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Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Insulin resistance is frequently associated with hypertension, but the mechanism underlying this association remains speculative. Although insulin is known to modify renal tubular functions, little is known about roles of insulin receptor substrates (IRS) in the renal insulin actions. For clarifying these issues, the effects of insulin on the rate of bicarbonate absorption (JHCO3-) were compared in isolated renal proximal tubules from wild-type, IRS1-deficient (IRS1-/-), and IRS2-deficient (IRS2-/-) mice. In wild-type mice, physiologic concentrations of insulin significantly increased JHCO3-. This stimulation was completely inhibited by wortmannin and LY-294002, indicating that the phosphatidylinositol 3-kinase pathway mediates the insulin action. The stimulatory effect of insulin on JHCO3- was completely preserved in IRS1-/- mice but was significantly attenuated in IRS2-/- mice. Similarly, insulin-induced Akt phosphorylation was preserved in IRS1-/- mice but was markedly attenuated in IRS2-/- mice. Furthermore, insulin-induced tyrosine phosphorylation of IRS2 was more prominent than that of IRS1. These results indicate that IRS2 plays a major role in the stimulation of renal proximal absorption by insulin. Because defects at the level of IRS1 may underlie at least some forms of insulin resistance, sodium retention, facilitated by hyperinsulinemia through the IRS1-independent pathway, could be an important factor in pathogenesis of hypertension in insulin resistance.

Angiotensin II (AngII) regulates renal proximal transport in a biphasic way. It has been recently shown that the basolateral type 1A receptor (AT(1A)) mediates the biphasic regulation of Na(+)-HCO(3)(-) cotransporter (NBC) by AngII. However, the receptor subtype(s) responsible for the luminal AngII actions remained to be established. To clarify this issue, the luminal AngII effects in isolated proximal tubules from wild-type (WT) and AT(1A)-deficient mice (AT(1A) KO) were compared. In WT, the rate of bicarbonate absorption (JHCO(3)(-)), analyzed with a stop-flow microspectrofluorometric method, was stimulated by 10(-10) mol/L luminal AngII but was inhibited by 10(-6) mol/L luminal AngII. Both stimulatory and inhibitory effects of AngII were completely blocked by valsartan (AT(1) antagonist) but unaffected by PD 123,319 (AT(2) antagonist). In AT(1A) KO, in contrast, luminal AngII (10(-10) - 10(-6) mol/L) did not change JHCO(3)(-). In WT, 10(-6) mol/L luminal AngII increased cell Ca(2+) concentrations ([Ca(2+)](i)), which was again blocked by valsartan but not by PD 123,319. However, luminal AngII did not increase [Ca(2+)](i) in AT(1A) KO. On the other hand, the addition of arachidonic acid similarly inhibited JHCO(3)(-) in WT and AT(1A) KO. Furthermore, the acute activation of protein kinase C by phorbol 12-myristate 13-acetate similarly stimulated JHCO(3)(-) in WT and AT1A KO, indicating that the inhibitory and stimulatory pathways necessary for the AngII actions were preserved in AT(1A) KO. These results indicate that the luminal AT(1A) mediates the biphasic regulation of bicarbonate absorption by luminal AngII, while no evidence was obtained for a role of AT(2).

Mutations in Na(+)-HCO(3)(-) cotransporter (NBC-1) cause proximal renal tubular acidosis (pRTA) associated with ocular abnormalities. One pRTA patient had increased serum amylase, suggesting possible evidence of pancreatitis. To further delineate a link between NBC-1 inactivation and pancreatic dysfunction, immunohistochemical analysis was performed on rat and human pancreas using antibodies against kidney-type (kNBC-1) and pancreatic-type (pNBC-1) transporters. In rat pancreas, the anti-pNBC-1 antibody labeled acinar cells and both apical and basolateral membranes of medium and large duct cells. In human pancreas, on the other hand, the anti-pNBC-1 antibody did not label acinar cells, although it did label the basolateral membranes of the entire duct system. The labeling by anti-kNBC-1 antibody was detected in only a limited number of rat pancreatic duct cells. To examine the effects of pRTA-related mutations, R342S and R554H, on pNBC-1 function, we performed functional analysis and found that both mutants had reduced transport activities compared with the wild-type pNBC-1. These results indicate that pNBC-1 is the predominant variant that mediates basolateral HCO(3)(-) uptake into duct cells in both rat and human pancreas. The loss of pNBC-1 function is predicted to have significant impact on overall ductal HCO(3)(-) secretion, which could potentially lead to pancreatic dysfunction.

