bluuebunny/crossref_metadata_2025 huggingface dataset:
Splanchnic and renal hemodynamic responses to intraportal infusion of glucagon
Authors: Premen A. J.
Published: December 2017

We compared the 60-min splanchnic and renal hemodynamic responses to intraportal (IPV) and intravenous infusion of glucagon (5 ng.kg-1.min-1) in anesthetized dogs and quantitated the importance of glucose in mediating the renal hemodynamic responses to intraportal infusion of glucagon. Intraportal glucagon elevated superior mesenteric (SMA) and renal (RA) artery blood flows by 8 and 16%, respectively, by minute 15. By minute 30, RA flow had increased by 23%. Thereafter, SMA flow returned to control, while RA flow remained elevated by 24%. Glomerular filtration rate (GFR) followed the same pattern as RA flow over 60 min. Intravenous glucagon elicited smaller hemodynamic responses. During intraportal and intravenous glucagon infusion, plasma glucose rose by 20-25%. Renal hemodynamics were not affected by incremental changes in blood glucose of up to 6.25 mmol/l. At an incremental change in glucose of 10.06 mmol/l, RA flow and GFR were elevated by 12 and 9%, respectively. We conclude that intraportal glucagon infusion increases splanchnic and renal hemodynamics, although the splanchnic response is evanescent. Importantly, hepatic release of glucose into the circulation during intraportal glucagon infusion does not have a significant effect on renal hemodynamics. Thus, similar to intravenous infusion of the hormone, renal hemodynamic responses to intraportal glucagon are independent of and dissociated from elevations in blood glucose produced during hormone infusion.
Potassium transport by medullary collecting tubule of rabbit: effects of variation in K intake
Authors: Wingo C. S.
Published: December 2017

These studies examine the effect of dietary K content on the rate efflux coefficient (KK) assessed both isotopically (42K) and by net K flux in segments dissected from the inner stripe of the outer medullary collecting tubule (MCT). Female New Zealand White rabbits were given either a K-replete diet (KCl diet) or one of two K-restricted diets with similar (142 meq K/kg diet) K content [basic (Na-deplete) diet or NaCl (Na-replete) diet]. After 7 days, renal K excretion (mmol/24 h) varied with K intake: KCl diet, 13.8 +/- 3.0; basic diet, 4.36 +/- 0.12; NaCl diet, 4.13 +/- 0.48. To determine whether alterations in KK of MCT could contribute to these changes in urinary K excretion, the MCT from the three groups of rabbits was perfused in vitro and volume reabsorption and KK were measured. The KK of tubules from the K-replete control group (KCl diet) was 82 +/- 14 nm/s but was significantly greater in tubules from either K-restricted group: 224 +/- 49 nm/s (basic diet group) or 193 +/- 34 nm/s (NaCl diet group). Net K flux in the presence of a 15 mM lumen-to-bath K gradient confirmed a significantly greater K reabsorptive flux and net rate coefficient in either K-restricted group (KNetK = 179 +/- 77 nm/s for the NaCl diet, KNetK = 170 +/- 27 nm/s for the basic diet) than the K-replete control group (KNetK = 12.0 +/- 18 nm/s for the KCl diet).(ABSTRACT TRUNCATED AT 250 WORDS)
Insulin stimulates ammoniagenesis in canine renal proximal tubular segments
Authors: Chobanian M. C. , Hammerman M. R.
Published: December 2017

To characterize the effect of insulin on ammoniagenesis in renal proximal tubule, ammonia productions were measured in suspensions of canine renal proximal tubular segments incubated with 10 mM L-glutamine and varying concentrations of insulin. Productions of ammonia were linear functions of time for 120 min. Insulin (10(-6) M) increased ammonia production at 2 h by 34 +/- 5%. At the same time, gluconeogenesis, as measured by glucose production, was decreased by 16 +/- 2%. Significant enhancement of ammoniagenesis occurred in suspensions of segments incubated with as little as 10(-9) M insulin. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M insulin. Oxidation of L-glutamine in cells within segments was also increased by insulin in a concentration-dependent manner. Insulin increased ammonia productions in segments incubated at pH 7.5 but not at 7.0. Under the former conditions, insulin enhanced ammoniagenesis in proximal tubular segments under conditions such that extracellular [Na+] was greater than intracellular [Na+], but not when extracellular [Na+] equaled intracellular [Na+]. We conclude that insulin stimulates ammonia production in suspensions of canine renal proximal tubular segments. Stimulation of ammonia production in vitro could reflect an action of insulin to enhance proximal tubular ammoniagenesis in vivo.
Characterization of amino acid metabolism by cultured rat kidney cells: study with 15N
Authors: Nissim I. , States B. , Yudkoff M. , Segal S.
Published: December 2017

