Partial recovery of in vivo function by improved incubation conditions of isolated renal proximal tubule.
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Isolated microperfused rabbit renal proximal tubule S2 segments, if incubated in conventional substrate containing HCO3–Ringer solution, exhibit lower cell membrane potentials (Vb) and elevated intracellular Na+ concentrations ([Na]i) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/or hormonal stimulation of the cells, we have used microelectrode techniques to test whether improving substrate supply and applying norepinephrine (NE, to compensate for the missing nerve supply) reverts Vb and [Na]i to values observed in vivo. Application of D-glucose (5.5 mmol/l) and additional application of pyruvate, lactate, or L-alanine (each 10 mmol/l), or bathing the tubules in Dulbecco’s modified Eagle’s tissue culture medium (DMEM) significantly increased Vb and, whenever tested, reduced [Na]i as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented – as tested in the case of pyruvate – by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, β-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 μmol/l) which increased Vb from –42.8 ± 1.3 mV (SEM) to –55.3 ± 2.5 mV (n = 11). Interestingly we noticed that under the latter conditions the Vb response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 ± 1.4 mV (in D-glucose Ringer solution) to +0.6 ± 0.7 mV (in DMEM plus NE) (n = 8), with some tubules showing even a small hyperpolarization. The latter implies partial restoration of the in vivo behaviour, since in experiments on rat proximal tubules in vivo amiloride regularly hyperpolarized the cells (by –3.4 ± 0.76 mV, n = 5). Obviously under conventional in vitro conditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vitro. In agreement with observations reported in the subsequent publication our results demonstrate that isolated proximal tubules undergo functional alterations which may be largely prevented by improved metabolic and stimulatory incubation conditions.
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