Rate Of Bicarbonate Reabsorption Research Articles

Physical and functional interaction between NHE3 and SGLT2 in the renal proximal tubule of male and female rats

Sodium-glucose cotransporter type 2 (SGLT2) and sodium/hydrogen exchanger-3 (NHE3) serve as the primary renal proximal tubule transporters responsible for the apical uptake of filtered glucose and NaHCO3/NaCl, respectively. Our previous studies demonstrated that chronic treatment with the selective SGLT2 inhibitor empagliflozin reduces NHE3 activity in the proximal tubule. Furthermore, we observed that the non-specific SGLT1/SGLT2 inhibitor, phlorizin, acutely inhibits NHE3 in the absence of luminal glucose, suggesting that the impact of SGLT inhibition on NHE3 may not be attributed to high tubular glucose concentrations. Additionally, our investigations revealed that SGLT2, but not SGLT1, colocalizes with NHE3 in the renal proximal tubule. This study aimed to gain insights into the functional and physical association of NHE3 and SGLT2 in the renal proximal tubule. In vivo microperfusion was conducted in male and female Wistar rats using glucose-free perfusates containing vehicle or empagliflozin (1 μM). Empagliflozin treatment significantly decreased the flow rate of bicarbonate reabsorption (JHCO3−) in both male (2.109±0.091 vs. 1.119±0.096 nmol/cm2∙s) and female rats (1.750±0.037 vs. 1.128±0.062 nmol/cm2∙s), indicating that SGLT2 inhibitors inhibit NHE3 activity through glucose-independent mechanisms. By employing nondenaturant detergents, we utilized blue native polyacrylamide gel electrophoresis (BN-PAGE) to preserve native protein-protein interactions. Double staining with antibodies against specific partner candidates revealed that bands attributed to NHE3, MAP17, PDZK1, and SGLT2 migrate similarly at ~480 kDa in samples from rat renal cortex, as supported by independent experiments. Rigorous controls were implemented to address concerns related to autofluorescence, antibody cross-reactivity, and signal specificity, confirming the validity of our results. Furthermore, complexes involving SGLT2 and NHE3 at an apparent ~480 kDa molecular weight were observed in male and female rats. Unlike SGLT2, SGLT1 bands in BN-PAGE did not overlap with the NHE3 signal, which is consistent with our earlier immunofluorescence experiments. Collectively, our data suggest that NHE3 and SGLT2 are functionally and physically associated with the renal proximal tubule. Future experiments will be undertaken to further characterize the physical association between NHE3 and SGLT2 and to determine whether the inhibition of NHE3 by SGLT2 inhibitors is mediated through the disruption of the NHE3-PDZK1-MAP17-SGLT2 multiprotein complex. Funding: São Paulo Research Foundation (FAPESP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in the activity and expression of protein phosphatase‐1 accompany the differential regulation of NHE3 before and after the onset of hypertension in spontaneously hypertensive rats

The Na(+) /H(+) exchanger NHE3 activity decreases in the proximal tubule of spontaneously hypertensive rats (SHRs) as blood pressure increases, and this reduction is correlated with higher NHE3 phosphorylation levels at the PKA consensus site serine 552. This study tested the hypothesis that this lowered NHE3 activity is associated with an increase in PKA activity and expression, and/or a decrease in protein phosphatase-1 (PP1) activity and expression. Proximal tubule NHE3 activity was measured as the rate of bicarbonate reabsorption by stationary microperfusion. NHE3 phosphorylation and protein expression were determined by immunoblotting. PKA and PP1 activities were determined using specific substrates under optimal enzymatic conditions. The PKA activator, 6-MB-cAMP, increased the phosphorylation levels of NHE3 at serine 552 in the renal cortex; this increase happens to a much greater extent in young pre-hypertensive SHRs (Y-SHRs) compared to adult SHRs with established hypertension (A-SHRs). Likewise, the inhibitory effect of 6-MB-cAMP on NHE3 transport activity was much more pronounced in the proximal tubules of Y-SHRs than in those of A-SHRs. Renal cortical activity of PKA was not significantly different between Y-SHRs and A-SHRs. On the other hand, Y-SHRs exhibited higher protein phosphatase 1 (PP1) activity, and their expression of the PP1 catalytic subunit PP1α in the renal cortex was also higher than in A-SHRs. Collectively, these results support the idea that the lower NHE3 transport activity and higher phosphorylation occurring after the development of hypertension in SHRs are due, at least in part, to reduced PP1-mediated dephosphorylation of NHE3 at serine 552.

Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR

Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. The Na(+)/H(+) exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 ± 0.10 vs. 0.41 ± 0.04 nmol/cm(2)×s), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 ± 0.05 vs. 1.26 ± 0.11 nmol/cm(2)×s). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. The molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.

Na+/H+ Exchanger NHE3 is differentially regulated in renal proximal tubules of pre‐hypertensive and hypertensive spontaneously hypertensive rats

The present work aimed to measure NHE3 in vivo transport activity and to define the mechanisms underlying NHE3 regulation in young pre‐hypertensive (5‐week‐old, systolic blood pressure (SBP) = 92 ± 2 mmHg) and adult hypertensive spontaneously hypertensive rats (SHR) (14‐week‐old, SBP = 187 ± 4 mmHg). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo microperfusion. NHE3‐mediated bicarbonate reabsorption was stimulated in 5‐week‐old SHR (44 ± 5%, P<0.01) and reduced in 14‐week‐old SHR (57 ± 4%, P<0.001) compared to Wistar Kyoto (WKY) rats at corresponding age. Stimulated NHE3 transport activity in young pre‐hypertensive SHR was associated with enhanced NHE3 microvillus expression, lower levels of phosphorylation at Ser552 and increased association with the enzyme dipeptidyl peptidase IV (DPPIV). Conversely, adult hypertensive SHR showed decreased NHE3 microvillus membrane content, higher relative ratio of phosphorylated Ser552, and lower amount of the NHE3‐DPPIV complex. No differences on renal cortical NHE3 protein content and mRNA levels were found between SHR and its WKY counterpart. We conclude that NHE3 is differentially regulated before and after development of hypertension in SHR by mechanisms that involve post‐translational modifications, subcellular redistribution and assembly with interacting proteins. Supported by FAPESP and CNPq

PH and bicarbonate in the ductuli efferentes testis of the rat.

The pH and bicarbonate concentrations of luminal fluids in the efferent ducts of the rat were estimated from pH measurements of samples in vitro under conditions of controlled temperature and carbon dioxide tension. The pH of scrotal blood was estimated to be more acidic than systemic blood (mean pH=7.44) at either of the putative scrotal carbon dioxide tensions (5% and 7%, pH, respectively,=7.42 and 7.28). For PCO2 tensions of 5% and 7%, respectively, the data indicated that the pH in the efferent ducts was significantly higher (distal initial zone pH=7.55 or 7.41; coni vasculosi pH=7.66 or 7.51; p < 0.01) than in fluid entering (rete testis fluid, pH=7.34 or 7.20) or leaving the ducts (zone 1a of the epididymal duct 7.26 or 7.11). Bicarbonate concentrations were also significantly higher (p < 0.01) in the efferent ducts (35.4 +/- 4.7 mM, distal initial zone; 45.2 +/- 7.6 mM, coni vasculosi) than in fluids entering (22.9 +/- 3.6 mM) or leaving (20.4 +/- 4.9 mM) the ducts. Estimates of the reabsorption of bicarbonate and fluid indicated that 96% of the testicular output of bicarbonate was reabsorbed in the efferent ducts, but there was also some secretion of bicarbonate into the ducts. It is concluded that luminal pH and bicarbonate levels in the efferent ducts of the rat are high relative to those found in the epididymis where low pH and bicarbonate contributes to sperm quiescence during storage. Nevertheless, the high rate of bicarbonate reabsorption in the efferent ducts is a major contributor to the establishment of the low pH and bicarbonate milieu of the epididymis.

Role of Cl- in electrogenic H+ secretion by cortical distal tubule.

