a 32P-labeled ANF cDNA. We had previously demonstrated these cultures to be enriched for the renal ANF synthetic and secretory cell type. However, we were unable to detect an ANF gene transcript in total rat kidney RNA using the above-mentioned polymerase chain reaction (PCR) conditions. Reanalysis of normal rat kidney PCR products by a second round of PCR amplification using nested primers successfully identified ANF mRNA. Similar to cultured kidney epithelial cells, normal rat kidney expresses ANF mRNA, but at a very low abundance, thus necessitating two rounds of PCR amplification. Further characterization of rat cortical distal tubular epithelia poly(A)+ RNA by Northern analysis revealed two ANF gene transcripts. A 1.0-kb message that comigrated with rat atrial ANF mRNA, and a second larger 1.4-kb transcript. These studies further substantiate the synthesis of ANF in the mammalian kidney. Unlike the mammalian heart, the kidney contains two ANF gene transcripts.
Cl- channels in basolateral renal medullary membranes, VI. Cl- conductance expression in Xenopus oocytes
Authors: Zimniak L. , Reeves W. B. , Andreoli T. E.
Published: December 2017

Cl- channels from basolateral membranes of the mammalian thick ascending limb of Henle differ significantly, in certain of their functional characteristics, from Cl- channels in apical membranes of secretory epithelia such as trachea or small intestine and from certain Cl- channels in the central nervous system. Yet there is no sequence information available about basolateral thick ascending limb Cl- channels. As an initial step in the isolation of a Cl- channel from the thick ascending limb of Henle's loop, we attempted a functional expression of a Cl- conductance in oocytes from Xenopus laevis. Oocytes injected with mRNA isolated from the outer medulla of rabbit kidney had a membrane conductance, measured using a two-electrode voltage clamp, which was sixfold greater than in water-injected oocytes (9.05 +/- 1.56 vs. 1.43 +/- 0.15 microS, respectively). Ion substitution experiments showed the conductance in the RNA-injected oocytes to be Cl- selective. In addition, the Cl(-)-channel blocker diphenylamine-2-carboxylate (1 mM) almost completely inhibited (79 +/- 6%) the increased conductance seen in the RNA-injected oocytes. Fractionation of the mRNA by sucrose gradient centrifugation revealed that peak Cl- channel activity was expressed using an mRNA fraction of 1.8–3.2 kb in size. These results demonstrate that a membrane Cl- conductance can be expressed in X. laevis oocytes injected with size-selected fractions of mRNA from rabbit outer renal medulla.
Mercury weakens membrane anchoring of Na-K-ATPase
Authors: Imesch E. , Moosmayer M. , Anner B. M.
Published: December 2017

Pages F837–F842: E. Imesch, M. Moosmayer, and B. M. Anner. ,Mercury weakens membrane anchoring of Na-K-ATPase.- Page F839: a line of type was inadvertently dropped from the legend to Fig. 3; it should read as follows: Enhanced trypsinolysis of Na-K-ATPase -subunit pretreated by mercury at 1 mg protein/ml. Na-K-ATPase (10 μg protein/10 μl) was incubated either in a solution stabilizing the E1 conformation [ A: containing (in mM) 50 NaCl, 50 KCl, 5 MgCl2, 5 ATP, 30 histidine, and 1 Tris-EDTA, pH 7.2] or in a solution stabilizing the E2 conformation ( B: 100 mM KCl, 5 mM MgCl2, 30 mM histidine, 1 mM Tris-EDTA, pH 7.2, for 30 min at 37°C) with HgCl2 concentrations ranging from 10 nM to 100 μM. Trypsinolysis (30 min at 20°C) and gel electrophoresis were performed as described in experimental procedures.
Electrophysiological properties of cultured outer medullary collecting duct cells
Authors: Pappas C. A. , Koeppen B. M.
Published: December 2017