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

Using a new stop-flow perfusion technique with microspectrofluorometric determination of luminal fluid pH, we have studied which substrates or incubation conditions allow isolated rabbit proximal tubules to attain in-vivo-like rates of HCO3- absorption (J(HCO3)) and maximal responses of J(HCO3) to norepinephrine (NE). Essentially three incubation media were tested: plasma-like HCO(3-)-Ringer solution containing 5 mmol/l D-glucose (G-Ringer sol.), the same solution also containing 10 mmol/l lactate and 5 mmol/l L-alanine, (LAG-Ringer sol.), and two tissue culture media (DMEM and RPMI 1640). Compared to G-Ringer sol., application of LAG-Ringer sol. in the bath and/or lumen, or application of DMEM or RPMI 1640 in the bath either slightly increased or decreased J(HCO3) with borderline significance. However, RPMI 1640 plus 1 mmol/l pyruvate stimulated J(HCO3) by 55%. While NE (10(-5) mol/l), if applied in G-Ringer sol., had no effect, in the presence of LAG-Ringer sol. it increased J(HCO3) by approximately =40%, and in the presence of DMEM or RPMI 1640 it increased J(HCO3) by approximately =100%. This stimulation by NE followed Michaelis-Menten kinetics with an EC50 value of 0.25 micromol/l and was probably mediated by alpha1-adrenergic receptors. Additional cell pH measurements suggest that NE stimulates the basolateral Na+-HCO3- cotransporter which then becomes susceptible to inhibition by cAMP. We conclude that incubation in tissue culture media allows isolated proximal tubules to maintain a better functional state than the commonly used solutions with unphysiologically high substrate concentrations.

In the present experiments on microdissected tubules of rabbit kidney we present a refined stop-flow method for determining the rate of HCO3- absorption (J(HCO3)) or H+ secretion (JH) that can be applied to isolated microperfused tubules. Using the pH-sensitive indicator dye BCECF (2',7'-bis [2-carboxyethyl]-5[6]-carboxyfluorescein) the luminal perfusate pH is continuously measured with a microspectrofluorometric set-up, and the pH change following a sudden stop of perfusion is analysed. Because the tubules partially collapse after stop-flow the calculation of fluxes requires a correction for volume loss. This is achieved by referring all fluxes to the remaining luminal volume, which can be estimated from the decay of the 440 nm reference fluorescence. During perfusion of the lumen with pure HCO3- Ringer solution, and of the bath with the same solution but containing 5.5 mmol/l D-glucose as metabolic substrate, J(HCO3) averaged 4.4+/-0.2 pmol cm(-1) x s(-1) (n=40) and 13.4+/-0.8 pmol x cm(-1) x s(-1) (n=5) in proximal straight tubules (PST) and in proximal convoluted tubules respectively. These values agree very well with data obtained in other laboratories with the picapnotherm technique. The present method has the advantage of requiring fewer micromanipulations and a shorter measuring time, thus allowing regulatory changes in J(HCO3) to be analysed. Moreover it does not involve measurements of radioactivity, and it also allows J(H) to be measured in HCO3(-) free solutions which in PST averaged 0.9 pmol x cm(-1) x s(-1) (n=8) in the present experiments.

We compare here the results of electrophysiological measurements on proximal tubular cells performed on rat kidney in vivo and on isolated rabbit and rat tubules in vitro. Based on different effects of carbonic anhydrase inhibitors in the in vivo and in vitro preparation, we conclude that NaHCO3 cotransport across the basolateral cell membrane functions as Na(+)-CO3(2-)-HCO3- cotransport in vivo, but as Na(+)-HCO3(-)-HCO3- cotransport in the classical in vitro preparation. The former, but not the latter, transport mode is characterized by generation of local disequilibrium pH/CO3(2-) concentrations that oppose fluxes if membrane-bound carbonic anhydrase is inhibited. In support of this conclusion, we find that overall transport functions with a HCO3- to Na+ stoichiometry of 3:1 in vivo (since each transported CO3(2-) eventually generates 2 HCO3- ions), but 2:1 in vitro. This has been deduced from various measurements, among them super-Nernstian and reverse nernstian, potential responses to changing ion concentrations which are characteristic of obligatorily coupled cation-anion cotransporters, but are not known in classical electrochemistry. The different transport modes in vivo and in vitro suggest that isolated proximal tubules have functional deficits compared to proximal tubules in vivo.