The presence of an electrogenic H+-ATPase has been described in the late distal tubule, a segment which contains intercalated cells. The present paper studies the electrogenicity of this transport mechanism, which has been demonstrated in turtle bladder and in cortical collecting duct. Transepithelial PD (Vt) was measured by means of Ling-Gerard microelectrodes in late distal tubule of rat renal cortex during in vivo microperfusion. The tubules were perfused with electrolyte solutions to which 2 x 10(-7) M bafilomycin or 4.6 x 10(-8) M concanamycin were added. No significant increase in lumen-negative Vt upon perfusion with these inhibitors as compared to control, was observed as well as when 10(-3) m amiloride, 10(-5) M benzamil or 3 mM Ba2+ were perfused alone or in combination. The effect of an inhibition of electrogenic H+ secretion, i.e., increase in lumen-negative Vt by 2-4 mV, was observed only when Cl- channels were blocked by 10(-5) M 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). This blocker also reduced the rate of bicarbonate reabsorption in this segment from 1.21 +/- 0.14 (n = 8) to 0.62 +/- 0.03 (8) nmol.cm-2.sec-1 as determined by stationary microperfusion and pH measurement by ion-exchange resin microelectrodes. These results indicate that: (i) the participation of the vacuolar H+ ATPase in the establishment of cortical late distal tubule Vt is minor in physiological conditions, but can be demonstrated after blocking Cl- channels, thus suggesting a shunting effect of this anion; and, (ii) the rate of H+ secretion in this segment is reduced by a Cl- channel blocker, supporting coupling of H+-ATPase with Cl- transport.

Role of protein kinase C in proximal bicarbonate absorption and angiotensin signaling

Using in vivo microperfusion in the proximal convoluted tubule (PCT) of the Munich-Wistar rat, we investigated the impact of varying protein kinase C (PKC) activity on the rate of bicarbonate reabsorption and on its regulation by angiotensin II. Activation of PKC with luminal perfusion of phorbol 12-myristate 13-acetate (PMA, 5 x 10(-7) M) caused bicarbonate absorption in the S1 PCT to increase by 25%, from 346 +/- 7 to 432 +/- 4 peq.mm-1.min-1 (P less than 0.001), without affecting intracellular cAMP level. Another PKC stimulator, dioctanoylglycerol, had the same effect. Inhibition of PKC activity with luminal perfusion of 5 x 10(-6) M sphingosine had the opposite effect, decreasing bicarbonate absorption by 45% to 190 +/- 2 peq.mm-1.min-1 (P less than 0.001). Pretreatment with PMA or with sphingosine each attenuated by approximately one-third the bicarbonate absorptive response usually observed following angiotensin II administration. Similar results for the action of PKC, but of smaller magnitude, were found in the S2 PCT. In conclusion, activation of PKC increases bicarbonate and water absorption in the S1 and S2 PCT in vivo, and PKC may participate in as much as one-third of the transport stimulation induced by angiotensin II.

Renal bicarbonate reabsorption in the rat. III. Distal tubule perfusion study of load dependence and bicarbonate permeability.

Using continuous microperfusion techniques, we studied the load dependence of bicarbonate reabsorption along cortical distal tubules of the rat kidney and their bicarbonate permeability. Net bicarbonate transport was evaluated from changes in tracer inulin concentrations and total CO2 measurements by microcalorimetry. Bicarbonate permeability was estimated from the flux of total CO2 along known electrochemical gradients into bicarbonate-and chloride-free perfusion solution containing 10(-4) M acetazolamide. Transepithelial potential differences were measured with conventional glass microelectrodes. Significant net bicarbonate reabsorption occurred at luminal bicarbonate levels from 5 to 25 mM, and at perfusion rates from 5 to 30 nl/min. Bicarbonate reabsorption increased in a load-dependent manner, both during increments in luminal bicarbonate concentration or perfusion rate, reaching saturation at a load of 250 pmol/min with a maximal reabsorption rate of approximately 75 pmol/min.mm. Rate of bicarbonate reabsorption was flow dependent at luminal concentrations of 10 but not at 25 mM. During chronic metabolic alkalosis, maximal rates of reabsorption were significantly reduced to 33 pmol/min.mm. The bicarbonate permeability was 2.32 +/- 0.13 x 10(-5) cm/s in control rats, and 2.65 +/- 0.26 x 10(-5) cm/s in volume-expanded rats. Our data indicate that at physiological bicarbonate concentrations in the distal tubule passive bicarbonate fluxes account for only 16-21% of net fluxes. At high luminal bicarbonate concentrations, passive bicarbonate reabsorption contributes moderately to net reabsorption of this anion.

