Rheogenic transport in the renal proximal tubule

The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.     

Sulphate and phosphate transport in the renal proximal tubule

Experiments performed on microperfused proximal tubules and brush-border membrane vesicles revealed that inorganic phosphate is actively reabsorbed in the proximal tubule involving a 2 Na+-HPO2-4 or H2PO-4 co-transport step in the brush-border membrane and a sodium-independent exit step in the basolateral cell membrane. Na+-phosphate co-transport is competitively inhibited by arsenate. The transtubular transport regulation is mirrored by the brush-border transport step: it is inhibited by parathyroid hormone intracellularly mediated by cyclic AMP. Transepithelial inorganic phosphate (Pi) transport and Na+-dependent Pi transport across the brush-border membrane correlates inversely with the Pi content of the diet. Intraluminal acidification as well as intracellular alkalinization led to a reduction of transepithelial Pi transport. Data from brush-border membrane vesicles indicate that high luminal H+ concentrations reduce the affinity for Na+ of the Na+-phosphate co-transport system, and that this mechanism might be responsible for the pH dependence of phosphate reabsorption. Contraluminal influx of Pi from the interstitium into the cell could be partly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). It is not, however, changed when dicarboxylic acids are present or when the pH of the perfusate is reduced to pH 6. Sulphate is actively reabsorbed, involving electroneutral 2 Na+-SO2-4 co-transport through the brush-border membrane. This transport step is inhibited by thiosulphate and molybdate, but not by phosphate or tungstate. The transtubular active sulphate reabsorption is not pH dependent, but is diminished by the absence of bicarbonate. The transport of sulphate through the contraluminal cell side is inhibited by DIDS and diminished when the capillary perfusate contains no bicarbonate or chloride. The latter data indicate the presence of an anion exchange system in the contraluminal cell membrane like that in the erythrocyte membrane.     

Amino acid transport in the renal proximal tubule

Summary. In the kidney the proximal tubule is responsible for the uptake of amino acids. This occurs via a variety of functionally and structurally different amino acid transporters located in the luminal and basolateral membrane. Some of these transporters show an ion-dependence (e.g. Na⁺, Cl⁻ and K⁺) or use an H⁺-gradient to drive transport. Only a few amino acid transporters have been cloned or functionally characterized in detail so far and their structure is known, while little is known about a majority of amino acid transporters. Only few attempts have been untertaken looking at the regulation of amino acid transport. We summarized more recent information on amino acid transport in the renal proximal tubule emphasizing functional and regulatory aspects. Received August 8, 1999; Accepted April 20, 2000     

An equation for flow in the renal proximal tubule

Approximate equations for epithelial solute and water transport have been combined with the relations of mass conservation to yield a single differential equation representing volume flow along the proximal tubule. This flow equation is first order, quasilinear and may be integrated directly. For the steady state, the result is an implicit relation between volume flow and distance along the tubule. For two time-dependent problems (step change of tubule inlet velocity or osmolality) the trajectories (distance as a function of transit time) of a fluid element starting at the inlet are obtained. Differentiation of the steady-state relation with respect to the inlet velocity yields a first-order differential equation relating inlet and outlet velocity. This equation is considered in detail, particularly with regard to the influence of solute-linked water reabsorption. Model calculations with parameters representing rat proximal tubule indicate that it will be difficult to discern coupled water flux in this epithelium from only outlet and inlet flows. Calculations using lower transport rates and lower permeabilities suggest that this equation may be useful in quantifying coupled water flow in proximal tubules from other species.     

