Lazaro J. Mandel

Intracellular respiratory dysfunction and cell injury in short-term anoxia of rabbit renal proximal tubules.

The effects of short-term anoxia and hypoxia were studied in a rabbit proximal renal tubule suspension in order to avoid the hemodynamic consequences of clamp-induced ischemia. The suspension was subjected to anoxia for 10-40 min and the effects on a number of cellular transport and respiratory parameters were monitored. Cellular respiration was measured upon addition of nystatin (Nys) to maximally stimulate Na pump activity. Mitochondrial respiration was measured in the tubules by addition of digitonin and ADP to obtain the state 3 respiratory rate. The release of lactate dehydrogenase (LDH) was measured as an index of plasma membrane damage. The cellular contents of K and Ca were also measured. Results show that 10 and 20 min of anoxia partially inhibited Nys-stimulated and mitochondrial respiration, and partially decreased the K contents, but all these effects were largely reversible after 20 min of reoxygenation. After 40 min of anoxia and 20 min of reoxygenation, all these variables remained irreversibly inhibited: Nys-stimulated respiration by 54%, mitochondrial respiration by 50%, K content by 42%, and LDH release was 40% of total. Ca content decreased slightly during anoxia, but increased up to fourfold during severe hypoxia; the excess Ca was released during the first 10 min of reoxygenation. The degree of respiratory impairment was identical during anoxia or hypoxia, suggesting that Ca accumulation was not associated with the impairment. Decreasing the extracellular Ca to 2.5 microM decreased LDH release significantly during anoxia, suggesting that plasma membrane damage during anoxia may be associated with increased intracellular free Ca. Addition of Mg-adenosine triphosphate during anoxia dramatically improved recovery of all the measured parameters after the anoxic period.

Intracellular glutathione in the protection from anoxic injury in renal proximal tubules.

Previous results (Weinberg, J. M., J. A. David, M. Abarzua, and T. Rajan. 1987. J. Clin. Invest. 80:1446-1454) have shown that GSH and glycine (GLY) are cytoprotective during anoxia when added extracellularly. The present studies investigate the role that intracellular GSH plays in this cytoprotection. Proximal renal tubules in suspension prepared with either high (11 +/- 1 nmol/mg protein) or low (6 +/- 1 nmol/mg protein) GSH contents were subjected to 40 min of anoxia and 40 min of reoxygenation. Low GSH tubules were protected from plasma membrane damage during anoxia by exogenous addition of 1 mM GSH or GLY, reducing lactate dehydrogenase (LDH) release from 42 +/- 7 to 14 +/- 1 and 10 +/- 1%, respectively. High GSH tubules were equally protected from anoxic damage without exogenous additions. Since the high GSH content approximates the in vivo values, it may be concluded that GSH may be cytoprotective during anoxia in vivo. However, it is not the intracellular GSH itself that is cytoprotective; rather, this protection resides in the ability to produce GLY, which appears to be the cytoprotective agent. Alanine was also shown to have similar cytoprotective properties, although higher concentrations were required. Sulfhydryl reducing agents such as cysteine and dithiothreitol offered less, but significant protection from anoxic damage. Protection by GSH, GLY, or alanine was not associated with higher ATP levels during anoxia. Tubules that were protected from membrane damage during anoxia recovered oxygen consumption and K and ATP contents significantly better during reoxygenation than unprotected tubules.

Mechanisms whereby exogenous adenine nucleotides improve rabbit renal proximal function during and after anoxia.

When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloride-treated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three- to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.

Inhibition of Renal Metabolism. Relative effects of arsenate on sodium, phosphate, and glucose transport by the rabbit proximal tubule.