What is the underlying defect in patients with isolated, proximal renal tubular acidosis?

Our aim in this article is to propose a new hypothesis concerning the etiology of renal tubular acidosis (RTA) in that subgroup of patients who have the isolated, primary type of proximal RTA. We suggest that their underlying disorder is a more alkaline intracellular pH of the proximal convoluted tubule. Increased alkalinity of proximal tubular cells would explain the low rate of bicarbonate reabsorption per liter glomerular filtration and the decreased rate of ammonium excretion despite a low urine pH and the presence of chronic metabolic acidosis. Additional diagnostic tests to evaluate this hypothesis in this specific subgroup of patients with proximal RTA are also outlined.

Bicarbonate transport by initial collecting tubule of aquatic- and land-phase amphibia.

The rate of bicarbonate reabsorption (JHCO3) by in vitro initial collecting tubules (ICT) from aquatic- and land-phase Ambystoma tigrinum was measured. These animals intrinsically have different levels of plasma bicarbonate, 17.6 and 27.1 mM, respectively. ICT from both phases reabsorbed bicarbonate at 11 pmol X mm-1 X min-1 when perfused in solutions equilibrated with 3% CO2. The induction of metabolic acidosis or alkalosis had no effect on JHCO3 by in vitro ICT. JHCO3 was abolished when ICT from aquatic animals were perfused with solutions equilibrated with 1% CO2 and returned toward the control value when 3% CO2 was reintroduced. The effect of plasma CO2 partial pressure on JHCO3 of the ICT may explain, at least in part, the observed difference in plasma bicarbonate of the aquatic- and land-phase animals. JHCO3 was reduced to 22% of control with amiloride and to 42% of control when a Na+-free perfusate was used, but was insensitive to ethoxzolamide. These data are consistent with the presence of a sodium-proton exchanger on the apical membrane and a carbonic anhydrase-independent mechanism of H+ secretion.

CAMP-associated inhibition of Na+-H+ exchanger in rabbit kidney brush-border membranes.

Adenosine 3',5'-cyclic monophosphate (cAMP) inhibits the rate of bicarbonate reabsorption and the rate of Na+-H+ exchange transport in the apical membrane of the proximal convoluted tubule. To study the relation between cAMP, cAMP-dependent protein kinase, and Na+-H+ exchange transport, brush-border membrane vesicles from the rabbit kidney were phosphorylated in vitro. The rate of proton gradient-stimulated amiloride-inhibitable 22Na+ uptake was measured as an index of Na+-H+ exchange transport activity. The inclusion of cAMP (10(-6) M) in a phosphorylating solution containing ATP decreased the 10-s uptake of amiloride-sensitive sodium from 2.25 +/- 0.21 nmol/mg protein in controls to 1.94 +/- 0.19 (P less than 0.001). Incubation of vesicles in the presence of purified catalytic subunit of cAMP-dependent protein kinase inhibited the amiloride-sensitive uptake of 22Na+ at 10 s from 2.35 +/- 0.49 nmol/mg protein to 2.05 +/- 0.44 (P less than 0.005). The inhibitory effect of both cAMP and catalytic subunit of cAMP-dependent protein kinase was blocked by the specific thermostable protein inhibitor of the kinase. These studies demonstrate that activation of endogenous membrane-bound cAMP-dependent protein kinase or exposure to exogenous catalytic subunit of cAMP-dependent protein kinase inhibits the rate of Na+-H+ exchange transport in the brush-border membrane of the rabbit kidney.

Atrial natriuretic factor ameliorates chronic metabolic alkalosis by increasing glomerular filtration.