The present study evaluates the metabolism of glutamine and glutamate by normal rat kidney (NRK) cells. The major aim was to evaluate the effect of acute acidosis on the metabolism of amino acid and ammonia formation by cultured NRK cells. Experiments at either pH 7.0 or 7.4 were conducted with phosphate-buffered saline supplemented with either 1 mM [5-15N]glutamine, [2-15N]glutamine, or [15N]glutamate. Incubation with either glutamine or glutamate as a precursor showed that production of ammonia and glucose was increased significantly at pH 7.0 vs. 7.4. The disappearance [corrected] of glutamine and glutamate was linear during a 60-min incubation at either pH. In experiments with [5-15N]glutamine, we found that approximately 57 and 43% of ammonia N was derived from 5-N of glutamine at pH 7.4 and 7.0, respectively. Experiments with [2-15N]glutamine or [15N]glutamate indicated that approximately 43 and 47% of 2-N glutamine and glutamate N utilization, respectively, was accounted for by ammonia production at pH 7.0. Similarly, 28 and 29% of NH3 was derived from 2-N of glutamine or glutamate N by activity of glutamate dehydrogenase at pH 7.4. In addition to 15NH3 formation, three major metabolic pathways of [2-15N]glutamine or [15N]glutamate disposal were identified: 1) transamination reactions involving the pH-independent formation of [15N] aspartate and [15N]alanine; 2) the synthesis of [6-15NH2]adenine nucleotide, a process more active at pH 7.4 vs. 7.0; and 3) glutamine synthesis from [15N]glutamate, especially at pH 7.4. The data indicate that NRK cells in culture consume glutamine and glutamate and generate ammonia and various amino acids, depending on the H+ concentration in the media. The studies suggest that these cell lines may provide a useful model for studying various aspects of the effect of pH on rat renal ammoniagenesis.
Differentiated transport functions in primary cultures of rabbit collecting ducts
Authors: Fejes-Toth G. , Naray-Fejes-Toth A.
Published: December 2017

Fragments of rabbit cortical collecting tubule (CCT) were isolated by solid-phase immunoadsorption with a monoclonal antibody against an ectoantigen on principal cells. Primary cultures, initiated from the "immunodissected" cells, were then grown to confluency in porous bottom dishes. The purity of the freshly isolated and cultured cells was verified by flow cytometry and immunocytochemistry, using a panel of cell-specific monoclonal antibodies and lectins. As a result of continued vectorial transport, the cultured epithelial monolayers generated pronounced transepithelial solute gradients. The composition of the luminal (L) and basolateral (B) media after 48 h of incubation was as follows: [Na], L 119 +/- 1.1, B 145 +/- 3.0; [K], L 11.9 +/- 0.5, B 2.0 +/- 0.3; [PO4], L 2.8 +/- 0.2, B 7.3 +/- 0.3; [Cl], L 134.1 +/- 2.7 B 117.8 +/- 2.2, [mM]; [Ca], L 42.1 +/- 1.2, B 812 +/- 26, [microM]; pH, L 5.52 +/- 0.12, B 7.15 +/- 0.08. The transepithelial osmotic concentration difference was 14.3 +/- 3.5 mosmol/l. The amount of glucose at the luminal side remained unaltered, whereas glucose concentration at the basolateral side decreased from 21 to 7.7 +/- 1.0 mM. Lactic acid accumulated predominantly at the basolateral side (L 1.0 +/- 0.15, B 23.5 +/- 1.7 mM). These data indicate that primary cultures of immunodissected CCT cells retain many of their differentiated transport functions and thus should prove useful for studying the mechanisms of solute transport in the collecting duct system.
Mechanism of adrenergic stimulation of anion secretion in cultured rat epididymal epithelium
Authors: Wong P. Y.
Published: December 2017

Primary monolayer cultures of the epithelial cells from the rat cauda epididymidis had a basal transepithelial potential of 1.24 +/- 0.11 mV, apical side negative, a short-circuit current (SCC) of 2.42 +/- 0.17 microA/cm2, and a transepithelial resistance of 503 +/- 29.6 omega/cm2. Epinephrine (0.23 microM) added to the basolateral side caused a rise in the SCC (inward flowing) that was partially reversed by addition of amiloride, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), bumetanide, and acetazolamide to the basolateral side. In the absence of Cl or HCO3, the SCC response to epinephrine was reduced by 65 and 66%, respectively. Removal of K reduced the SCC response by 40%, whereas Na removal completely abolished it. A23187 and phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated the SCC when added to the apical side. Addition of the Cl channel blockers, anthracene-9-carboxylate and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) to the apical side reduced the SCC in a dose-dependent manner. It is concluded that anion secretion by the epididymis shares common mechanisms with other Cl-secreting epithelia.
Renal ammoniagenic response to chronic acid loading: role of glucocorticoids
Authors: Welbourne T. C. , Givens G. , Joshi S.
Published: December 2017

Adrenalectomized (ADX) animals exhibit a blunted renal response to chronic acid loading. To determine whether this response truly reflects impaired renal ammoniagenesis from glutamine, urinary ammonium excretion was compared with acid intake in ADX, intact, and ADX rats supplemented with either a low dose (4 micrograms.100 g-1.day-1) or a high dose (40 micrograms.100 g-1.day-1) of triamcinolone. ADX rats consumed similar amounts of acid as did intact controls yet excreted only 37% of the load as ammonium; in contrast intact controls returned 86% and triamcinolone-supplemented animals returned 98 and 88% for low and high doses, respectively. Nor could the reduced ammonium excretion be attributed to increased renal venous release, since total ammonia production, the sum of renal venous and urine ammonium, was reduced to 49% of the intact controls; low- and high-dose triamcinolone restored and markedly increased the production rate. Underlying the impaired ammonia production rate in ADX rats was a reduced rate of glutamine extraction, 350 +/- 49 vs. 896 +/- 102 and 1,260 +/- 247 and 1,448 +/- 112 nmol.min-1.100 g-1 for intact and low and high doses, respectively. Unlike intact acidotic and glucocorticoid-supplemented ADX acidotic rats, glutamine extraction was disassociated from the delivered glutamine load consonant with the role of glucocorticoid in coupling cellular glutamine transport to its metabolic utilization.
Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder
Authors: Hoch B. S. , Ast M. B. , Fusco M. J. , Jacoby M. , Levine S. D.
Published: December 2017

