Ammonia production by isolated mouse proximal tubules perfused in vitro. Effect of metabolic acidosis.

We examined the effects of metabolic acidosis in vivo and reduced bath and luminal pH in vitro on total NH3 (NH3 + NH+4) production rates by isolated mouse proximal tubule segments. Midproximal tubule segments were obtained from mice with NH4Cl-induced metabolic acidosis and from nonacidotic controls. The segments were perfused with modified Krebs-Ringer bicarbonate (KRB) buffer, incubated in KRB buffer containing 0.5 mM L-glutamine and 1.0 mM sodium acetate, and gassed with 95% O2 and 5% CO2. Isolated unperfused and perfused proximal tubules from acidotic mice produced total NH3 at higher rates than corresponding tubules from nonacidotic mice. Perfusion of the tubular lumen stimulated total NH3 production by tubules from both acidotic and nonacidotic mice. In contrast, lowering the bath pH to 7.0 by lowering the HCO3- concentration increased total NH3 production rates by tubules from nonacidotic mice but not by tubules from acidotic mice. Reducing the HCO3- concentration of the bath buffer to 10 mM while maintaining a pH of 7.4 had no significant effect on total NH3 production by tubules from nonacidotic mice. Lowering the luminal fluid pH by reducing the perfusate HCO-3 from 25 mM to 10, 5, or 1.2 mM while maintaining a bath pH of 7.4 lowered collected luminal fluid pH but had no effect on total NH3 production by proximal tubules from nonacidotic mice. These observations demonstrated that metabolic acidosis in vivo stimulated total NH3 production in isolated mouse proximal tubule segments and that low peritubular pH and HCO-3 stimulated total NH3 production by proximal tubule segments from nonacidotic mice in vitro.     

Effect of in vitro metabolic acidosis on luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport in rabbit kidney proximal tubules.

The aim of this study was to evaluate the role of the kidney in mediating the signals involved in adaptive changes in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (A) or control (C) media (pH 6.9 vs 7.4, 5% CO2) for 2 h. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of Na+/H+ exchange and Na+:HCO3- cotransport. Influx of 1 mM 22Na at 10 s (pH6 7.5, pH(i) 6.0) into BBM vesicles was 68% higher in group A compared to group C. The increment in Na+ influx in the group A was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.1 mM in C vs 9.2 in A and Vmax of 31 nmol/mg protein per min in group C vs 57 in A. Influx of 1 mM 22Na at 10 s (pH0 7.5, pH(i) 6.0, 20% CO2, 80% N2) into BLM vesicles was 83% higher in the group A compared to C. The HCO3-dependent increment in 22Na uptake in group A was 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid sensitive, suggesting that Na+:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 11.4 mM in C vs 13.6 in A and Vmax of 35 nmol/mg protein per min in C vs 64 in A. The presence of cyclohexamide during incubation in A medium had no effect on the increments in 22Na uptake in group A. We conclude that the adaptive increase in luminal Na+/H+ exchange and basolateral Na+:HCO3- cotransport systems in metabolic acidosis is acute and mediated via direct signal(s) at the level of renal tubule.     

Potassium depletion increases luminal Na+/H+ exchange and basolateral Na+:CO3=:HCO3- cotransport in rat renal cortex.

Most HCO3- reabsorption in proximal tubules occurs via electroneutral Na+/H+ exchange in brush border membranes (BBMS) and electrogenic Na+:CO3=:HCO3- cotransport in basolateral membranes (BLMS). Since potassium depletion (KD) increases HCO3- reabsorption in proximal tubules, we evaluated these transport systems using BBM and BLM vesicles, respectively, from control (C) and KD rats. Feeding rats a potassium deficient diet for 3-4 wk resulted in lower plasma [K+] (2.94 mEq/liter, KD vs. 4.47 C), and higher arterial pH (7.51 KD vs. 7.39 C). KD rats gained less weight than C but had higher renal cortical weight. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0, 10% CO2, 90% N2) into BLM vesicles was 44% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was DIDS sensitive, suggesting that Na+:CO3=:HCO3- cotransport accounted for the observed differences. Kinetic analysis of Na+ influx showed a Km of 8.2 mM in KD vs. 7.6 mM in C and Vmax of 278 nmol/min/mg protein in KD vs. 177 nmol/min/mg protein in C. Influx of 1 mM 22Na+ at 5 s (pHo 7.5, pHi 6.0) into BBM vesicles was 34% higher in the KD group compared to C with no difference in equilibrium uptake. The increment in Na+ influx in the KD group was amiloride sensitive, suggesting that Na+/H+ exchange was responsible for the observed differences. Kinetic analysis of Na+ influx showed a Km of 6.2 mM in KD vs. 7.1 mM in C and Vmax of 209 nmol/min/mg protein in KD vs. 144 nmol/min/mg protein in C. Uptakes of Na(+)-dependent [3H]glucose into BBM and [14C]succinate into BLM vesicles were not different in KD and C groups, suggesting that the Na+/H+ exchanger and Na+:CO3=:HCO3- cotransporter activities were specifically altered in KD. We conclude that adaptive increases in basolateral Na+:CO3=:HCO3- cotransport and luminal Na+H+ exchange are likely responsible for increased HCO3- reabsorption in proximal tubules of KD animals.     