Whole cell patch-clamp techniques were used to characterize the electrophysiological properties of cells from the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture. With pipette and bathing solutions mimicking intracellular and extracellular fluid, the resting membrane voltage was -30 to -40 mV. The whole cell conductance exhibited slight outward rectification, and at the resting membrane voltage the cell conductance averaged 2.58 +/- 0.49 nS (n = 17). The major conductive ion species was Cl-. The Cl- conductance was also found to have a significant permeability to HCO3- and was inhibited by the Cl(-)-channel blockers diphenylamine carboxylic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. A small K+ conductance was also present, but no Na+ conductance was detected. Current generated by the H(+)-adenosinetriphosphatase (H(+)-ATPase) was quantitated. This current was dependent on the presence of ATP in the pipette. Dicyclohexylcarbodiimide, N-ethylmaleimide, and bafilomycin A1, inhibitors of the vacuolar H(+)-ATPase, also reduced this outward current in an ATP-dependent manner. The inhibitor-sensitive component of the outward current, a measure of the current generated by the H(+)-ATPase, was in the range of 35-100 pA/cell.
Beta-adrenergic regulation of H+ secretion by cultured outer medullary collecting duct cells
Authors: Manger T. M. , Pappas C. A. , Koeppen B. M.
Published: December 2017

The effects of the beta-adrenergic agonist isoproterenol (Iso) on cells of the inner stripe portion of the rabbit outer medullary collecting duct (OMCDi) grown in primary culture were examined using whole cell patch-clamp techniques and measurements of intracellular pH (pHi) and Ca2+. Iso (10(-6) M) increased the cellular Cl- conductance, and this effect was mimicked by treatment of the cells with dibutyryladenosine 3',5'-cyclic monophosphate (cAMP, 10(-5) M) or protein kinase A (PKA, 0.4 U/ml). Iso did not alter the baseline pHi, but it did increase the activity of both the Cl-/HCO3- antiporter and the H(+)-adenosinetriphosphatase (H(+)-ATPase). The increase in Cl-/HCO3- antiporter rate was mimicked by dibutyryl-cAMP plus 3-isobutyl-1-methylxanthine (cAMP + IBMX, 10(-4) M + 10(-5) M). However, the Iso-induced stimulation of the H(+)-ATPase activity was not mimicked by cAMP + IBMX. Measurements of intracellular Ca2+ showed that Iso also increased intracellular Ca2+ levels. This response was not dependent on extracellular Ca2+, nor did cAMP + IBMX appreciably alter intracellular Ca2+. Consequently, we postulate that beta-adrenergic agonists are potential stimulators of OMCDi H+ secretion. These agonists stimulate cellular HCO3- efflux through a signal transduction pathway involving cAMP and PKA. However, a different signal transduction pathway appears to mediate the stimulation of cellular H+ efflux. This second pathway may involve an elevation of intracellular Ca2+.
Activation of endothelin ETB receptors increases glomerular cGMP via an L-arginine-dependent pathway
Authors: Edwards R. M. , Pullen M. , Nambi P.
Published: December 2017

The effects of endothelins (ET) on guanosine 3',5'-cyclic monophosphate (cGMP) levels in intact rat glomeruli were examined. ET-3 produced a rapid approximately fivefold increase in cGMP levels with the maximum effect occurring at 1 min. The ET-3-induced increase in cGMP accumulation occurred in the absence and presence of 3-isobutyl-1-methylxanthine. ET-1, ET-2, ET-3, and the structurally related toxin, sarafotoxin S6c, all increased glomerular cGMP levels in a concentration-dependent manner and with similar potencies (EC50 approximately 15-30 nM). The L-arginine analogue, N omega-nitro-L-arginine (L-NNA), reduced basal levels of cGMP and also totally inhibited ET-induced increases in cGMP as did methylene blue, an inhibitor of soluble guanylate cyclase. The effect of L-NNA was attenuated by L-arginine but not by D-arginine. The stimulation of cGMP accumulation by ET-3 was dependent on extracellular Ca2+ and was additive to atriopeptin III but not to acetylcholine. The ETA-selective antagonist, BQ 123, had no effect on ET-3-induced formation of cGMP. Glomerular membranes displayed high-affinity (Kd = 130-150 pM) and high-density (approximately 2.0 pmol/mg) binding sites for 125I-ET-1 and 125I-ET-3. ET-1, ET-3, and sarafotoxin S6c displaced 125I-ET-1 binding to glomerular membranes with similar affinities. BQ 123 had no effect on 125I-ET-1 binding. We conclude that ET increases cGMP levels in glomeruli by stimulating the formation of a nitric oxide-like factor that activates soluble guanylate cyclase. This effect of ET appears to be mediated by activation of ETB receptors and may serve to modulate the contractile effects of ET.
A mathematical model of the rabbit cortical collecting tubule
Authors: Strieter J. , Stephenson J. L. , Giebisch G. , Weinstein A. M.
Published: December 2017