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. “High dose” cycloheximide (200 micrograms/ml) inhibited flow immediately. “Low dose” cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of changes in extracellular Cl on intracellular Cl activity in frog skin
Authors: Drewnowska K. , Biber T. U.
Published: December 2017

Intracellular Cl activity was measured in isolated frog skin (Rana pipiens) with double-barrel microelectrodes. The initial rate of Cl uptake was measured in Cl-depleted cells on reexposure to Cl on apical or basolateral side. In skins with high and low conductance, cell CL activity increased 1.33 and 0.14 mM/s with apical reexposure and 5.03 and 0.30 mM/s with basolateral reexposure, respectively. The initial Cl uptake was reduced on the apical side by 93% with 10(-3) M DIDS (4,4'-diisothiocyanostilbene-2,2ߗ-disulfonic acid) and on the basolateral side by 99% with 10(-3) M SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid) plus 10(-5) M bumetanide. The initial rate of Cl loss was measured when Cl was removed from the bath: addition of HCO3 to Cl- and HCO3-free solution caused an acceleration of Cl loss in absence but not in presence of DIDS on apical side. In contrast, Cl loss across the basolateral side was not enhanced by HCO3. In conclusion, Na-transporting cells have a substantial Cl permeability on both sides. HCO3-stimulated Cl loss provides evidence for Cl-HCO3 exchange and permits localization of this process in apical cell membranes of granular cells.
Effect of atrial natriuretic factor on renal function in rats with nephrotic syndrome
Authors: Hildebrandt D. A. , Banks R. O.
Published: December 2017

This study was designed to evaluate the renal effects of atrial natriuretic factor [ANF(8-33)] in rats with aminonucleoside (AMN)-induced nephrotic syndrome. AMN (100 mg/kg iv) was administered to adult female rats either 2 (AMN 2, n = 7), 4 (AMN 4, n = 7), 6 (AMN 6, n = 7), or 14 (AMN 14, n = 6) days before clearance experiments; untreated (UNT, n = 7) animals served as controls. During clearance experiments, rats were anesthetized with pentobarbital sodium. Protein excretion rates were similar between UNT and AMN 2 but increased stepwise in AMN 4, AMN 6, and AMN 14 rats. The glomerular filtration rate (GFR) was similar in UNT and AMN 2, lower in AMN 4 and AMN 14, and lowest in AMN 6 rats. Basal sodium excretion (UNaV) was not different among the five groups. An ANF primer (1.0 micrograms/kg iv) plus a constant infusion (0.1 micrograms.kg-1.min-1) for 1 h produced a significantly lower increase in UNaV in AMN 2 and AMN 14 than in UNT and was not natriuretic or diuretic in AMN 4 or AMN 6 rats. The ANF-induced increase in UNaV was similar between AMN 2 and AMN 14 rats. ANF had no effect on the GFR in any group. A higher ANF bolus (5.0 micrograms/kg iv) was then infused. This ANF bolus increased UNaV only in UNT and AMN 2 rats. Finally, a bolus of furosemide (4.0 mg/kg iv) was given; UNaV increased similarly in UNT, AMN 2, and AMN 14, and to a lesser extent in AMN 4 and AMN 6 rats. Thus, there is an attenuated natriuretic and diuretic response to ANF in rats with AMN-induced nephrotic syndrome. This altered responsiveness to ANF may contribute to the sodium and water retention characteristic of this disorder.
Influence of calcium on phosphate transport in cultured kidney epithelium
Authors: Caverzasio J. , Bonjour J. P.
Published: December 2017

Renal epithelia cultured from opossum kidney (OK) cell line were exposed to high- and low-Ca media (HCaM and LCaM, respectively). Changing the extracellular Ca concentration from 2.0 mM to 0.02 mM stimulated sodium-dependent Pi transport (NaPiT) of OK epithelia. The effect was detectable after 1 h of LCaM exposure and reached a maximal value at 4 h (HCaM, 1.49 +/- 0.02; LCaM, 2.71 +/- 0.02 nmol.mg prot-1.4 min-1, P less than 0.001), this difference remaining constant up to 16 h. The Na-dependent transports of both alanine and methyl-alpha-D-glucopyranoside, a glucose analogue, were not affected by LCaM. Kinetic analysis of NaPiT revealed an increase in Vmax with no change in the apparent Km. The cellular adenosine 3',5'-cyclic monophosphate production was not affected by LCaM. In OK epithelia pretreated with either cycloheximide or actinomycin D the LCaM stimulatory effect was completely abolished. In the presence of 10(-9) to 10(-7) M bovine parathyroid hormone (1-34), the effect of LCaM on NaPiT was markedly blunted. In conclusion, this study in cultured kidney epithelia demonstrates that the renal NaPiT can be directly and selectively stimulated by lowering the extracellular Ca concentration. This effect required the de novo synthesis of proteins. The results also indicate that parathyroid hormone antagonizes the stimulatory effect of extracellular Ca on the renal transport of Pi.
Electrophysiology of renal capillary membranes: gel concept applied and Starling model challenged
Authors: Wolgast M. , Ojteg G.
Published: December 2017

