A B S T R A C T Insulin influences certain metabolic and transport renal functions and is avidly degraded by the kidney, but the relative contribution of the luminal and basolateral tubular membranes to these events remains controversial. We studied 125I-insulin degradation [TCA and immunoprecipitation (IP) methods] and the specific binding of the hormone by purified luminal (L) and basolateral (BL) tubular membranes. These were prepared from rabbit kidney cortical homogenates by differential and gradient centrifugation and ionic precipitation steps in sequence, which resulted in enrichment vs. homogenate of marker enzymes' activities (sodium-potassium-activated adenosine triphosphatase for BL and maltase for L) of 8-and 12-fold, respectively. Both fractions degraded insulin avidly and bound the hormone specifically without saturation even at pharmacologic concentrations (10 lM). At physiologic insulin concentrations (0.157 nM) BL membranes degraded substantial amounts of insulin (44.2±2.6 and 40.7±2.2 pg/mg protein per min by the TCA and IP methods, respectively), even though at lesser rates (P <0.001) than the luminal fraction (67.2±2.3 and 75±6.2 pg/mg protein per min, respectively); the rate of insulin catabolism by BL membranes was significantly higher (P < 0.001) than that which could be attributed to their contamination by luminal components [12.2±1.9 pg/mg per min (TCA method), or 13.7±1.9 pg/mg per min (IP method)]. Competition experiments suggested that insulin-degrading activity in both fractions includes both specific and nonspecific components. In contrast to degradation, insulin binding by both membranes was highly specific for native insulin and was severalfold higher in BL than L membranes [17.5±1.3 vs. 4.5±0.4 fmol/mg protein (P < 0.001) at physiologic insulin concentrations]. Despite the marked difference in the binding capacity for insulin by the two membranes,

Renal acidification in renal proximal tubule is thought to be mediated by luminal Na-H antiporter and the HCO3- generated by this antiporter is removed from the cell by a basolateral Na-HCO3 cotransporter. To study the effect of respiratory acid-base disorders on these transport systems, we have measured the Na-HCO3 cotransport in basolateral membranes and Na-H antiporter in luminal membranes in control rabbits, rabbits exposed to 10% CO2 (chronic hypercapnia), and rabbits exposed to 10% O2-90% N2 (chronic hypocapnia). The Vmax of HCO3(-)-dependent 22Na uptake was significantly higher in chronic hypercapnia than controls (2.54 +/- 0.03 vs. 1.18 +/- 0.21 nmol.mg protein-1.3 s-1, P less than 0.001). Likewise, the Vmax of the Na-H antiporter was also increased compared with controls (924.9 +/- 42.1 vs. 549.1 +/- 62.8 fluorescence units (FU).300 micrograms protein-1.min-1). In chronic hypocapnia, the Vmax of Na-HCO3 cotransport was lower than controls (0.72 +/- 0.11 vs. 1.18 +/- 0.21 nmol.mg protein-1.3 s-1, P less than 0.05). There was no difference, however, in the Vmax of the Na-H antiporter between hypocapnia and control (524.2 +/- 24.3 vs. 549.1 +/- 62.8, FU.300 micrograms protein-1.min-1). The Vmaxs of the Na-HCO3 cotransport and of the Na-H antiporter in hypocapnic, control, and hypercapnic rabbits were linearly related (r = 0.81), suggesting a simultaneous adaptation of the two systems in respiratory acid-base disorders.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of [2‐14C]pyruvate into adipose tissue triglycerides was measured in fasted, diabetic and triamcinolone treated rats and related to the activity of enzymes active in the elaboration of glyceride glycerol. Incorporation of pyruvate into glyceride glycerol was increased in fasting and diabetes. The activity of phosphoenolpyruvate carboxykinase increased in parallel with the enhanced glyceroneogenesis, whereas the activity of pyruvate carboxylase changed in the opposite direction. After triamcinolone treatment, the activity of both enzymes was decreased as was the incorporation of [2‐14C]pyruvate into glyceride glycerol. In all experimental animals glutamate‐pyruvate transaminase activity in adipose tissue decreased, whereas the activity of glutamate‐oxaloacetate transaminase increased slightly. These results were interpreted as implying that the enhanced glyceroneogenesis in diabetes and fasting is related to the increase in phosphoenolpyruvate carboxykinase activity, whereas pyruvate carboxylase activity seems associated with changes in fatty acid synthesis. In contrast to adipose tissue, in the liver the activity of both enzymes of the dicarboxylic acid shuttle and of transaminases increased in the situations associated with enhanced gluconeogenesis. This finding is discussed in the light of possible differences in the availability of precursors for gluco‐ and glyceroneogenesis in the respective tissues. The role of glyceroneogenesis is pointed out, as one of the factors maintaining the balance between synthesis and breakdown of triglycerides in adipose tissue, particularly in conditions associated with rapid lipolysis.