The epithelium of the cortical collecting tubule of the rabbit is represented as four well-stirred compliant compartments corresponding to principal cell, alpha- and beta-intercalated cells, and lateral interspace. Model variables include the concentrations of Na, K, Cl, and HCO3, pH, cell volume, and electrical potential. The model equations specify mass conservation and chemical equilibrium for buffer reactions. Ionic conductance is represented by the Goldman constant-field equation. For the intercalated cells, phenomenological expressions describing the proton pumps are structured to agree with data of O. S. Andersen, J. E. N. Silveira, and P. R. Steinmetz (J. Gen. Physiol. 86: 215-234, 1985) in the turtle bladder. Coupled transport via Na/H and Cl/HCO3 exchangers is represented according to the formalism of linear nonequilibrium thermodynamics. To construct the tubule model, the flat epithelium is wrapped into a cylinder, creating a luminal compartment. Luminal variables include volume flow, hydrostatic pressure, electrical potential, and ionic concentrations. A specific aim of this investigation was to simulate the capability of the epithelium to maintain Na reabsorption in the presence of low luminal salt concentration. In this regard, critical features of the model include tight junctional conductance and the apical Na permeability of the principal cell. In particular, we examine a principal cell apical Na permeability inversely dependent on luminal and intracellular Na concentrations (M. M. Civan and R. J. Bookman. J. Membr. Biol. 65: 63-80, 1982). This concentration-dependent permeability together with a low junctional conductance produces three results congruent with experimental data: 1) dilution of luminal Na and maintenance of reabsorptive Na transport despite a steep transtubular gradient, 2) a relatively constant level of K secretion over a wide range of luminal Na concentrations, and 3) a relatively constant transepithelial potential over this range of luminal Na.
Expression of mRNA (D2) encoding a protein involved in amino acid transport in S3 proximal tubule
Authors: Kanai Y. , Stelzner M. G. , Lee W. S. , Wells R. G. , Brown D. , Hediger M. A.
Published: December 2017

A rat kidney- and intestine-specific cDNA (D2) that induces high-affinity, Na(+)-independent uptake of cystine and dibasic and neutral amino acids into cRNA-injected Xenopus oocytes was recently isolated by expression cloning in our laboratory (R. G. Wells and M. A. Hediger. Proc. Natl. Acad. Sci. USA 89: 5596-5600, 1992). At present it is not known whether the D2-encoded protein functions as a transporter or as a transporter activator. To gain more insight into the role of D2 in renal amino acid transport, we studied the site of its expression in the kidney. This was determined by Northern blot analysis and by using a combination of in situ hybridization and immunocytochemistry with antibodies that recognize specific proximal tubule segments. D2 antisense RNA hybridized to the same tubular segments that were strongly positive for anti-ecto-adenosinetriphosphatase but negative for carbonic anhydrase type IV and the facilitated glucose transporter GLUT2. We conclude that D2 mRNA is strongly expressed in the rat kidney proximal tubule S3 segment, although there is weak hybridization to the S1 and S2 segments. The signal is absent in all other parts of the kidney. The S3 specific expression of D2 mRNA coincides with the site of high-affinity transport of cystine and other amino acids, consistent with the proposed involvement of D2 in these processes.
Adenosine receptor gene expression in rat kidney
Authors: Weaver D. R. , Reppert S. M.
Published: December 2017

Adenosine is an important modulator of renal function. Adenosine produced and released within the kidney is thought to participate in the metabolic regulation of glomerular filtration (tubuloglomerular feedback), as well as in regulating renal excretory function and renin secretion. The recent cloning of cDNAs encoding the A1 and A2a adenosine receptors from rat brain allows direct examination of potential sites of adenosine action within the rat kidney. Northern blot analysis of rat kidney poly(A)+ RNA revealed that A1 adenosine receptor mRNA was more abundant in kidney than the A2a adenosine receptor transcript. In situ hybridization with 35S-labeled cRNA probes was used to localize A1 and A2a adenosine receptor mRNAs within the kidney. A1 adenosine receptor mRNA was most abundant in the collecting ducts of the papilla and inner medulla. Collecting ducts in the outermost portion of the inner stripe of the outer medulla and cells of the juxtaglomerular apparatus also expressed A1 adenosine receptor mRNA. A2a adenosine receptor mRNA was localized to the renal papilla. The distribution of A1 and A2a adenosine receptor mRNAs within the rat kidney supports previously postulated roles for adenosine in the regulation of renal hemodynamics, excretory function, and renin secretion.
Streptozotocin-induced renal hemodynamic changes in isogenic Lewis rats: a kidney transplant study
Authors: Churchill P. , Churchill M. , Bidani A. , Dunbar J.
Published: December 2017