In the classical Starling model the hydrostatic pressure in the pores is generally lower than that in capillary plasma, a phenomenon that necessitates the assumption of a rigid porous membrane. In flexible gel membranes, the capillary pressure is suggested to be balanced by a gel swelling pressure generated by negative fixed charges. Regarding the fluid transfer, the transmembranous electrical potential gradient will generate a net driving electroosmotic force. This force will be numerically similar to the net driving Starling force in small pores, but distinctly different in large pores. From previous data on the hydrostatic and colloid osmotic forces, the fixed charge density at the two interfaces of 1) the glomerular and 2) the peritubular capillary membrane were calculated and used to predict the flux of a series of charged protein probes. The close fit to the experimental data in both the capillary beds is in line with the gel concept presented. The gel concept (but hardly a rigid membrane) explains the ability of capillary membranes to alter their permeability in response to external forces. Gel membranes can furthermore be predicted to have a self-rinsing ability, as entrapped proteins will increase the local fixed charge density, leading to fluid entry into the region between the particle and the pore rim, which by consequent widening of the channel will facilitate extrusion of trapped proteins.
Functional differences of six forms of renin in rats
Authors: Kim S. H. , Lloyd M. C. , Sessler F. M. , Feng J. Q. , Malvin R. L.
Published: December 2017

Six forms of renin with different isoelectric points (pIs) have been described in rats. This study was designed to determine if any of the renin forms have different biological activities. Each form of rat renin was semipurified and injected intravenously or intraventricularly in Sprague-Dawley rats anesthetized with pentobarbital sodium or Inactin. Changes in blood pressure (BP), renal function, sodium, and water excretion were observed, before and following equipressor doses; the peak response of BP was similar for all forms. However, the half-lives were significantly different. Form 4 (pI = 5.2) caused a significant increase in urine flow, Na, and K excretion, and urinary osmolality when given intravenously. The other forms were without significant effect. Infusion of converting enzyme inhibitor not only completely blocked the BP response, but also prevented the natriuresis and diuresis. This was observed in rats anesthetized with pentobarbital sodium or Inactin. Intraventricular infusions resulted in a diuresis and natriuresis when form 6 (pI = 4.8) was infused, but not with other forms. BP remained unchanged throughout. This study presents evidence that functional differences exist between renin forms.
Juxtaglomerular interstitial hypertonicity in Amphiuma: tubular origin-TGF signal
Authors: Persson B. E. , Sakai T. , Marsh D. J.
Published: December 2017

One of the mechanisms mediating renal vascular autoregulation in mammals senses tubular flow rate-dependent changes in luminal NaCl concentrations and signals renal arterioles to change diameter. A similar mechanism operates in the salamander, Amphiuma means. To trace the signal, we measured chloride activity in juxtaglomerular interstitial spaces in Amphiuma during perfusion of the early distal tubule belonging to the same nephron. Interstitial Cl- activity exceeded systemic levels and increased when perfusion rate in the adjacent early distal tubule was increased, reaching values more than five times isotonic. Bumetanide, which inhibits NaCl transport by the early distal tubule, eliminated the hypertonicity. Regions of the interstitial space not a part of the juxtaglomerular apparatus (JGA) were not hypertonic. The Cl- concentration was 80% greater than isotonic in the JGA of nephrons studied under free-flow conditions. Single-nephron blood flow, measured by counting the flux of erythrocytes labeled with a fluorescent molecule, showed typical feedback inhibition with maximum sensitivity to the same rates of tubular perfusion that caused the maximum change in JGA interstitial hypertonicity. Juxtaglomerular interstitial hypertonicity could be an important part of the signal for renal autoregulation.
Characterization of amino acid metabolism by cultured rat kidney cells: study with 15N
Authors: Nissim Itzhak , States Beatrice , Yudkoff Marc , Segal Stanton
Published: December 2017

Page F1243: Itzhak Nissim, Beatrice States, Marc Yudkoff, and Stanton Segal. “; Characterization of amino acid metabolism by cultured rat kidney cells: study with 15N.”Page F1243: the sentence beginning on the 14th line of the abstract should read: The disappearance of glutamine and glutamate was linear during a 60-min incubation at either pH. Page F1245: the last sentence in the legend of Figure 1 should read: Each data point is the mean ± SE of 4–6 experiments. Page F1246: Table 2, unit of measure was omitted, should be nmol·min-1·mg protein-1 Page F1249: the first full sentence in the left column should read: The data of Fig. 8 indicate that the formation of [6-amino]-15N-labeled adenine nucleotide is dependent on the H+ concentration of the incubation medium.
Role of angiotensin II in experimental membranous nephropathy
Authors: Gabbai F. B. , Wilson C. B. , Blantz R. C.
Published: December 2017