These studies examine the inhibitory effects of arsenate on the transport of sodium, phosphate, glucose, and para-aminohippurate (PAH) as well as oxidative metabolism by proximal convoluted tubules from the rabbit kidney. Transport rates were measured with radioisotopes in isolated and perfused segments. Metabolic activity was monitored through oxygen-consumption rates and HADH fluorescence in parallel studies in suspensions of cortical tubules. The addition of 1mM arsenate to the perfusate reduced fluid absorption rates from 1.24 +/- 0.17 to 0.66 +/- 0.19 nl/nm.min (P < 0.01) and lumen-to-bath phosphate transport from 9.93 +/- 3.47 to 4.25 +/- 1.08 pmol/mm.min (P < 0.01). Similar concentrations of arsenate reduced glucose transport only slightly from 66.1 +/- 6.0 to 56.8 +/-4 4.6 pmol/mm.min (P < 0.05) and had no effect of PAH secretion. Removing phosphate from the perfusate did not affect the net transport of sodium or glucose. In suspensions of tubules, arsenate increased oxygen consumption rates by 20.5 +/- 2.9% and decreased NADH fluorescence by 10.8 +/- 1.5%. These effects on metabolism were concentration dependent and magnified in the presence of ouabain. The data indicate that arsenates main effect is to uncouple oxidative phosphorylation, and that graded uncoupling of oxidative metabolism causes graded reductions in the net transport of both sodium and phosphate. Glucose transport is inhibited only slightly and PAH secretion is not affected. Thus, partial as opposed to complete inhibition of metabolism reveals that different relationships exist between net sodium transport and the transport of phosphate, glucose, and PAH by the proximal renal tubule.

On the cross-reactivity of amiloride and 2,4,6 Triaminopyrimidine (TAP) for the cellular entry and tight junctional cation permeation pathways in epithelia

Summary2,4,6 Triaminopyrimidine (TAP) has been previously shown to inhibit the passive tight junctional cation permeation pathway in various “leaky” epithelia. Amiloride has been shown to be an effective inhibitor of the cation cellular entry pathway in “tight” epithelia. In this paper we demonstrate that TAP and amiloride at appropriate concentrations are able to block either of these epithelial cation permeation pathways. TAP was found to block the Na entry pathway in frog skin with the following characteristics: it (1) inhibits from the external solution only, (2) is completely reversible, (3) increases the transepithelial resistance, (4) is active in the monoprotonated form, (5) is noncompetitive with Na, (6) displays saturation kinetics which obey a simple kinetic model (KI=1×10−3m), (7) is independent of external calcium, (8) is dependent on external buffering capacity, and (9) is competitive with amiloride. Amiloride inhibition of the junctional permeation in gallbladder had the following characteristics: it (1) increases the transepithelial resistance, (2) decreases cation conductance without affecting the anion conductance, (3) displays saturation kinetics which obey a simple kinetic model (KI=1×10−3m), and (4) possesses inhibitory activity in both its protonated and unprotonated form. These results not only indicate that a similar inhibitory site may exist in both of these cation permeation pathways, but also provide information on the chemical nature and possible location of these inhibitory sites.

Metabolic Requirement for Inorganic Phosphate by the Rabbit Proximal Tubule: EVIDENCE FOR A CRABTREE EFFECT

These studies examine the effects of acute changes in the availability of inorganic phosphate on the function of isolated proximal renal tubules from rabbit kidney. We removed phosphate from the extracellular fluids and measured fluid absorption rates in isolated perfused tubules and oxygen consumption rates in suspensions of cortical tubules. In proximal convoluted tubules, the selective removal of phosphate from the luminal fluid reduced fluid absorption rates from 1.11+/-0.12 to -0.01+/-0.08 nl/mm . min. This effect on fluid absorption was dependent on the presence of glucose transport and metabolism. The addition of phlorizin to the phosphate-free luminal fluid preserved fluid absorption rates (1.12+/-0.12 nl/mm . min) as did the substitution of nonmetabolized alpha-methyl d-glucopyranoside for glucose (1.05+/-0.21 nl/mm . min) or the addition of 2-deoxyglucose, an inhibitor of glycolysis, to the bathing medium (1.01+/-0.15 nl/mm . min). There was no effect on fluid absorption if phosphate was removed from the bath only. Additionally, removal of phosphate from the luminal fluid of proximal straight rather than convoluted tubules had no effect on fluid absorption rates. Oxygen consumption rates in suspensions of cortical tubules were reduced from 18.9+/-0.6 to 10.6+/-0.6 nmol O(2)/mg tubular protein . min by the removal of phosphate from the medium. This inhibition was prevented by the substitution of alpha-methyl d-glucopyranoside for glucose in the phosphate-free medium. The data indicate that under certain conditions, proximal convoluted tubules require the presence of phosphate in the luminal fluid to preserve tubular function. In the absence of intraluminal phosphate, glucose metabolism causes a reduction in both oxidative metabolism and fluid absorption. This response is analogous to the Crabtree effect and suggests limitations on the intracellular availability of inorganic phosphate.