Differential effects of histamine H2 receptor antagonists on amantadine uptake in the rat renal cortical slice, isolated proximal tubule and distal tubule

The interaction between amantadine and two histamine receptor antagonists was examined in the rat kidney. Amantadine (10 microM, 30 sec) was actively accumulated by cortical slices (slice/medium ratio = 0.4 +/- 0.3 [3.3 +/- 0.3 at 4 min], mean +/- S.E.M.), isolated proximal tubules (tubule/medium ratio = 35 +/- 1) and distal tubules (tubule/medium ratio = 19 +/- 2). In cortical slices, low cimetidine concentrations facilitated amantadine accumulation, whereas higher concentrations produced inhibition. Uptake in proximal tubules was enhanced by cimetidine and reached a maximum at approximately 100 microM. Cimetidine (20 microM) decreased the apparent Km (88 +/- 5 to 55 +/- 3 microM, P less than .005) without altering Vmax (6.8 +/- 0.5 to 5.8 +/- 0.6 nmol/mg/min). Conversely, cimetidine did not enhance uptake in distal tubules but elicited competitive inhibition at concentrations greater than 1 mM. Although this may partially delineate the differences observed between the cortical slice and proximal tubule data, such a discrepancy may also implicate additional sites of interaction in other segments of the cortical nephron and/or cimetidine inhibition of the relatively more significant luminal amantadine efflux in the proximal tubules. Ranitidine did not enhance amantadine accumulation but produced inhibition at high concentrations. In proximal and distal tubule preparations, ranitidine (10 mM) increased Km from 86 +/- 7 to 121 +/- 8 and 95 +/- 5 to 160 +/- 10 microM, respectively (P less than .05), whereas Vmax was not changed (8.9 +/- 0.7 to 7.9 +/- 0.8 and 4.3 +/- 0.1 to 3.8 +/- 0.2 nmol/mg/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of adaptation to chronic respiratory acidosis in the rabbit proximal tubule.

The hyperbicarbonatemia of chronic respiratory acidosis is maintained by enhanced bicarbonate reabsorption in the proximal tubule. To investigate the cellular mechanisms involved in this adaptation, cell and luminal pH were measured microfluorometrically using (2",7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein in isolated, microperfused S2 proximal convoluted tubules from control and acidotic rabbits. Chronic respiratory acidosis was induced by exposure to 10% CO2 for 52-56 h. Tubules from acidotic rabbits had a significantly lower luminal pH after 1-mm perfused length (7.03 +/- 0.09 vs. 7.26 +/- 0.06 in controls, perfusion rate = 10 nl/min). Chronic respiratory acidosis increased the initial rate of cell acidification (dpHi/dt) in response to luminal sodium removal by 63% and in response to lowering luminal pH (7.4-6.8) by 69%. Chronic respiratory acidosis also increased dpHi/dt in response to peritubular sodium removal by 63% and in response to lowering peritubular pH by 73%. In conclusion, chronic respiratory acidosis induces a parallel increase in the rates of the luminal Na/H antiporter and the basolateral Na/(HCO3)3 cotransporter. Therefore, the enhanced proximal tubule reabsorption of bicarbonate in chronic respiratory acidosis may be, at least in part, mediated by a parallel adaptation of these transporters.     