Glomerular hemodynamics measurements in rats with experimental membranous nephropathy [passive Heymann nephritis (PHN)] have demonstrated that the appearance of proteinuria 5 days after administration of anti-Fx1A antibody is temporally related to changes in the glomerular ultrafiltration coefficient (LpA). Previous studies in other models of glomerular injury have suggested a significant role for angiotensin II (ANG II) in the glomerular hemodynamic abnormalities. To evaluate the possible role of ANG II in the LpA decrease, converting enzyme inhibitor (CEI) was administered acutely or chronically (5 days before and after induction of PHN) to rats with PHN. Acute ANG II blockade produced a fall in mean arterial pressure (MAP), single-nephron glomerular filtration rate (SNGFR), absolute proximal reabsorption (APR), single-nephron plasma flow, single-nephron blood flow, and glomerular capillary hydrostatic pressure (PG); however, no changes in LpA were detected. Chronic administration of CEI (MK421, 5 mg.kg-1.day-1) in the drinking water was associated with a fall in MAP; however, both SNGFR and APR increased. PG and the transcapillary hydrostatic pressure gradient were unchanged, and LpA remained depressed. These results suggest that reduction of LpA in rats with PHN is ANG II independent and that other mechanisms are required to explain these changes in glomerular hemodynamics.
In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP
Authors: Homma S. , Gapstur S. M. , Yusufi A. N. , Dousa T. P.
Published: December 2017

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, “semipermeabilization,” increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band “ A ” of apparent molecular mass (Mr) approximately equal to 66 kDa, and band “B” of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.
Interaction between ANP and amiloride in renal tubular sodium handling in anesthetized rabbits
Authors: Nushiro N. , Abe K. , Seino M. , Yoshinaga K.
Published: December 2017

To examine whether amiloride, an inhibitor of a conductive sodium channel in the distal tubule, modifies a possible tubular action of atrial natriuretic peptide (ANP), alpha-human ANP (0.05 micrograms.kg-1.min-1) was infused intravenously with or without pretreatment of amiloride (5 mg/kg + 0.04 mg.kg-1.min-1) in anesthetized rabbits. No significant changes in arterial pressure and renal blood flow were observed between two groups after ANP infusion. However, increases in filtered sodium load (FNa) and net tubular sodium reabsorption (RNa) induced by ANP were attenuated after the amiloride pretreatment. These attenuations in FNa and RNa could be due to the failure of increase in glomerular filtration rate, although the reason is not clear. Amiloride pretreatment abolished the ANP-induced kaliuresis, whereas ANP-induced increases in urinary sodium and fractional sodium excretion were additive after amiloride pretreatment. Thus, ANP elicited a further natriuretic effect even when the amiloride-sensitive component was blocked. This suggests that there is another mechanism by which ANP induces natriuresis, in addition to any ANP effects mediated by interference with sodium conductive channels.
Role of cardiac atria in the human renal response to changing plasma volume
Authors: Myers B. D. , Peterson C. , Molina C. , Tomlanovich S. J. , Newton L. D. , Nitkin R. , Sandler H. , Murad F.
Published: December 2017

We examined the role of cardiac atria in the renal response to sequential volume expansion and contraction, during and directly following water immersion. In immersed healthy volunteers (group 1, n = 9) atrial diameter, plasma levels of atrial natriuretic peptide (ANP), and natriuresis increased, whereas renal vascular resistance (RVR) and filtration fraction declined. Each parameter changed in an opposite direction postimmersion. An analysis of transglomerular dextran transport suggests that transglomerular hydraulic pressure difference (delta P) changed in parallel with filtration fraction. Baseline atrial diameter, plasma ANP, RVR, and filtration fraction were significantly elevated in nine recipients of denervated cardiac allografts (group 2). Atrial diameter and plasma ANP changed in parallel with group 1 during and after immersion. However, corresponding reciprocal changes in RVR were smaller and filtration fraction remained constant throughout. From transglomerular dextran transport, we compute that delta P increased progressively during and after immersion, suggesting predominant efferent arteriolar tone. The postimmersed state was associated also with enhanced sodium retention despite sixfold higher plasma ANP than in group 1. These findings are consistent with an effect of cardiac denervation to leave unopposed efferent sympathetic nervous traffic to the kidney. They suggest that the latter is an important modulator of the renal response to changing plasma volume in humans.
Bicarbonate secretion modulates ammonium absorption in rat distal colon in vivo
Authors: Cohen R. M. , Stephenson R. L. , Feldman G. M.
Published: December 2017

Although the mammalian colon is thought to absorb large quantities of total ammonia, principally in the form of NH3, quantitative support for this hypothesis is lacking. In rat distal colon, we observed that NH3 was approximately 400 times more permeant than NH+4. In addition, colonic HCO-3 secretion influenced total ammonia (NH3 plus NH+4) absorption; that is, alteration of HCO-3 secretion caused a parallel change in total ammonia absorption. Perfusion with total ammonia also caused net HCO-3 secretion to switch to net absorption, and, in the setting of preexisting HCO-3 absorption, perfusate containing total ammonia enhanced HCO-3 absorption. These events suggest that colonic HCO-3 secretion titrates luminal NH+4 to NH3, permitting NH3 to diffuse from the lumen, while HCO-3 is titrated to carbon dioxide and also diffuses from the lumen. In support of titration of NH+4 and HCO-3, the magnitude of induced HCO-3 absorption approximated total ammonia absorption. This titration relationship suggests that, in kinetic studies, total ammonia absorption will be limited by a fixed rate of HCO-3 secretion. A model was developed that simulated these events.
Alterations in rabbit renal microvascular prostanoid synthesis in acute renal failure
Authors: Chaudhari A. , Kirschenbaum M. A.
Published: December 2017