Binding of the anion-exchange inhibitor 3Hz-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3Hz]DIDS of 5.5 pM and 19.3 pM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolaterdl and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 3 5 S 0 4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed C1 gradient, OH gradient or HCOJ gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HC03-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 pM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport. . Lucci and Warnock [6] have shown inhibition of volume reabsorption by furosemide and 4-acetamido-4-isothiocyano-2,2'-stilbene disulfonic acid (SITS) when added to the luminal perfusion solution in microperfusion studies of superficial proximal convoluted tubules of rat. In addition, the disulfonic stilbene SITS has been shown to inhibit H C 0 3 and fluid reabsorption by the proximal tubule when added only to the peritubular fluid [7], and to inhibit SO, transport when added to either bath or perfusate [8]. Taken together these data suggest the existence of an anion exchanger in both the luminal and in the peritubular side of the proximal tubular cell. Therefore, the purpose of the present study was to characterize specific binding of [3H2]DIDS by highly purified luminal and basolateral renal tubular membranes and to assess the role of this Correspondence to Z . Talor, Section of Nephrology (m/c 793)

Chronic hypercapnia is associated with increased proximal HCO3 reabsorption that is thought to be mediated by a Na-H antiporter. We hypothesized that chronic hypercapnia would be associated either with increased Vmax or with decreased Km of the Na-H antiporter. To test this hypothesis we made rabbits hypercapnic for 48 h by exposure to 10% CO2. In both control and hypercapnic animals, cortical luminal membranes were enriched over the homogenate 16-fold in alkaline phosphatase and 10-fold in maltase activity. The kinetic activity of the Na-H antiporter was measured by the dissipation of the quenching of acridine orange by addition of different Na concentrations. Chronic hypercapnic rabbits had significantly higher Vmax of the Na-H antiporter of luminal membranes than controls (593 +/- 81 vs. 252 +/- 40 arbitrary fluorescence units X min-1 X 300 micrograms protein-1, P less than 0.01). The Km, however, was not different between control and hypercapnic rabbits. 22Na uptake in presence of an outwardly directed pH gradient was significantly higher in vesicles from hypercapnic rabbits than controls. Amiloride inhibited the Na-H antiporter (as assessed by acridine orange quenching or 22Na uptake) to the same degree in membranes from both control and hypercapnic rabbits, suggesting that the increase in Vmax is mediated by the electroneutral component of the Na-H antiporter. In addition, under voltage clamp conditions by K and valinomycin the Vmax was still increased in membranes from hypercapnic animals, again suggesting that the increase in Vmax is mediated by the electroneutral component of the Na-H antiporter. The uptake of D-[3H]glucose by luminal membranes was not different between control and hypercapnic rabbits, indicating a specific enhancement of the Na-H antiporter. Acute hypercapnia (4 h) failed to increase the Vmax of the Na-H antiporter despite comparable increase in PCO2. Thus chronic hypercapnia, but not acute hypercapnia, induces a selective and specific increase in the Vmax of Na-H antiporter, and this may mediate the adaptation to chronic hypercapnia.(ABSTRACT TRUNCATED AT 250 WORDS)

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A 60-year-old woman who had been instructed to increase her water intake because of nephrolithiasis developed the syndrome of inappropriate secretion of antidiuretic hormone when treated with chlorthalidone for mild hypertension. Serum osmolality was 235 mOsm/kg with concomitant urine osmolality of 490 mOsm/kg. When serum sodium decreased to 110 mEq/liter, plasma antidiuretic hormone (ADH) was elevated at 30 pg/ml. The syndrome resolved when chlorthalidone was discontinued together with fluid intake restriction. Plasma ADH returned to normal (less than 0.5 pg/ml) after three days of treatment. The favorable outcome in this patient is attributed to early recognition of the syndrome, which might occur even with nonthiazide diuretics such as chlorthalidone.

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