Stereoselective inhibition of amantadine accumulation by quinine and quinidine in rat renal proximal tubules and cortical slices

The renal organic cation transport system was examined. The accumulation of a nonchiral cation, amantadine, by rat renal proximal tubules and cortical slices was investigated, together with the effects of two diastereoisomers, quinine and quinidine. The proximal tubules actively concentrated amantadine with a tissue/medium ratio of 96.3 +/- 1.7 (mean +/- S.E.M., n = 18). Apparent Km was 85 +/- 2 microM and Vmax was 8.0 +/- 0.2 nmol/mg of tubular protein per min. Amantadine accumulation was inhibited competitively by quinine and quinidine with Ki values of 32 +/- 3 and 84 +/- 11 microM, respectively (n = 4). Amantadine was also concentrated by renal cortical slices with tissue/medium ratio of 3.3 +/- 0.3 (n = 4). Apparent Km and Vmax were 94.0 +/- 5.2 microM and 1.27 +/- 0.08 nmol/mg of tubular protein per min, respectively (n = 10). Quinine and quinidine again inhibited amantadine accumulation competitively by the slices, with Ki values of 368 +/- 28 and 780 +/- 84 microM, respectively (n = 4). A similar affinity (Km) for amantadine was observed in both preparations. However, the lower Vmax value in the slice system may be due to additional amantadine transport sites with lower capacity, lesser luminal accumulation and/or limited substrate(s) penetration in the cortical slices. In either preparation, quinine and quinidine functioned as competitive inhibitors and stereoselectivity was observed for the (-)-isomer, quinine, over the (+)-isomer, quinidine. Additional transport sites, reduced luminal substrate accumulation and/or diffusional restraints in the slices are also feasible mechanisms in explaining the differences in Ki values between the two preparations, and their relative contributions await further investigation.     

Spontaneous luminal disequilibrium pH in S3 proximal tubules. Role in ammonia and bicarbonate transport.

We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.     

The excretion of carbonic anhydrase isozymes CA I and CA II in the urine of apparently healthy subjects and in patients with kidney disease

Carbonic anhydrase isozymes CA I and CA II were assayed by a radio-immunosorbent technique in the plasma and urine of apparently healthy subjects and of patients with renal disease. The concentrations (mean +/- SD, n = 8) of CA I and CA II in the plasma of healthy subjects were 2.3 +/- 2.3 and 0.8 +/- 0.5 mg/l, respectively. The urinary excretion values were 3.8 +/- 2.0 and 3.5 +/- 1.9 micrograms/24 h, and the apparent renal clearances were 21 +/- 17 and 52 +/- 44 microliters/min, respectively, values that are similar to those of other low molecular weight proteins. CA I and CA II have mol. wt of 28,850 and 29,300, respectively, they are globular in shape and have a Stoke-Einstein radius of 25 A. They could, therefore, be expected to be filtered at the glomeruli and thereafter reabsorbed by the proximal tubules. CA II is also present in the cytoplasm of renal proximal and distal tubular cells. A study of the pattern of urinary excretion of CA I and CA II could permit detection of damage to renal tubular cells in two ways--either from defective reabsorption of filtered CA I and CA II by the proximal tubular cells, or from leakage of CA II from the proximal or distal tubules into the urine. Some patients with hypercalcuria and renal tubular acidosis showed increased excretion of these enzyme proteins and of beta 2-microglobulin (BMG) into the urine, but the prevalence was rather low (27%). Further studies of patients with more severely damaged kidneys are required.     

Effect of acidosis on chloride transport in the cortical thick ascending limb of Henle perfused in vitro.

The present studies examined the effect of acute in vitro acidosis on chloride reabsorption in the rabbit cortical thick ascending limb of Henle (cTALH). Four protocols were used: hypercapnic acidosis; "isocapnic" peritubular acidosis (bath bicarbonate reduction to 10 mM); isocapnic luminal acidosis (luminal bicarbonate reduction to 10 mM); isocapnic peritubular acidosis in the absence of luminal potassium. Transepithelial voltage (VT) decreased during hypercapnic acidosis and increased with recovery. Chloride reabsorption (pmol X mm-1 X min-1) decreased from 50.3 +/- 8.4 to 15.7 +/- 5.6, then increased to 45.6 +/- 11.1 with recovery. Likewise, VT was decreased reversibly during isocapnic peritubular acidosis, and chloride reabsorption decreased by 60%. Chloride reabsorption was greater (28.3 +/- 3.6) when tubules were perfused at normal luminal pH than at an acidotic luminal pH (11.4 +/- 4.5; P less than 0.05). Luminal potassium removal reduced chloride transport, and acidosis had no significant additional effect. Decreased chloride reabsorption in the cTALH during acidosis could contribute to the chloruresis associated with systemic acidosis. The symmetrical nature of this effect suggests that acidosis inhibits chloride reabsorption through an effect on cytosolic pH.     

Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (lumen) and Cl- removal (lumen, bath). After a brief exposure to 20 mM NH4Cl, pHi fell by approximately 0.7 and recovered at a rate of 0.89 +/- 0.15 pH/min in the nominal absence of Na+, HCO3-, organic anions, and SO4(2-) (lumen, bath). 1 mM N,N'-dicyclohexylcarbodiimide (lumen), 1 mM N-ethylmaleimide (lumen), 0.5 mM colchicine (bath), and 0.5 mM iodoacetic acid (lumen, bath) inhibited the Na+-independent pHi recovery rate by 73%, 55%, 77%, and 86%, respectively, whereas 1 mM KCN (lumen, bath) did not inhibit pHi recovery. Reduction of intracellular, but not extracellular chloride, also decreased the Na+-independent pHi recovery rate. In conclusion, the S3 proximal tubule has an apical Na+/H+ antiporter with a Michaelis constant for Na+ of 29 mM and a maximum velocity of 0.47 pH/min. S3 tubules also possess a plasma membrane H+-ATPase that can regulate pHi, has a requirement for intracellular chloride, and utilizes ATP derived primarily from glycolysis.     

Interference with renal organic cation transport by (-)- and (+)-nicotine at concentrations documented in plasma of habitual tobacco smokers.

Nicotine is the principal psychoactive Nicotiana alkaloid in tobacco. In the present study, we used amantadine as a marker and investigated the potential ability of nicotine and cotinine to interfere with renal organic cation transport in vitro. [3H]Amantadine is concentrated actively by isolated proximal tubules, distal tubules and cortical slices. In proximal tubules, the addition of (-)- or (+)-nicotine (0.1-100 microM) facilitated amantadine (10 microM) accumulation. Apparent Km for amantadine uptake was decreased by a clinically relevant concentration of (-)- and (+)-nicotine (0.4 microM), from 78 +/- 2 to 52 +/- 2 and 61 +/- 5 microM, respectively (mean +/- S.E.M., P less than .05), whereas Vmax was not altered (6.6 +/- 0.1 to 6.3 +/- 0.1 and 6.5 +/- 0.2 nmol/mg/min). The addition of (-)-cotinine (0.4-100 microM) also facilitated amantadine uptake, but with lesser efficacy. Possible mechanisms underlying the present enhancement of uptake include facilitation of amantadine influx and/or attenuation of efflux. Efflux data indicate a prominent hindrance of amantadine egress from preloaded tubules in the presence of 0.4 microM (-)- and (+)-nicotine (51 +/- 4 to 32 +/- 8 and 27 +/- 4 pmol/mg/30 sec, P less than .05) and are supportive of the latter notion. In distal tubules, (-)- or (+)-nicotine produced inhibition only a high concentrations (greater than or equal to 100 microM). Km was increased by 400 microM (-)- and (+)-nicotine from 76 +/- 5 to 124 +/- 9 and 116 +/- 17 microM, and Vmax was moderately decreased from 3.4 +/- 0.5 to 3.0 +/- 0.4 and 3.0 +/- 0.4 nmol/mg/min (P less than .05). The incorporation of (-)-cotinine did not alter amantadine uptake. Enhancement of uptake by (-)- or (+)-nicotine was absent in cortical slices, in which tubular luminal transport has ben proposed to be insignificant, and only low affinity inhibition was apparent. The present data indicate potent interference of renal proximal tubular transport of amantadine by nicotine at concentrations equivalent to those documented in plasma of habitual tobacco smokers and suggest potential alterations in renal organic cationic drug elimination in these subjects.     

Glomerular ultrafiltration of IGF-I may contribute to increased renal sodium retention in diabetic nephropathy.