Vasodepressor prostanoids have been suggested to regulate renal hemodynamics after nephrotoxic injury and thus protect the kidney against the effects of prolonged ischemia. This study assessed whether changes in two microvascular vasodilator prostanoids would correlate with changes seen in renal hemodynamics in rabbits with nephrotoxic renal injury produced by either uranyl nitrate or mercuric chloride. Rabbits were killed at 3, 24, and 72 h after the nephrotoxin injections and 6-ketoprostaglandin (PG) F1 alpha and PGE2 synthesis was measured in vitro in isolated renal microvessels. At the end of 24 h, synthesis of both prostanoids was significantly increased in all nephrotoxin-treated animals, an observation not noted at the end of 3 h. At 72 h, 6-keto-PGF1 alpha production remained elevated. Pretreatment with mepacrine blocked the increased prostanoid production seen in uranyl nitrate-treated animals. Thus, renal microvascular vasodilator prostanoid biosynthesis is increased 24-72 h after nephrotoxin administration. These data suggest that the biosynthesis of prostacyclin and PGE2 may contribute to the maintenance of renal blood flow in the first few days after acute renal injury and further suggest that a mechanism for this increase may be stimulation of phospholipase A2.
Time-dependent actions of aldosterone and amiloride on Na+-K+-ATPase of cortical collecting duct
Authors: Hayhurst R. A. , O'Neil R. G.
Published: December 2017

The actions of aldosterone on Vmax Na+-K+-ATPase activity and length of latent period were assessed for the rabbit cortical collecting duct (CCD). Initially, animals were moderately aldosterone depleted and then treated with a constant infusion of physiological doses of aldosterone. Aldosterone administration had no influence after 3 h but caused a detectable increase with 6 (borderline significance) or more hours. An apparent plateau was reached between 24 and 48 h at twice the initial activity. This aldosterone-induced stimulation could be abolished by simultaneous treatment of the animals with amiloride, demonstrating a Na+-dependent modulation of the Vmax Na+-K+-ATPase activity. The aldosterone-stimulated enzyme had kinetic properties similar to those reported by others, but the latent period for aldosterone action on the Vmax Na+-K+-ATPase activity averaged near 6 h in the present study, as opposed to the highly variable period (from 1 h to several days) seen by others. This latent period variability was shown to be directly related to the initial Vmax Na+-K+-ATPase activity in the CCD and could be likened to an "end product dependent" latent period, i.e., the lower the initial end product (Vmax Na+-K+-ATPase activity) the shorter the latent period. Hence aldosterone's actions on the Na+-K+-ATPase of the CCD would be consistent with a single mechanism of action, i.e., increased synthesis, but with a variable modulation of this synthesis, which is dependent on the initial Vmax Na+-K+-ATPase activity of the CCD cells and/or the initial aldosterone status of the animal.
Impaired control of renal hemodynamics and renin release during hyperkalemia in rabbits
Authors: Lin H. B. , Young D. B.
Published: December 2017

We analyzed the changes in control of renal hemodynamics and renin release resulting from hyperkalemia in the rabbit. Plasma potassium activity was maintained at a controlled elevated level by intravenous infusion of KCl. The potassium activity of the control group (n = 23) averaged 3.20 +/- 0.06 meq/l and that of the hyperkalemic group (n = 13) averaged 5.80 +/- 0.13 meq/l. Renal blood flow (RBF), glomerular filtration rate (GFR), and renin release were measured over a range of renal perfusion pressures achieved by constriction of the suprarenal aorta. The control group's RBF and GFR exhibited excellent autoregulatory capability from 100 to 80 mmHg. However, significant impairment of autoregulation was apparent in the hyperkalemic group. At 100 mmHg, RBF and GFR in the hyperkalemic group averaged 33 and 34% greater, respectively, than those of the control group (P less than 0.005); both variables in the hyperkalemic group were greater than the values of the control group over the autoregulatory range (100-80 mmHg). The renin release values for the two groups were not different at the 100-mmHg pressure level, although renin release of the hyperkalemic group increased to higher levels than those of the control group as perfusion pressure was reduced. At the 70- and 60-mmHg levels renin release from the hyperkalemic group averaged approximately 300% greater than that of the control group (P less than 0.05). However, when expressed as percentage change, the stimulatory effect of hyperkalemia was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)
Enhanced vasopressin (V2-receptor)-induced sodium retention in mineralocorticoid hypertension
Authors: Jeffries W. B. , Wang Y. , Pettinger W. A.
Published: December 2017

The deoxycorticosterone acetate (DOCA)-Na model of hypertension requires the presence of vasopressin for expression of high blood pressure. In the present study, the effects of vasopressin V2-receptor stimulation were examined in kidneys from rats receiving 1 wk of DOCA-Na or control (olive oil-tap water) treatment. The dose response to vasopressin (10(-10) to 10(-6) M) was tested in microdissected cortical collecting tubule (CCT) segments and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was significantly increased in segments from DOCA-Na rats vs. controls, confirming our previous study. In other experiments, kidneys from DOCA-Na and control rats were perfused with a modified Krebs-Henseleit buffer (37 degrees C, pH 7.4) and treated with either vehicle or 0.21-2.1 pM 1-desamino-8-D-arginine vasopressin (DDAVP). DDAVP caused significant (P less than 0.05) dose-related reductions in urine excretion (UV) and urinary sodium excretion (UNaV) in both DOCA-Na and control kidneys in the absence of changes in renal hemodynamics. However, DDAVP produced earlier and significantly greater reductions in UV and UNaV in kidneys from DOCA-Na vs. control rats. Percent fractional excretion of sodium was reduced significantly only in the DOCA-Na group (2.1 pM DDAVP). A small degree of antikaluresis was seen with DDAVP in both groups. Thus, DOCA-Na treatment augmented cAMP accumulation in the CCT, accompanied by a significant enhancement of DDAVP-stimulated urinary sodium and water reabsorption at the level of the intact kidney.(ABSTRACT TRUNCATED AT 250 WORDS)
Cortical interstitium as a site for solute polarization during tubular absorption
Authors: Williams J. C. , Schafer J. A.
Published: December 2017