Streptozotocin (STZ)-induced diabetes mellitus is associated with decreased renal clearances of inulin and p-aminohippurate (PAH). The present experiments were designed to determine whether STZ-induced renal hemodynamic changes are due to the drug per se, rather than to the diabetic state that it induces. Isogenic Lewis rats with native right and transplanted left kidneys were studied. In one group, kidney donors received 50 mg STZ/kg body wt on day 1 and transplantation was performed on day 4 (untreated recipients). On day 29, the inulin and PAH clearances of these nondiabetic recipients were, respectively, 0.94 +/- 0.04 and 2.58 +/- 0.11 ml.min-1 x g-1 for the transplanted left kidney (previously exposed to STZ) and 0.95 +/- 0.07 and 2.54 +/- 0.14 ml.min-1 x g-1 for the native right kidney (never exposed to STZ). In another group, recipients received STZ on day 1 and transplantation was performed on day 4 (untreated donors). On day 29, the inulin and PAH clearances of these diabetic recipients were, respectively, 0.62 +/- 0.04 and 1.46 +/- 0.11 ml.min-1 x g-1 for the transplanted left kidney (never exposed to STZ) and 0.61 +/- 0.05 and 1.42 +/- 0.08 ml.min-1 x g-1 for the native right kidney (previously exposed to STZ). We conclude that the diabetic state, rather than STZ, is responsible for the decreased renal clearances of inulin and PAH in this experimental model.
Identification of a novel K-channel gene (KC22) that is highly expressed in distal tubule of rabbit kidney
Authors: Desir G. V. , Velazquez H.
Published: December 2017

The Shaker gene family encodes voltage-gated K channels. Five partial-length Shaker-like cDNAs (KC2, 4, 10, 19, and 22) were previously isolated from rabbit kidney using polymerase chain reaction (PCR) [G. V. Desir, E. Hamlin, A.H. Puente, R.F. Reilly, F. Hiledebrandt, and P. Igarashi. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F151-F157, 1992]. We now report the cloning of another Shaker-like cDNA (KC6) from rabbit kidney and the identification of one isoform that is highly expressed in rabbit distal tubule cells grown in culture. A partial-length cDNA (859 bp) for KC6 was isolated by PCR amplification of rabbit kidney cDNA using Shaker-specific degenerate primers. KC6 was most similar to the rat brain clone RBK2 (77% amino acid identity) and to the rabbit clone KC19 (78% amino acid identity). Transcript levels for KC2, 4, 6, 10, 19, and 22 were quantified using the ribonuclease protection assay. Transcripts for all six isoforms were detected in renal tissues. KC22 was the most abundant isoform in kidney cortex and medulla (20- to 40-fold greater than the other isoforms). Furthermore, KC22 expression levels were fivefold higher in primary cultures of rabbit distal convoluted tubules and connecting tubules than in whole kidney cortex. Although the partial-length sequence for KC22 represents the most conserved regions in the Shaker gene family it only has 35-88% amino acid identity with other Shaker channels, suggesting that KC22 represents a novel isoform. In contrast, KC4 and KC19 (less abundant in kidney than KC22) are highly homologous to the rat brain clones RBK1 and RBK2, respectively (97% amino acid identity).(ABSTRACT TRUNCATED AT 250 WORDS)
KCl and angiotensin responses in isolated rat renal arterioles: effects of diltiazem and low-calcium medium
Authors: Conger J. D. , Falk S. A.
Published: December 2017