The kidney maintains the elevated plasma concentration of bicarbonate that occurs in chronic metabolic alkalosis. A reduction in the glomerular filtration rate (GFR) can maintain the filtered bicarbonate load at a normal level so that a normal rate of bicarbonate reabsorption suffices to prevent urinary excretion of this anion. It is also possible that bicarbonate reabsorption might increase so as to maintain the alkalosis if GFR were not reduced. To examine this latter possibility, atrial natriuretic factor was used in alkalotic rats to restore a more normal GFR and to increase the amount of bicarbonate filtered by the glomerulus. Proximal bicarbonate reabsorption remained relatively static. Higher than normal amounts of bicarbonate were then delivered out of the proximal tubule, bicarbonate appeared in the urine, and the plasma concentration of bicarbonate fell. A reduction in GFR is thus necessary for the maintenance of chronic metabolic alkalosis. Normalizing GFR induces bicarbonaturia and initiates repair of the alkalosis.

Application of the disequilibrium pH method to investigate the mechanism of urinary acidification.

DuBose, Thomas D., Jr. Application of the disequilibrium pH method to investigate the mechanism of urinary acidification. Am. J. Physiol. 245 (Renal Fluid Electrolyte Physiol. 14): F535-F544, 1983.--The cellular mechanism of renal bicarbonate reabsorption has been debated for four decades. Recent technological advances have allowed distinction between primary bicarbonate reabsorption and a proton secretory mechanism. The disequilibrium pH method has been applied widely for this purpose and has supported the latter hypothesis uniformly. The demonstration of elevated values for PCO2 in tubular and vascular structures of the renal cortex has not altered this view. Indeed, by employing a newly developed method for measurement of equilibrium pH in vivo that permits contact with the environment within the tubule lumen to continue, we demonstrated an acid disequilibrium pH in the proximal tubule after carbonic anhydrase inhibition equal to -0.68 pH units. A spontaneous disequilibrium pH was not present in the distal tubule during control conditions or during metabolic alkalosis but was demonstrated during combined respiratory acidosis-metabolic alkalosis. This finding agrees qualitatively with observed rates of bicarbonate reabsorption in the perfused distal tubule in vivo. With use of similar techniques, an acid disequilibrium pH in conjunction with elevated values for PCO2 was observed in the papillary collecting duct. Thus, proton secretion appears to be the predominant mechanism of bicarbonate reabsorption in superficial nephrons and explains, as well, the means by which the urine-to-blood PCO2 gradient in alkaline urine is established.

Evaluation of bicarbonate transport in rat distal tubule: effects of acid-base status.

Previous micropuncture studies utilizing indirect methods to estimate bicarbonate transport in the rat superficial distal tubule have indicated that the distal bicarbonate reabsorptive process normally operates well below the saturation level. Recent studies from our laboratory failed to demonstrate a spontaneous acid disequilibrium pH in this segment, implying that the bicarbonate reabsorptive rate was less than previously estimated. The purpose of the present experiments were 1) to measure the rate of absolute bicarbonate reabsorption by the rat superficial distal tubule while controlling bicarbonate delivery, and 2) to examine the effects of alterations in acid-base status on the rate of bicarbonate reabsorption. Five groups of rats in different states of acid-base balance were studied. No significant bicarbonate reabsorption was detected in the control hydropenic, combined respiratory acidosis-metabolic alkalosis, acute respiratory acidosis, or acute metabolic acidosis groups. In contrast, metabolic acidosis of 3 days duration resulted in a significant bicarbonate reabsorptive rate of 52.6 +/- 13.9 pmol . mm-1 . min-1. The observation of significant bicarbonate reabsorption in the distal tubule only during chronic metabolic acidosis of 3 days duration is compatible with adaptation of this normally low-capacity segment to chronic changes in systemic acid-base states.