A mechanism of S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-l-cysteine toxicity to rabbit renal proximal tubules☆

S-(1,2,3,4,4-Pentachloro-1,3-butadienyl)-L-cysteine (PCBC) has been identified as the penultimate compound responsible for hexachlorobutadiene-induced nephrotoxicity. The primary goal of these studies was to determine the mechanism of PCBC-induced toxicity in rabbit renal proximal tubules by examining the early changes in tubular physiology. PCBC (20-500 microM) induced a specific sequence of toxic events. Following 15 min of exposure, 200 microM PCBC increased basal (25%) and ouabain-insensitive (78%) respiration. This was followed by a decrease in basal (46%), nystatin-stimulated (54%), and ouabain-insensitive (21%) respiration and a decrease in glutathione content (79%). Finally, there was a decrease in cell viability as measured by a decrease in LDH retention at 60 min. Direct probing of mitochondrial function revealed that the initial increase in respiration resulted from the uncoupling of oxidative phosphorylation, while the late changes in respiration appeared to result from gross mitochondrial damage characterized by inhibited state 3 respiration, inhibited cytochrome c-cytochrome oxidase, and inhibited electron transport. Studies utilizing tubules with decreased glutathione content revealed that glutathione plays little if any role in the early events of PCBC-induced toxicity. These results suggest that PCBC-induced mitochondrial dysfunction may initiate the renal proximal tubule injury.

Phenamil: an irreversible inhibitor of sodium channels in the toad urinary bladder

SummarySeveral new amiloride analogues and two reported photoaffinity analogues were tested for irreversible inhibition of short-circuit current,Isc, in toad bladder. Bromoamiloride, a photoaffinity analogue, induced 40% irreversible inhibition at 500 μm after irradiation with ultraviolet light ≥320 nm. Iodoamiloride caused no irreversible inhibition. Of the new analogues tested, only 3,5-diamino-6-chloro-N-[(phenylamino) aminomethylene] pyrazinecarboxamide,phenamil, irreversibly inhibitedIsc at concentrations of 0.05 to 5 μm when added to the mucosal solution. Irreversible inhibition ofIsc by phenamil may be attributed to specific blockage of the mucosal sodium channels, which depended on: 1) time of exposure; 2) mucosal pH: 3) mucosal sodium concentration. For example, 5 μm phenamil irreversibly inhibitedIsc by 38% in 103mm Na at pH 8.6 and nearly 75% in 30mm Na at pH 6.4 after a 40-min exposure. Irreversible inhibition occurred in two phases with time constants of ≤10 min and approximately 140 min. Due to its irreversible nature, phenamil may be used to measure channel density.

Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules.

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3(200/215). To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3(200/215) but no Ank3(170) or Ank3(120). Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3(200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3(200/215) and Na-K-ATPase abundance measured a lower Ank3(200/215)-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1 cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3(200/215) suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.

Potassium transport in the rabbit renal proximal tubule: Effects of barium, ouabain, valinomycin, and other ionophores

SummaryPotassium fluxes in a suspension of rabbit proximal tubules were monitored using a potassium-sensitive extracellular electrode. Ouabain (10−4m) and barium (5mm) were used to selectively quantitate the potassium efflux pathway (105±5 nmol K+·mg protein−1·min−1) and the sodium pump-related potassium influx (108±7), respectively. These equal and opposite fluxes suggest that potassium accumulation in the cell occurs mainly through the sodium pump and that potassium efflux occurs mainly through barium-sensitive potassium channels. Thus the activity of the sodium pump (Na, K-ATPase) in the basolateral membrane of the proximal tubule is balanced by the efflux of potassium, presumably across the basolateral membrane, which has a high potassium permeability. In addition, the effect of valinomycin and other ionophores was examined on potassium fluxes and several metabolic parameters [oxygen consumption (QO2), ATP content]. The addition of valinomycin to the tubules produced a net efflux of potassium which was quantitatively equivalent to the efflux produced by the addition of ouabain. The valinomycin-induced efflux was mainly due to the activity of valinomycin as a mitochondrial uncoupler, which indirectly inhibited the sodium pump by allowing a rapid reduction of the intracellular ATP. Amphotericin, nystatin, and monensin all produced large net releases of intracellular potassium. The action of the ionophores could be localized to the plasma or mitochondrial membrane and classified into three groups, as follows: (a) those which demonstrated full mitochondrial uncoupler activity (FCCP, valinomycin), (b) those which had no uncoupler activity (amphotericin B, nystatin); and (c) those which displayed partial uncoupler activity (monensin, nigericin).