The possibility that significant concentration differences could exist between the interstitial fluid and capillary plasma was investigated by modeling the renal cortex as the following three compartments: tubular lumen, interstitium, and capillary lumen. A simple analysis of this system suggests that for the interstitium surrounding a proximal tubule, the concentration in the interstitium of a solute like glucose could be well over 1 mM greater than in the peritubular capillary if the solute permeability of the peritubular capillary were like that measured in other organs (i.e., less than 10 micron/s). The effect of varying capillary permeability on the interstitial concentrations of several solutes was examined using a modification of a model of the proximal tubule, and results were found to be similar to those obtained with the simpler analysis for glucose. This model was also used to see if placing an osmotic difference between the tubule lumen and capillary could cause significant solute polarization within the interstitium, as might occur in an experiment to measure the osmotic water permeability (Pf) of the proximal tubule in vivo. The results show that an apparent Pf calculated from the difference between the osmolalities of tubular perfusate and peritubular plasma is likely to underestimate the true Pf of the proximal tubule. Even for a high value of capillary permeability (10 micron/s, which allows relatively rapid diffusion between capillary and interstitium), the model predicts that the apparent Pf may underestimate the true value by half. Thus the analyses presented suggest that if the permeability of renal peritubular capillaries is similar to that measured in other organs the composition of the interstitium may be significantly different from capillary plasma, a situation that would have great impact on our view of the mechanism of volume absorption in the proximal tubule in vivo.
Direct evaluation of the microvascular actions of ANP in juxtamedullary nephrons
Authors: Veldkamp Peter J. , Carmines Pamela K. , Inscho Edward W. , Navar L. Gabriel
Published: December 2017

Page F440: Peter J. Veldkamp, Pamela K. Carmines, Edward W. Inscho, and L. Gabriel Navar. “Direct evaluation of the microvascular actions of ANP in juxtamedullary nephrons.” Page F442: because of the disappointing quality of Fig. 1, the figure is reproduced here.

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Do osmotic forces play a role in renin secretion?
Authors: Skott O.
Published: December 2017

Secretory granules swell during exocytosis. Swelling may follow fusion and assist in extrusion of the granular content, or swelling may cause granular fusion with the plasmalemma. A granular proton gradient has been suggested to be involved in such preexocytic granular swelling. Exocytosis of renin from juxtaglomerular cells of isolated preparations is very sensitive to changes in the extracellular osmolality. Extracellular hyposmolality causes swelling of secretory granules, fusions between peripherally located granules and plasmalemma, and an increased number of release episodes. Induction of granule swelling at constant extracellular osmolality also stimulates renin release. Newly recruited renin granules are osmosensitive, and a high extracellular osmolality blocks secretion induced by other means (low calcium). Dissipation of granular proton gradients inhibits renin release without affecting the osmosensitivity. Thus, in renin release in vitro, a granular swelling precedes fusion and exocytosis, and a granular proton gradient may contribute to preexocytic swelling when extracellular osmolality is constant. The osmosensitivity may be important for macula densamediated renin release.
Effects of maternal hyperglycemia on fetal renal function in sheep
Authors: Smith F. G. , Lumbers E. R.
Published: December 2017

The effects of maternal hyperglycemia on fetal renal function were investigated in 10 chronically catheterized fetal sheep after the infusion of 100 g of glucose into the ewe over 30 min. Fetal blood glucose levels rose (P less than 0.001) within 15 min of completing the glucose infusion from 15.75 +/- 2.8 to 195.4 +/- 18 (SE) mg/dl (n = 10). There was a significant increase in fetal glomerular filtration rate (P less than 0.05) from 2.73 +/- 0.41 to 3.65 +/- 0.40 (SE) ml/min (n = 10) within 1.5 h of the infusion of glucose into the ewe. Urine flow rate increased from 0.38 +/- 0.06 to 0.63 +/- 0.12 (SE) ml/min (n = 10, P less than 0.001), and sodium excretion increased from 18.42 +/- 7.21 to 38.4 +/- 13.7 (SE) mumol/min (n = 10, P less than 0.002) within 2.5 h of the infusion of glucose into the ewe. The fraction of the filtered load that was excreted (urine flow rate divided by glomerular filtration rate) also increased (P less than 0.01) as did the fraction of the osmolar load (P less than 0.002). Glycosuria occurred in all fetuses within 30 min of the infusion of glucose into the ewe, and glucose excretion reached 26.16 +/- 12.36 (SE) micrograms/min (n = 8) after 1.5 h. These findings of diuresis, natriuresis, and glycosuria in response to hyperglycemia are evidence that an increased delivery of fluid into the amniotic cavity might occur after a rise in fetal plasma glucose levels.
Immunocytochemical localization of band 3 protein in the rat collecting duct
Authors: Verlander J. W. , Madsen K. M. , Low P. S. , Allen D. P. , Tisher C. C.
Published: December 2017

