We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.

Abstract-TheT he proximal tubule is the major site of sodium and water reabsorption in the mammalian nephron. Paracrine regulation of sodium reabsorption in the proximal tubule by the renin/angiotensin system occurs via several angiotensin receptor subtypes (AT 1 , AT 2 , and AT 4 ). [1][2][3][4][5] The activation of angiotensin II type 1 (AT 1 ) receptors by angiotensin II increases sodium transport, whereas the activation of AT 2 and AT 4 receptors decreases sodium reabsorption in this nephron segment. [1][2][3][4][5] However, in physiological conditions, the major effect of angiotensin II on sodium transport is stimulatory, via AT 1 receptors. 1,2,6 The dopaminergic system also exerts a paracrine regulatory role on renal sodium transport in the proximal tubule. 7,8 Dopamine receptors, like the angiotensin II receptors, are expressed in brush border and basolateral membranes of RPTs. 8 -11 In contrast to the stimulatory effect of angiotensin II on sodium transport in RPTs, the major consequence of the activation of dopamine receptors is an inhibition of sodium transport. 7,8 Inhibition of renal proximal tubular angiotensin II production or blockade of AT 1 receptors increases the natriuretic effect of the D 1 -like agonist, fenoldopam. 11 D 1 -like and D 2 -like receptor agonists also antagonize the stimulatory effect of angiotensin II, acting via AT 1 receptors, on renal proximal tubular luminal sodium transport. 12,13 The 2 D 1 -like (D 1 and D 5 ) and the 3 D 2 -like (D 2 , D 3 , and D 4 ) receptors are expressed in specific segments of the mammalian kidney. 7,8,14 -19 Whereas the D 4 receptor is expressed mainly in collecting ducts, the D 3 receptor, the major D 2 -like receptor, like the D 1 and D 5 receptors, is expressed in the proximal tubule. [7][8][9][10] The distribution of D 2 receptor protein along the nephron is still uncertain. 8,19 The effect of D 2 receptors on renal sodium transport is also not clear because of the lack of agonists that are highly selective to the D 2 over the D 3 receptor. 16,17 However, 7-OH-DPAT, a ligand with a 50-fold selectivity to the D 3 over the D 2 receptor, 16 increases sodium excretion in rats. 17 Moreover, D 3 receptor-null mice have a decreased ability to excrete an acute sodium load, whereas no such limitation is found in D 2 receptor-null mice. 18,19 We surmise that the D 3 receptor may be the D 2 -like subtype receptor that interacts with the AT 1 receptor in rat RPTs.Angiotensin and dopamine receptors are expressed in immortalized rat RPT cells. 20,21 These RPT cells have charOriginal

The pancreatic variant of the sodium bicarbonate cotransporter, pNBC1, mediates basolateral bicarbonate influx in the exocrine pancreas by coupling the transport of bicarbonate to that of sodium, with a 2 HCO3−:1 Na+ stoichiometry. The kidney variant, kNBC1, mediates basolateral bicarbonate efflux in the proximal tubule by coupling the transport of 3 HCO3− to 1 Na+. The molecular basis underlying the different stoichiometries is not known. pNBC1 and kNBC1 are 93 % identical to each other with 41 N‐terminal amino acids of kNBC1 replaced by 85 distinct amino acids in pNBC1. In this study we tested the hypothesis that the differences in stoichiometry are related to the difference between the N‐termini of the two proteins. Mouse renal proximal tubule and collecting duct cells, deficient in both pNBC1‐ and kNBC1‐mediated electrogenic sodium bicarbonate cotransport function were transfected with either pNBC1 or kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The stoichiometry was calculated from the reversal potential by measuring the current‐voltage relationships of the cotransporter at different Na+ concentration gradients. Our data indicate that both kNBC1 and pNBC1 can exhibit either a 2:1 or 3:1 stoichiometry depending on the cell type in which each is expressed. In proximal tubule cells, both pNBC1 and kNBC1 exhibit a 3 HCO3−:1 Na+ stoichiometry, whereas in collecting duct cells, they have a 2:1 stoichiometry. These data argue against the hypothesis that the stoichiometric differences are related to the difference between the N‐termini of the two proteins. Moreover, the results suggest that as yet unidentified cellular factor(s) may modify the stoichiometry of these cotransporters.

Epithelial cell lines from the proximal tubule of SHR and WKY rats were generated by microdissection, cell growth on 3T3 cell feeder layers, and transduction of the SV40 large T-antigen gene. The cell lines that formed confluent, electrically-resistive monolayers (basal conductance 1 to 20 mS/cm2) were selected for further study. Of these, cell lines generated from one rat did not show evidence of T-antigen expression or integration, and apparently immortalized spontaneously. Cell lines from three other rats expressed high levels of T-antigen, and showed evidence of integration of one or more copies of T-antigen. All cell lines formed polarized monolayers with apical microvilli, tight junctional complexes, and convolutions of the basolateral plasma membrane. Most cell lines grew in the absence of extracellular glucose indicating a capacity for gluconeogenesis. Sodium succinate cotransport and P2-purinergic receptor mediated signaling were demonstrated in all lines tested. The cell lines also showed that Na/H exchanger activity is regulated by angiotensin II. The results indicate that these cell lines express a proximal tubular phenotype, and are morphologically and functionally similar to primary cultures. These rat cell lines represent a new, potentially useful cell model for elucidating the cellular and molecular mechanisms of genetic differences in proximal tubule Na+ reabsorption.