Studies in intact renovascular models have shown that calcium entry blockers inhibit angiotensin (ANG II)-induced vasoconstriction in afferent (AA) but not efferent arterioles (EA), suggesting that increases in smooth muscle cell cytosolic calcium, the initiating intracellular message, result from entry through potential-operated channels in AA, but from organelle storage mobilization or entry through nonpotential-operated channels in EA. The present study examined the effects of diltiazem (10(-5) M) on the constrictor responses to KCl (50 mM) and half-maximal constricting concentrations (EC50) of ANG II and the effects of low-calcium bathing medium on EC50 ANG II responses in isolated rat AA and EA. KCl caused slightly greater decreases in lumen diameter in AA than in EA (P < 0.05) that were completely inhibited by diltiazem in both. Vasoconstriction to ANG II was significantly inhibited by diltiazem (29 +/- 12 vs. 67 +/- 31%; P < 0.02) in AA. However, constrictor response to ANG II in EA was unchanged by diltiazem (42 +/- 32 vs. 41 +/- 31%). Constriction to ANG II of AA in low-calcium medium was significantly attenuated (8 +/- 13 vs. 54 +/- 12%; P < 0.01); however, EA constrictor response was not affected (43 +/- 22 vs. 51 +/- 19%). These data indicate that EC50 ANG II-induced AA constriction requires calcium entry primarily through potential-operated channels. While potential-operated calcium entry channels can be functionally expressed in EA, intracellular calcium mobilization is the primary mechanism for ANG II-induced constriction.
Role of mesangial cell in glomerular response to volume and angiotensin II
Authors: Blantz R. C. , Gabbai F. B. , Tucker B. J. , Yamamoto T. , Wilson C. B.
Published: December 2017

We have examined the physiological role of the mesangial cell in the regulation of glomerular hemodynamics utilizing mesangial cell lysis by the administration of antithymocyte antibody serum (ATS) 24 h before micropuncture evaluation. Plasma volume expansion (PVE) in normal NaCl-depleted rats increased single-nephron glomerular filtration rate (SNGFR) by 30% because of increases in single-nephron plasma flow (SNPF), whereas glomerular capillary hydrostatic pressure (PG) remained constant. SNGFR did not increase with PVE in NaCl-depleted ATS rats despite increases in SNPF, and PG increased significantly (51 +/- 2 to 67 +/- 3 mmHg) because of afferent arteriolar dilation, whereas efferent resistance remained elevated. Angiotensin II (ANG II) infusion in normal rats decreased SNGFR because of reductions in SNPF and the glomerular ultrafiltration coefficient (LpA), whereas the hydrostatic pressure gradient (delta P) increased. In ATS rats ANG II infusion did not change SNGFR, LpA, or delta P. These in vivo studies suggest that the mesangial cell plays an important role in the regulation of LpA, efferent arteriolar resistance, and the regulation of PG, whereas this cell exerts little effect on the afferent arteriole.
Differences in dynamic autoregulation of renal blood flow between SHR and WKY rats
Authors: Chen Y. M. , Holstein-Rathlou N. H.
Published: December 2017

In halothane-anesthetized Wistar-Kyoto (WKY) rats the single-nephron blood flow and the proximal tubule pressure oscillate at a frequency of 35-50 mHz because of the operation of the tubuloglomerular feedback (TGF) mechanism. In spontaneously hypertensive rats (SHR) the oscillations are replaced by chaotic fluctuations. We sought to determine whether this change was associated with a change in the dynamic autoregulation of renal blood flow. In halothane-anesthetized 250- to 320-g SHR and WKY rats, renal blood flow was measured during "white noise" forcing of arterial blood pressure. The frequency response of renal vascular admittance was estimated by the method of autoregressive-moving averages. In the frequency band below 60-70 mHz there was a significant difference in the transfer functions between the two strains of rats. This was due mainly to an increased phase difference, but also to a decreased magnitude of the admittance in SHR at frequencies below 20-30 mHz. Above 70 mHz there was no significant difference in the transfer functions. Because TGF is active in the low frequency band (below approximately 100 mHz), whereas the myogenic mechanism also acts in the higher frequency band, we conclude that the change in the dynamics of TGF leads to a change in the dynamic autoregulation of renal blood flow between SHR and WKY rats. This change results in a more efficient dynamic autoregulation of renal blood flow in the SHR compared with the WKY rats. The functional consequences of this, in terms of the regulation of salt and water excretion, are not presently known.
Hypertonicity stimulates transcription of gene for Na(+)-myo-inositol cotransporter in MDCK cells
Authors: Yamauchi A. , Uchida S. , Preston A. S. , Kwon H. M. , Handler J. S.
Published: December 2017