Bicarbonate reabsorption and potassium handling in isolated perfused kidneys of Bufo marinus

Average rates of bicarbonate reabsorption, normalized for glomerular filtration rates, rose linearly with perfusate [HCO3-] of 6-21 mM with a constant bicarbonate/creatinine clearance ratio of 0.06, similar to the in vivo value. Maximal rates of bicarbonate reabsorption occurred at 20-25 mM HCO3- perfusate. At 3 mM perfusate [K+], normalized rates of potassium reabsorption were constant from filtered perfusates mimicking metabolic acidosis, and from control perfusates; during perfusion with alkalotic Ringer solutions net potassium secretion was observed. Elevation of perfusate [K+] to 10 mM in alkalotic Ringer significantly decreased maximal rates of bicarbonate reabsorption. Overall, the pattern of bicarbonate reabsorption in toad kidneys perfused at 6-21 mM HCO3- was similar to that observed in acid-base disturbances of metabolic origin in mammals, while the pattern of potassium excretion showed significant differences in these two vertebrate classes.

Effects of lysine on bicarbonate and fluid absorption in the rat proximal tubule.

The effects of lysine on bicarbonate and fluid reabsorption in the rat proximal tubule were studied by luminal and capillary perfusion in situ. The proximal tubule and peritubular capillaries were perfused with bicarbonate Ringer solution containing [14C]inulin. The rate of bicarbonate reabsorption (JHCO3) was estimated to be 124 +/- 9.5 peq.min-1.mm-1 using a pH membrane glass electrode. The rate of net fluid reabsorption (Jv) was 2.6 +/- 0.21 nl.min-1.mm-1. When 10 mM L-lysine was added to the luminal perfusate, a 35% reduction in JHCO3 and no change in Jv were observed. Increase of L-lysine concentration in the luminal perfusate to 20 mM did not reduce JHCO3 further nor did it influence Jv.l When 10 mM L-lysine was added to the capillary perfusate, a 13% reduction in JHCO3 was observed (NS). Increase of lysine concentration in the capillary perfusate to 20 mM significantly reduced JHCO3 by 26% (P less than 0.01). There was no significant change in Jv under both conditions. The effect of L-lysine in the lumen was related to its reabsorption kinetics, D-Lysine, which was not reabsorbed significantly, did not affect bicarbonate reabsorption in the proximal tubule. These results indicate that the inhibitory effect of L-lysine is related to the entry of lysine into the cell from the lumen.

Bicarbonate reabsorption in the papillary collecting duct of rats.

Using the technique of capillary perfusion and simultaneous luminal stop flow microperfusion the reabsorption of bicarbonate and glycodiazine from the papillary collecting duct was evaluated. Starting with equal H14CO3- and 3H-glycodiazine concentrations in the luminal and peritubular perfusates, the decrease in the luminal concentration at 10 and 45 s contact time was measured. In control rats with 25 mmol/l HCO3- in the perfusates the rate of HCO3- reabsorption calculated from the 10 s values was 0.34 nmol cm-2 s-1. In acute metabolic acidosis, the rate of bicarbonate reabsorption was 2,3 times higher. In metabolic alkalosis, the rate of bicarbonate absorption dropped to 13% of the control values. Also the 45 s values of acidotic and alkalotic animals differed significantly from each other. With 25 mmol/l glycodiazine in both perfusates the rate of buffer reabsorption as calculated from the 10 s values was 0.76 nmol cm-2 s-1 in control rats and did not deviate significantly from this value in acidotic and alkalotic animals. In control rats the bicarbonate reabsorption in % was the same, no matter whether both luminal and capillary perfusate contained 25 mmol/l bicarbonate or 10 mmol/l. In acidotic rats the rate of HCO3- reabsorption did not change significantly if all Na+ in the perfusates was replaced by choline (0.88 versus 0.79 nmol cm-2 s-1 at 25 mmol/l HCO3-). When in acidotic rats. 0.1 mmol/l acetazolamide or 1 mmol/l SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) was added to both perfusates the rate of HCO3- reabsorption dropped by 75 and 58%, respectively. A potassium deficient diet for one week and DOCa administration had no influence on the bicarbonate reabsorption of rats which were on standard diet. The data indicate that (1) the buffer reabsorption from the papillary collecting duct is rather due to H+ ion secretion than to buffer anion reabsorption. (2) The adaptation to metabolic acidosis and alkalosis is specific for bicarbonate and not seen with glycodiazine. (3) Within the concentration range tested the HCO3- reabsorption rises linearly with the HCO3- concentration. (4) The HCO3- reabsorption in the papillary collecting duct is Na+-independent, it can be inhibited by acetazolamide and SITS, but is not influenced by K+-deficient diet plus DOCA.