Localization of spectrin isoforms in the adult mouse heart

The distribution of two isoforms of spectrin in the adult mouse heart was investigated by Western blotting and immunocytochemistry by use of monospecific antibodies to erythrocyte spectrin and nonerythroid brain spectrin (240/235). Western blotting revealed proteins analogous to both isoforms of -spectrin in adult heart. Light-microscopic immunocytochemistry indicated that erythroid spectrin was distributed throughout the myocardium, with immunofluorescence localized to plasma membranes, Z-lines, and intercalated discs. Antibodies to brain spectrin (240/235) exhibited staining throughout the heart, with a generally diffuse distribution except for the prominent immunoreactivity associated with the intercalated discs. Nonerythroid spectrin immunofluorescence was detected in the endothelial cells of the endocardium and the mesothelial cell lining of the epicardium. Erythrocyte spectrin was not detected in the endocardium or the epicardium. The identification and localization of spectrin isoforms in the mammalian heart suggest the importance of spectrin proteins in the structural integrity and proper function of cardiac cells and tissues. This is the first demonstration of two different -spectrin subunits in the mammalian heart.     

Innervation of the renal proximal convoluted tubule of the rat

Experimental data suggest the proximal tubule as a major site of neurogenic influence on tubular function. The functional and anatomical axial heterogeneity of the proximal tubule prompted this study of the distribution of innervation sites along the early, mid, and late proximal convoluted tubule (PCT) of the rat. Serial section autoradiograms, with tritiated norepinephrine serving as a marker for monoaminergic nerves, were used in this study. Freehand clay models and graphic reconstructions of proximal tubules permitted a rough estimation of the location of the innervation sites along the PCT. In the subcapsular nephrons, the early PCT (first third) was devoid of innervation sites with most of the innervation occurring in the mid (middle third) and in the late (last third) PCT. Innervation sites were found in the early PCT in nephrons located deeper in the cortex. In juxtamedullary nephrons, innervation sites could be observed on the PCT as it left the glomerulus. This gradient of PCT innervation can be explained by the different tubulovascular relationships of nephrons at different levels of the cortex. The absence of innervation sites in the early PCT of subcapsular nephrons suggests that any influence of the renal nerves on the early PCT might be due to an effect of neurotransmitter released from renal nerves reaching the early PCT via the interstitium and/or capillaries.     

Ion exchangers mediating NaCl transport in the renal proximal tubule

We have studied the mechanisms of NaCl transport in the mammalian proximal tubule. Studies of isolated brush-border membrane vesicles confirmed the presence of Na+-H+ exchange and identified Cl(-)-formate and Cl(-)-oxalate exchangers as possible mechanisms of uphill Cl- entry. We found that formate and oxalate each stimulate NaCl absorption in microperfused proximal tubules. Stimulation of NaCl absorption by formate was blocked by the Na+-H+-exchange inhibitor EIPA, whereas stimulation by oxalate was blocked by omission of sulfate from the perfusion solutions. These observations were consistent with recycling of formate from lumen to cell by H+-coupled formate transport in parallel with Na+-H+ exchange and recycling of oxalate by oxalate-sulfate exchange in parallel with Na+-sulfate cotransport. Using isoform-specific antibodies, we found that NHE1 is present on the basolateral membrane of all nephron segments, whereas NHE3 is present on the apical membrane of cells in the proximal tubule and the loop of Henle. The inhibitor sensitivity of Na+-H+ exchange in renal brush-border vesicles and of HCO3- absorption in microperfused tubules suggested that NHE3 is responsible for most, if not all, apical membrane Na+-H+ exchange in the proximal tubule. The role of NHE3 in mediating proximal tubule HCO3- absorption and formate-dependent Cl- absorption was confirmed by studies in NHE3 null mice. Finally, we cloned and functionally expressed CFEX, an anion transporter expressed on the apical surface of proximal tubule cells and capable of mediating Cl(-)-formate exchange.     