Insulin-like growth factor-I (IGF-I) is found in plasma at relatively high levels (approximately 40 nmol/L) but <1% is present in the free form and >99% is bound to specific binding proteins to form high-molecular-weight complexes of approximately 50 and approximately 150 kd. We hypothesized that in rats with diabetic nephropathy but not in normal animals, IGF-I-containing binding protein complexes undergo glomerular ultrafiltration, allowing the peptide to interact with IGF-I receptors in apical tubular membranes. By this route, ultrafiltered IGF-I may increase tubular epithelial cell sodium absorption in overt diabetic nephropathy. In serum samples from diabetic rats, IGF-I levels (227 +/- 34 ng/mL) were reduced as compared with control levels (319 +/- 33 ng/mL, P = .05), and IGF-binding protein-2 (IGFBP-2) is increased about 2-fold. In diabetic rats, IGF-I undergoes glomerular ultrafiltration and is present in proximal tubular fluid that was collected by nephron micropuncture at 2.54 +/- 0.54 nmol/L but is below the detection limit in tubular fluid from normal rats. IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4 are all present in diabetic rat glomerular ultrafiltrate, but IGFBP-2 levels are greater than those of each of the other three IGFBPs. Neither recombinant human IGF-I (1 nmol/L) nor diabetic rat glomerular ultrafiltrate affect sodium transport in cultured mouse proximal tubular cells. In contrast, rhIGF-I and diabetic rat glomerular ultrafiltrate increase the apical-to-basolateral transport of 22Na+ in distal tubule-like A6 cells through mechanisms involving apical IGF-I receptors. In normal rats, luminal infusion with rhIGF-I or with diabetic rat glomerular ultrafiltrate into late proximal tubules increases distal tubular Na+ absorption. These findings indicate that diabetic glomerular sclerosis causes glomerular ultrafiltration of IGF-I, and they suggest that tubular fluid IGF-I may contribute to sodium (and fluid) retention that is commonly observed in patients with severe diabetic nephropathy.     

Acidosis during early reperfusion prevents myocardial stunning in perfused ferret hearts.

Cellular calcium overload figures prominently in the pathogenesis of the contractile dysfunction observed after brief periods of ischemia (myocardial stunning). Because acidosis is known to antagonize Ca influx and the intracellular binding of Ca, we reasoned that acidosis during reperfusion might prevent Ca overload and ameliorate functional recovery. We measured developed pressure (DP) and 31P-nuclear magnetic resonance spectra in 26 isovolumic Langendorff-perfused ferret hearts. After 15 min of global ischemia, hearts were reperfused either with normal solution (2 mM [Ca]o, Hepes-buffered, pH 7.4 bubbled with 100% O2; n = 6) or with acidic solutions (pH 6.6 during 0-3 min, pH 7.0 during 4-6 min) before returning to the normal perfusate (n = 7). Ventricular function after 30 min of reperfusion was much greater in the acidic group (105 +/- 5 mmHg at 2 mM [Ca]o) than in the unmodified reperfusion group (79 +/- 7 mmHg, P less than 0.001); similar differences in DP were found over a broad range of [Ca]o (0.5-5 mM, P less than 0.001) and during maximal Ca2+ activation (P less than 0.001). Intramyocardial pH (pHi) was lower in the acidic group than in the unmodified group during early reperfusion, but not at steady state. Phosphate compounds were comparable in both groups. To clarify whether the protective effect of acidosis is due to intracellular or extracellular pH, we produced selective intracellular acidosis during early reperfusion by exposure to 10 mM NH4Cl for 6 min just before ischemia (n = 6). For the first 12 min of reperfusion with NH4Cl-free solution (pH = 7.4), pHi was decreased relative to the unmodified group. Recovery of DP was practically complete, and maximal Ca2+-activated pressure was comparable to that in a nonischemic control group (n = 5). These results indicate that transient intracellular acidosis can prevent myocardial stunning, presumably owing to a reduction of Ca influx into cells and/or competition of H+ for intracellular Ca2+ binding sites during early reperfusion.     

Chronic hypercapnia stimulates proximal bicarbonate reabsorption in the rat.