Myo-inositol is a major compatible osmolyte accumulated in the hypertonic renal medulla and in Madin-Darby canine kidney (MDCK) cells cultured in hypertonic media. Myo-inositol is taken up by MDCK cells on a Na(+)-coupled transporter whose activity increases sixfold 24 h after cells are switched to hypertonic medium. To investigate the mechanism of regulation of the cotransporter by hypertonicity, we used the cDNA encoding the canine Na(+)-myo-inositol cotransporter that we recently cloned to measure the abundance of the mRNA for the cotransporter and its rate of transcription after changes in osmolality. When MDCK cells were switched from isotonic to hypertonic medium, cotransporter mRNA abundance rose 10-fold in 16 h. Transcription of the cotransporter gene also rose and 16 h after the switch reached a peak approximately 15-fold that in isotonic cells. When cells were switched back to isotonic medium, mRNA abundance and transcription of the gene returned to isotonic levels in 8 h and transport rate reached isotonic levels in 48 h. Thus transcription appears to be the primary step in regulation of myo-inositol transport by hypertonicity.
Potassium adaptation in hypothyroidism: changes in transport adenosinetriphosphatases
Authors: Salem M. M. , Chen Y. , Mujais S. K.
Published: December 2017

To evaluate whether hypothyroidism alters the adaptive responses of renal transport adenosine-triphosphatases (ATPases) to modifications in dietary K content, we examined the activities of Na-K pump and H-K pump in hypothyroid rats under basal conditions and after dietary K changes. Hypothyroidism led to a decline in Na-K pump activity in all three nephron segments examined [proximal convoluted tubule from 2,333 +/- 103 to 1,099 +/- 32, medullary thick ascending limb from 4,344 +/- 119 to 1,613 +/- 61, and cortical collecting tubule (CCT) from 1,133 +/- 45 to 640 +/- 38 pmol.mm-1 x h-1; all P < 0.01 vs. euthyroid] along with morphological changes manifest in a decrease in tubule diameter. K loading led to an increase in Na-K pump activity in the CCT of both euthyroid (from 1,133 +/- 45 to 2,269 +/- 74, pmol.mm-1 x h-1, P < 0.01) and hypothyroid (from 640 +/- 38 to 1,118 +/- 67 pmol.mm-1 x h-1, P < 0.01) animals. Furthermore, in euthyroid rats, 3 wk of K depletion led to a major increase in H-K pump activity in both the CCT (from 203 +/- 14 to 331 +/- 22 pmol.mm-1 x h-1, P < 0.01) and medullary collecting tubule (MCT, from 137 +/- 9 to 210 +/- 14 pmol.mm-1 x h-1, P < 0.01). Hypothyroidism was associated with a decline in H-K pump activity in the CCT and MCT (to 94 +/- 6 and 55 +/- 5 pmol.mm-1 x h-1, respectively; both P < 0.01 vs. euthyroid).(ABSTRACT TRUNCATED AT 250 WORDS)
PGE2 inhibits Na-K-ATPase activity and ouabain binding in MDCK cells
Authors: Cohen-Luria R. , Rimon G. , Moran A.
Published: December 2017

In the present study we report on a direct effect of prostaglandin E2 (PGE2) on ouabain binding and Na-K-adenosinetriphosphatase (Na-K-ATPase) activity in a clone of Madin-Darby canine kidney cells, a renal cell line with collecting duct properties. Incubation of the cells with low concentrations (pM) of PGE2 produced a concomitant reduction of approximately 50% in the activity of Na-K-ATPase in the cell homogenate and in ouabain binding to the intact cells (half-maximal inhibition of approximately 0.1 pM). The inhibition was apparent within 10 min of preincubation of the cells with PGE2. Scatchard analysis of the binding demonstrated that the treatment with PGE2 reduced the number of ouabain binding sites without a change in the dissociation constant. PGE1 and PGF2 alpha (10 nM) did not affect ouabain binding or Na-K-ATPase activity. The fast, potent, and specific effect of PGE2 suggests that the diuretic/natriuretic effect of prostaglandins of the E series in the collecting tubule, in addition to the interference with the activity of arginine vasopressin, may result from a direct reduction in the number of the Na-K-ATPase active units, via a prostaglandin receptor.