CFTR mediated chloride secretion in the avian renal proximal tubule

In primary cell cultures of the avian (Gallus gallus) renal proximal tubule parathyroid hormone and cAMP activation generate a Cl⁻-dependent short circuit current (I_SC) response, consistent with net transepithelial Cl⁻ secretion. In this study we investigated the expression and physiological function of the Na-K-2Cl (NKCC) transporter and CFTR chloride channel, both associated with Cl⁻ secretion in a variety of tissues, in these proximal tubule cells. Using both RT-PCR and immunoblotting approaches, we showed that NKCC and CFTR are expressed, both in proximal tubule primary cultures and in a proximal tubule fraction of non-cultured (native tissue) fragments. We also used electrophysiological methods to assess the functional contribution of NKCC and CFTR to forskolin-activated I_SC responses in filter grown cultured monolayers. Bumetanide (10 μM), a specific blocker of NKCC, inhibited forskolin activated I_SC by about 40%, suggesting that basolateral uptake of Cl⁻ is partially mediated by NKCC transport. In monolayers permeabilized on the basolateral side with nystatin, forskolin activated an apical Cl⁻ conductance, manifested as bidirectional diffusion currents in the presence of oppositely directed Cl⁻ gradients. Under these conditions the apical conductance appeared to show some bias towards apical-to-basolateral Cl⁻ current. Two selective CFTR blockers, CFTR Inhibitor 172 and GlyH-101 (both at 20 μM) inhibited the forskolin activated diffusion currents by 38-68%, with GlyH-101 having a greater effect. These data support the conclusion that avian renal proximal tubules utilize an apical CFTR Cl⁻ channel to mediate cAMP-activated Cl⁻ secretion.     

Membrane receptors involved in endocytosis in rat renal proximal tubule

In conclusion the present study has demonstrated the localization of 3 receptors for endocytosis in rat renal proximal tubule cells. The three receptors are located in the membranes of the vacuolar compartment involved in endocytosis in these cells and in addition in dense apical tubules responsible for membrane recycling in the proximal tubule.     

Heterogeneity of pH in the aqueous cytoplasm of renal proximal tubule cells

Heterogeneity of cytosolic pH was studied with compounds that distribute between the cytosol and mitochondrial matrix in fundamentally different ways, i.e., according to the extent of ionization or according to the function of H+-coupled transport systems. Results show that the average cytosolic pH is considerably more alkaline than the region to which mitochondria are exposed. Because mitochondria are localized predominantly in the basal region, the results are consistent with a transcellular pH gradient within the cytosol of proximal tubule cells. Experiments analyzing the effects of inhibiting efflux of HCO3- at the basal surface and Na+-H+ exchange at the apical surface support the interpretation that the function of these systems contributes to the transcellular pH gradient. The existence of a heterogeneity in pH within the cytosol has important implications concerning the function and regulation of numerous cell processes.     

The transepithelial shunt pathway in the renal proximal tubule of newt kidney

The transepithelial shunt pathway of newt proximal tubule was examined with glass micro-electrode and electron microscopic methods. The input resistance of the peritubular (basal) membrane and tubular wall were found to be $\text{4.2 ± 0.1 · 10}{}^6$ ($\text{mean ± S.E.}\text{, n = 16}$) and $\text{11.4 ± 0.2 · 10}{}^4\text{(n = 11)}$, respectively. The input resistance of the peritubular membrane was approximately 40-times larger than that of the tubular wall. When the kidneys were perfused in a lanthanum solution, the lanthanum ions were then observed in the junctional complexes and in the intercellular spaces on both the basal and apical sides. The results indicate that the electrical shunt pathway corresponds to the apical junctional complexes and the intercellular spaces, and that the tight junctions are not truly ‘tight’ for the transepithelial movement of small ions in the proximal tubule of the newt kidney.     

Arachidonic acid release in renal proximal tubule cell injuries and death

Arachidonic acid release and the effect of phospholipase inhibitors on various types of cell injuries and death to rabbit renal proximal tubule suspensions were determined. Proximal tubules were exposed to the mitochondrial inhibitor antimycin A (0.1 μM), the protonophore carbonyl cyanide ρ-trifluoromethoxypheitylhydrazone (1 μM FCCP), the oxidant tertbutyl hydroperoxide (0.5 mM TBHP), or the calcium ionophore ionomycin (5 μM) in the absence or presence of the putative phospholipase inhibitors dibucaine, mepacrine, chlorpromazine, or U-26384. The phospholipase inhibitors had no effect on the proximal tubule lactate dehydrogenase (LDH) release (a marker of cell death) produced by FCCP, antimycin A, or ionomycin after 1,2, or 2 hours of exposure, respectively. Only dibucaine and mepacrine decreased LDH release in TBHP-treated proximal tubules without decreasing TBHP-induced lipid peroxidation. Antimycin A and ionomycin did not release arachidonic acid from proximal tubules prelabeled with [1-¹⁴C] arachidonic acid. In contrast, TBHP released arachidonic acid from proximal tubules prior to the onset of cell death, and dibucaine and mepacrine decreased the TBHP-induced release. Thus, phospholipase inhibitors were cytoprotective in those injuries that produced arachidonic acid release. These results suggest that arachidonic acid release and phospholipase A₂ activation play a contributing role in oxidant-induced renal proximal tubule cell injury and death but not in mitochondrial inhibitor- or calcium ionophore-induced proximal tubule cell injury and death.     