Band 3 protein is the major anion transport protein of the erythrocyte cell membrane where it catalyzes the exchange of HCO3- for Cl-. There is evidence that band 3 protein is present in the collecting duct of both the rat and rabbit kidney. We used colloidal-gold immunocytochemistry to determine the ultrastructural location of band 3 protein in the rat cortical (CCD) and outer medullary collecting ducts (OMCD). Kidneys of normal Sprague-Dawley rats were fixed by intravascular perfusion with 1% glutaraldehyde and embedded in Lowicryl K4M. Two polyclonal antibodies raised in rabbits were used as the primary antibody in separate experiments, one against the 43-kDa fragment of the cytoplasmic domain of human erythrocyte band 3 protein and the other against rat erythrocyte band 3 protein. This was followed by exposure to gold-conjugated goat anti-rabbit immunoglobulin G. Transmission electron microscopy revealed gold particles along the basal and lateral plasma membranes of all intercalated cells of the OMCD. In the CCD, the basal and lateral plasma membranes of the type A intercalated cells only were labeled with gold particles. The type B intercalated cells and principal cells were devoid of gold particles, as were all cells of the proximal tubule, the distal convoluted tubule, and the thick ascending limb of the loop of Henle. We conclude that a Cl(-)-HCO3- transporter is present in the basal and lateral plasma membranes of the intercalated cells in the OMCD and the type A intercalated cells in the CCD. These findings provide further evidence that these intercalated cells are involved in H+ secretion in the OMCD and CCD of the rat. We have no evidence for the presence of band 3 protein in the type B intercalated cells of the CCD, which supports the hypothesis that type B cells are functionally and structurally distinct from type A cells.
NH3 permeation through the apical membrane of MDCK cells is via a lipid pathway
Authors: Golchini K. , Kurtz I.
Published: December 2017

The pathway for NH3 permeation across the apical membrane of MDCK cells was determined by measuring the effect of membrane fluidizing agents, protein reactive agents, and temperature on cellular NH3 influx. The rate of NH3 influx was calculated from the time course of increase in intracellular pH (pHi), measured with 2,7-biscarboxyethyl-5(6)-carboxyfluorescein, when MDCK cell monolayers were exposed to NH4Cl. The apical membrane NH3 permeability was 7.13 +/- 0.37 x 10(-3) cm/s (n = 12) at 37 degrees C and 1.23 +/- 0.07 x 10(-3) cm/s (n = 7) at 18 degrees C. In comparison, apical membrane permeability at 37 degrees C to the weak acids, valeric acid and acetic acid, were 1.39 +/- 0.11 x 10(-2) cm/s (n = 4) and 6.93 +/- 0.11 x 10(-3) cm/s (n = 4), respectively. The activation energy for NH3 permeation was 15.0 +/- 1.0 kcal/mol (17.5 degrees C-37.5 degrees C). In the presence of the membrane fluidizing agents, heptanol or chloroform, NH3 permeability increased in a dose-dependent manner. Heptanol (15 mM) significantly decreased the activation energy for NH3 permeation to 4.4 +/- 0.6 kcal/mol, P less than 0.001. The carboxyl reactive agent (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluensulfonic acid 1 mM), aminoreactive agents (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid 50 microM; picrylsulphonic acid 1 mM), the sulphydryl reactive agent (p-chloromercuriphenylsulfonic acid 1 mM), and the nonspecific membrane protein cleaving agent pronase (1 mg/ml) had no effect on the NH3 influx. The results suggest that NH3 permeates the plasma membrane of MDCK cells via a lipid pathway.
Evidence for active dipeptide transport in isolated proximal straight tubules
Authors: Barfuss D. W. , Ganapathy V. , Leibach F. H.
Published: December 2017

Transport of the dipeptide glycylsarcosine (Gly-Sar) was examined in isolated proximal straight tubules of the rabbit kidney by an in vitro microperfusion technique to determine whether it can be actively transported intact. The unidirectional lumen-to-bath flux of Gly-Sar was measured by two separate methods, namely its appearance rate (JA) in the bathing fluid and its disappearance rate (JD) from the luminal fluid. In addition, the cell Gly-Sar concentration was measured immediately after the last flux period. Mean luminal fluid Gly-Sar concentration was 0.22 mM. Transepithelial Gly-Sar flux (260.0 fmol.min-1.mm-1) was greater than could be accounted for by passive leakage, whereas cellular Gly-Sar accumulation (2.72 mM) was greater than could be attributed to passive equilibration across the luminal membrane. High-pressure liquid chromatographic analysis of cellular extract indicated that 63% of the transported Gly-Sar was hydrolyzed within the cell. Analysis of the bath solution revealed that 47% of the radioactivity that crossed the tubule cell was in the form of intact dipeptide, whereas the remainder of the radioactivity was in the form of hydrolytic and metabolic products of Gly-Sar. This indicates that the dipeptide Gly-Sar is actively transported intact at the luminal membrane into the cytosol of proximal straight tubule cells with subsequent hydrolysis. It then exits across the basolateral membrane as intact Gly-Sar and its hydrolytic and metabolic products.