The hyperbicarbonatemia of chronic respiratory acidosis might be maintained by a reduction in filtration rate or an enhancement of tubular bicarbonate reabsorption. To investigate this question, 12 Munich-Wistar rats were exposed to a 10% CO2 atmosphere for 6-8 d. Chronic respiratory acidosis developed, with arterial pH 7.30 +/- 0.01, partial pressure of CO2 (pCO2) 80 +/- 2 mmHg, and total CO2 concentration 45 +/- 1 mM. Single nephron glomerular filtration rate was normal (42 +/- 1 nl/min). Chronic hypercapnia caused absolute proximal reabsorption to be significantly stimulated (1,449 +/- 26 pmol/min) as compared with reabsorption previously observed in normal animals (1,075 +/- 74 pmol/min) or in animals subjected to acute hypercapnia (1,200 +/- 59 pmol/min). This is the first demonstration that proximal bicarbonate reabsorption can be stimulated above normal euvolemic values. When eight animals were subsequently allowed to return toward a normocapnic state (arterial pCO2 46 +/- 1 mmHg) over the course of 1-1.5 h, bicarbonate reabsorption was still significantly higher (1,211 +/- 34 pmol/min) than in similarly alkalotic, normocapnic control groups (994 +/- 45 pmol/min). In conclusion, chronic, but not acute, hypercapnia stimulates absolute proximal bicarbonate reabsorption to exceed the level found in normal euvolemic rats.     

Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading.

Cellular cystine loading with cystine dimethyl ester inhibits volume absorption, transepithelial potential difference, glucose transport, and bicarbonate transport in proximal convoluted tubules perfused in vitro. This study examined the roles of ATP and NaK ATPase in this in vitro model of the Fanconi syndrome of cystinosis. Intracellular ATP was measured using the luciferin-luciferase assay. Intracellular ATP was reduced by 60% in proximal convoluted tubules incubated with 0.5 mM cystine dimethyl ester for 15 min at 37 degrees C (P less than 0.001). Incubation of cystine loaded tubules with 1 mM exogenous ATP increased intracellular ATP to levels not significantly different than that of controls. On the other hand, Vmax NaK ATPase activity was unchanged even though the incubation times and the concentration of cystine dimethyl ester were doubled to 30 min and 1 mM, respectively. In proximal convoluted tubules perfused in vitro, 0.5 mM cystine dimethyl ester resulted in an 89% inhibition in volume absorption (0.81 +/- 0.14 to 0.09 +/- 0.09 nl/mm.min), while there was only a 45% inhibition in volume absorption (P less than 0.01) due to cellular cystine loading in the presence of 1 mM lumen and bath ATP (0.94 +/- 0.05 to 0.52 +/- 0.11 nl/mm.min). These data demonstrate that proximal tubule cellular cystine loading decreases cellular ATP concentration, but does not directly inhibit NaK ATPase activity. The inhibition in transport and decrease in intracellular ATP due to cellular cystine loading was ameliorated by exogenous ATP. These data are consistent with cellular ATP depletion playing a major role in the inhibition of proximal tubule transport due to intracellular cystine loading.     

Renal metabolic rate during changes in bicarbonate-dependent sodium reabsorption in the proximal tubules

Previous studies indicate that water and at least 2 mol NaCl are reabsorbed in the proximal tubules for each mol NaHCO3 reabsorbed. To examine the effect on cortical energy metabolism of variations in this bicarbonate-dependent sodium reabsorption, the cortical metabolic rate was examined in anaesthetized dogs by the heat production technique during continuous infusion of saline and ethacrynic acid. Sodium reabsorption was altered either by intravenous infusion of a large dose of acetazolamide (500 mg/kg body wt) or by changing plasma Pco2 during metabolic alkalosis. Acetazolamide reduced bicarbonate reabsorption by 71 +/- 2%, sodium reabsorption by 54 +/- 2% and cortical heat production by 21 +/- 3%. A rise in Pco2 to 16.4 +/- 1.3 kPa during metabolic alkalosis increased sodium reabsorption by 25 +/- 3% and cortical heat production by 14 +/- 2%. A similar elevation of plasma Pco2 during metabolic acidosis had no effect on electrolyte reabsorption or the cortical metabolic rate. A reduction in Pco2 to 2.3 +/- 0.3 kPa reduced sodium reabsorption by 40 +/- 3% and cortical heat production by 19 +/- 2%. We conclude that a rise in proximal tubular reabsorption requires energy. However, the changes in energy requirement are small, accounting for previous failures to observe significant changes in cortical energy metabolism during less extensive changes of sodium reabsorption in the proximal tubules.     

cAMP-adenosine pathway in the proximal tubule

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methyleneadenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A1, A2A, and A3 receptors on both apical and basolateral proximal tubular membranes, with A1 and A2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.     