Intracellular calcium chelators and oxidant-induced renal proximal tubule cell death

The effect of intracellular calcium chelators on rabbit renal proximal tubule (RPT) cell death induced by t-butyl hydroperoxide (TBHP) and H₂O₂ was examined. Preincubation of RPT suspensions with 50 μM QUIN 2/AM completely prevented TBHP (0.5 mM) and H₂O₂ (2 mM) induced cell death [i.e., release of lactate dehydrogenase (LDH)]. QUIN 2/AM, BAPTA/AM, EGTA/AM, and FURA 2/AM, at 5 μM, decreased LDH release (at 6 hr) from 41% to 4%, 21%, 26%, and 33%, and decreased lipid peroxidation (at 1 hr) from 1.0 to 0.1, 0.4, 0.6, and 0.8 nmol MDA/mg protein, respectively, after TBHP exposure. Since oxidant-induced lipid peroxidation and cell death are iron-dependent in this model, these results suggest that the intracellular calcium chelators inhibit cell death by chelating iron.     

Evidence for water channels in renal proximal tubule cell membranes

Summary Water transport mechanisms in rabbit proximal convoluted cell membranes were examined by measurement of: (1) osmotic (P _f ) and diffusional (P _d ) water permeabilities, (2) inhibition ofP _f by mercurials, and (3) activation energies (E _a ) forP _f .P _f was measured in PCT brush border (BBMV) and basolateral membrane (BLMV) vesicles, and in viable PCT cells by stopped-flow light scattering;P _d was measured in PCT cells by proton NMR T_i relaxation times using Mn as a paramagnetic quencher. In BLMV,P _f (0.019 cm/sec, 23°C) was inhibited 65% by 5mm pCMBS and 75% by 300 m HgCl₂ (K _l =42 m);E _a increased from 3.6 to 7.6 kcal/mole (15–40°C) with 300 m HgCl₂. In BBMV,P _f (0.073 cm/sec, 23°C,E _a =2.8 kcal/mole, <33°C and 13.7 kcal/mole, >33°C) was inhibited 65% with HgCl₂ withE _a =9.4 kcal/mole (15–45°C). Mercurial inhibition in BLMV and BBMV was reversed with 10 m mercaptoethanol. Viable PCT cells were isolated from renal cortex by Dounce homogenization and differential seiving. Impedence sizing studies show that PCT cells are perfect osmometers (100–1000 mOsm). Assuming a cell surface-to-volume ratio of 25,000 cm^–1,P _f was 0.010±0.002 cm/sec (37°C) andP _d was 0.0032 cm/sec.P _f was independent of osmotic gradient size (25–1000 mOsm) withE _a 2.5 kcal/mole (<27°C) and 12.7 kcal/mole (>27°C). CellP _f was inhibited 53% by 300 m HgCl₂ (23°C) withE _a 6.2 kcal/mole. These findings indicate that cellP _f is not restricted by extracellular or cytoplasmic unstirred layers and that cellP _f is not flow-dependent. The high BLMV and BBMVP _f , inhibition by HgCl₂, lowE _a which increases with inhibition, and the measuredP _f /P _d >1 in cells in the absence of unstirred layers provide strong evidence for the existence of water channels in proximal tubule brush border and basolateral membranes. These channels are similar to those found in erythrocytes and are likely required for rapid PCT transcellular water flow.