All the relevant literature reports indicate that net rates of salt and water absorption and cell membrane potentials (Vb) are lower, but intracellular Na+ concentration is higher in rabbit renal proximal tubule in vitro than in rat proximal tubule in vivo. Since the different driving forces should influence basolateral Na(+)-HCO3- cotransport we have studied the operation of the cotransporter in isolated rabbit renal proximal tubule in vitro with special emphasis on the stoichiometry of flux coupling (q). Using conventional and ion-selective intracellular microelectrodes three series of experiments were performed: (a) we determined the Vb response to a 2:1 reduction of bath HCO3- or Na+ concentration, (b) we determined initial efflux rates of HCO3- or Na+ ions in response to a sudden 10:1 reduction of bath HCO3- concentration, and (c) we collapsed the tubules and determined electrochemical driving forces of Na+ and HCO3- across the basolateral cell membrane under conditions approaching zero net flux in the control state in the presence of Ba2+- and in Cl(-)-free solutions. All measurements concurrently yielded a coupling ratio of approximately two HCO3- ions to one Na+ ion (q = 2). This result contrasts with the ratio q = 3, which we have previously observed in similar experiments on rat renal proximal tubule in vivo [Yoshitomi et al. (1985) Pflügers Arch 405:360] and which was also observed on rabbit renal basolateral cell membrane vesicles in vitro [Soleimani et al. (1987) J Clin Invest 79:1276].(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of the carbonic anhydrase inhibitor acetazolamide (ACZ) was investigated on HCO3- transport mechanisms in the basolateral cell membrane of rabbit renal proximal tubule. Experiments were performed on isolated S2 segments using double-barrelled microelectrodes to measure cell membrane potential (Vb) and cell pH (pH(i)) during step changes in bath perfusate ion concentrations. Peritubular application of ACZ (1 mmol/l) reduced the initial Vb response to 10:1 reduction of bath HCO3- concentration only slightly, from +53.8 +/- 4.2 mV to +49.1 +/- 0.3 mV (n = 5), but caused an intermittent overshooting repolarization in the secondary Vb response. In conjunction with these effects it left the initial pH(i)) response virtually unchanged but induced a secondary slow acidification. These observation indicate that--under the present experimental conditions--ACZ does not block the Na(+)-HCO3- cotransporter but acts via inhibition of cytosolic carbonic anhydrase. This was confirmed by studying the effect of elevated intracellular HCO3- concentrations under reduced flux conditions and by comparing the concentration dependence of the Vb response with the inhibition kinetics of cytosolic carbonic anhydrase. In contrast, peritubular ACZ inhibited Na(+)-independent Cl-/HCO3- exchange in the basolateral cell membrane of S2 segments directly in a similar way to that described in the preceding publication for S3 segments.

Isolated microperfused S3 segments of rabbit renal proximal tubule were investigated with pH-sensitive double-barrelled intracellular microelectrodes to determine whether the Cl-/base exchanger, which we have previously identified in the basolateral cell membrane of this segment requires HCO3- or can also work in CO2/HCO3- free conditions. Cell pH (pHi) was measured in response to sudden substitution of bath Cl- by gluconate. In control solutions containing 25 mmol/l HCO3 pHi increased initially by 5.0 +/- 0.3 x 10(-3) unit/s but after perfusion with CO2/HCO3(-)-free solutions pHi of the same cells increased only by 1.3 +/- 0.2 x 10(-3) unit/s in response to Cl- substitution. From measurements of the cellular buffering power it was calculated that the control base flux had fallen drastically from 3.7 +/- 0.3 to 0.3 +/- 0.1 x 10(-12) mol/s.cm tubule length. To test whether the remaining flux might have resulted from metabolic CO2, oxidative metabolism was poisoned with cyanide (5 mmol/l). This abolished the pH change (delta pHi) in CO2/HCO3(-)-free solutions, but did not affect the pH shift in the presence of HCO3-. The data indicate that basolateral Cl-/base exchange in S3 segment requires HCO3- to operate. A model in which HCO3- absorption proceeds in form of OH- and CO2 can be largely excluded.