Renal proximal tubular acidification. Role of brush-border and cytoplasmic carbonic anhydrase.

Carbonic anhydrase is found in the cytoplasm and brush border membranes of renal proximal tubular cells. Both the soluble and the membrane-bound enzyme have been assigned roles for the secretion of hydrogen ions into the tubular fluid and hence also for the reabsorption of bicarbonate. Attempts were made to differentiate between the roles of these enzymes for the rate of proximal tubular acidification. Proximal tubules of rats were instilled and perfused with bicarbonate solutions containing carbonic anhydrase inhibitors, especially designed to be impermeable to cell membranes. The acidification rate was measured with an antimony micro-electrode system--the only instantly responding micro-pH electrode. The membrane impermeable inhibitors had no effect on this rate in contrast to acetazolamide, which markedly inhibited the acidification rate when administered intraluminally. It is therefore concluded that the cytoplasmic carbonic anhydrase is the important enzyme for the proximal tubular acidification rate, and hence the rate of bicarbonate reabsorption. The function of the brush border enzyme remains an outstanding problem.

Glomerular-tubular balance for bicarbonate in the dog.

Previous work that apparently showed a functional relationship between GFR and maximum bicarbonate reabsorption was done at a time when the effects on the latter of several factors (PCO2, plasma potassium concentration, and extracellular fluid volume expansion) were not recognized. The present study re-examines this relationship, while controlling these factors. In 14 hydropenic dogs, bicarbonate reabsorption per unt GFR increased linearly with increases in plasma bicarbonate concentration. At any level of plasma bicarbonate concentration,the absolute rate of bicarbonate reabsorption was functionally related to the GFR. In six volume-expanded dogs, bicarbonate reabsorption remained stable at 20-22 mmol/liter GFR as plasma bicarbonate was raised to greater than 40mM. The absolute rate of bicarbonate reabsorption increased with large increases in GFR induced by methylprednisolone and high-protein diet. In a third group of dogs, bicarbonate reabsorption varied directly with increases in GFR, while plasma bicarbonate concentration was held relatively constant above the threshold. We conclude there is a close functional relationship between the absolute rate of bicarbonate reabsorption and GFR in individual dogs.

On the influence of extracellular fluid volume expansion and of uremia on bicarbonate reabsorption in man.

The patterns of bicarbonate reabsorption during increasing plasma concentrations were studied in subjects with a range of glomerular filtration rates (GFR) from 170 to 2 ml/min. In a group of five subjects with GFR values above 30 ml/min, paired bicarbonate titration studies were performed first under conditions which minimized extracellular fluid (ECF) volume expansion, and second under conditions which were conducive to exaggerated expansion of ECF volume. In patients with GFR values below 30 ml/min, a single protocol was employed. Studies also were performed on two patients with far advanced renal disease who were nephrotic and exhibited a sodium-retaining state. When ECF volume expansion was minimized in the nonuremic subjects, values for bicarbonate reabsorption were well in excess of the usually accepted Tm level and over the range of plasma bicarbonate concentrations employed, no evidence of a Tm phenomenon was observed. A similar pattern emerged in the two nephrotic patients despite the presence of uremia. However, with both exaggerated expansion of ECF volume (GFR greater than 30) and in patients with advanced renal disease in the absence of exaggerated ECF volume expansion a tendency towards saturation kinetics for bicarbonate reabsorption was demonstrable. In comparing the minimized with the exaggerated expansion studies, evidence emerged for a decrease in both bicarbonate reabsorption per unit of GFR and the absolute rate of bicarbonate reabsorption. When ECF volume expansion was exaggerated in uremic patients after stable rates of bicarbonate reabsorption had been achieved, a decrease in reabsorption per unit of GFR and in absolute bicarbonate reabsorption occurred. The possible relationship of the factors controlling sodium excretion to the observed patterns of bicarbonate reabsorption is considered in the text.