Passive permeability of salicylic acid in renal proximal S2 and S3 tubules

The role of nonionic diffusion in the transport of salicylic acid across rabbit proximal S2 and S3 segments was investigated using the in vitro isolated perfused tubule technique. The [14C] salicylic acid apparent reabsorptive permeability (P'I-b, 10(-5) cm/s) was measured at 19 degrees C with luminal solutions kept at different pH and bath maintained at pH 7.4. In S2 tubules, P'I-b was 25.0 +/- 3.5 when luminal pH was 6.0; P'I-b decreased to 8.1 +/- 1.4 and to 4.4 +/- 1.2 at a luminal pH of 6.5 and 7.0, respectively. In S3 tubules, P'I-b was 17.6 +/- 2.4, 5.3 +/- 1.1 and 3.4 +/- 1.1 at a luminal pH of 6.0, 6.5 and 7.0, respectively. There was a close correlation between P'I-b and the calculated proportion of nonionized salicylic acid present at each pH, indicating that only the nonionized molecule could diffuse in our conditions. We calculated the apparent permeability of nonionic salicylic acid and found 0.248 +/- 0.032 cm/s for S2 and 0.176 +/- 0.022 cm/s for S3 tubules. These calculated permeabilities were independent of pH.     

Anion transport processes in the mammalian superficial proximal straight tubule.

The experiments reported in this paper were designed to evaluate some of the characteristics of anion transport processes during fluid absorption from superficial proximal straight tubules isolated from rabbit kidney. We measured net chemical C1- flux during fluid absorption from tubules perfused and bathed with Krebs-Ringer buffers containing 113.6 mM C1-, 10 mM acetate, and 25 mM HCO-/3 at pH 7.4; assessed the effects of carbonic anhydrase inhibitors on net fluid absorption in the presence and absence of CO2; and evaluated the influx and efflux coefficients for [14C]-acetate transport at 37degreesC, at 21degreesC, and in the presence of carbonic anhydrase inhibitors. The experimental data shown that, for this nephron segment, net C1- flux accompanies approximately 27.5% of net Na+ absorption; and net C1- absorption may be accounted for by a passive transport process, primarily diffusional in nature. Fluid absorption in this nephron segment is reduced 40-60% by carbonic anhydrase inhibitors, but only when the tubules are exposed to 95% O2-5% CO2 rather than 100% O2. Thus, it seems probably that approximately half of Na+ absorption in these tubules may be rationalized in terms of a carbonic anhydrase-dependent CO2 hydration process. In addition, there may occur in these isolated proximal tubules an acetazolamide-insensitive moiety of HCO-/3 absorption comparable to that observed for proximal tubules in vivo. Finally, we provide evidence that net efflux of luminal acetate is due to metabolic energy-dependent processes other than CO2 hydration and may, under appropriate conditions, account for approximately one-fourth of net Na+ absorption.     

Inhibitory effect of acetazolamide on renal tubular reabsorption of NaHCO3 and NaCl in dogs varies inversely with plasma pH

To examine the effect of carbonic anhydrase inhibition on proximal tubular electrolyte reabsorption, plasma pH was altered before and after acetazolamide administration in six volume-expanded dogs during continuous infusion of ethacrynic acid to inhibit transcellular NaCl reabsorption. Plasma pH was altered by changing PCO2, keeping plasma bicarbonate concentration and glomerular filtration rate constant. Linear inverse relationships were obtained between electrolyte reabsorption and plasma pH. Before acetazolamide administration, a change in plasma pH of 0.1 unit from pH 7.4 altered bicarbonate reabsorption by about 10% and sodium and chloride reabsorption remaining during ethacrynic acid infusion by about 6.5%. Administration of acetazolamide (30 mg/kg b.wt.) caused a reduction in electrolyte reabsorption at all plasma pH levels examined. A further reduction occurred after increasing the dose to 100 mg/kg b.wt. The absolute inhibitory effects were almost twice as large during hypercapnia as during hypocapnia whereas the reduction in fractional reabsorption was the same at all plasma pH levels. Both variations in plasma pH and administration of acetazolamide altered the reabsorption of bicarbonate, chloride and sodium in molar ratios of about 1:2:3. Hence, acetazolamide inhibits a constant fraction of the NaHCO3 reabsorption and the associated NaCl reabsorption in the proximal tubules independent of changes in plasma pH.