Cisplatin nephrotoxicity: role of filtration and tubular transport of cisplatin in isolated perfused kidneys.

Isolated perfused rat kidneys were used to determine the contribution of filtration and tubular transport of cisplatin to its nephrotoxicity. Perfusion of kidneys with 0.5 mM cisplatin concomitantly reduced tubular reabsorption of electrolytes and glomerular filtration rate in a time-dependent manner. These renal functional changes were similar to those obtained following in vivo cisplatin treatment (10 mg/kg). In vitro exposure to cisplatin reduced the renal clearance of organic ions without reducing renal perfusate flow, suggesting that renal hemodynamic changes do not mediate cisplatin-induced proximal tubular dysfunction. Inhibition of organic ion transport also was observed in non-filtering perfused kidneys treated with 0.5 mM cisplatin, implying that filtration of cisplatin is not a prerequisite for induction of toxicity. These data also suggest that cisplatin transport from a basolateral site may be important in the development of acute nephrotoxicity.

Age-related differences in susceptibility to renal ischemia in rats.

These experiments were designed to determine the influence of age on the response of the kidney to ischemia. Renal ischemia was induced in female Fischer-344 rats, 3-4 or 37-38 months old, by renal arterial and venous occlusion followed by 0, 1, 24, or 96 hr of reflow. Age-matched controls were sham operated but were not subjected to ischemia. A transient postischemic increase in blood urea nitrogen (BUN) and serum creatinine was observed in young rats. In old rats, BUN and serum creatinine remained markedly elevated through 96 hr postischemia. In vitro renal cortical slice accumulation of organic ions was inhibited to a greater extent in old rats than in young rats 96 hr postischemia. Histologically, renal tubular damage was more severe in old than in young rats 24 and 96 hr postischemia. Tubular regenerative activity was similar in old and young rats at 96 hr, but restoration of tubular architecture was more complete in young rats. Organic ion accumulation by renal cortical slices from naive old rats was inhibited by in vitro anoxia (treatment with 100% N2) to a greater extent than tissue from young rats. These data suggest that old rats are more susceptible to renal ischemia than are young rats and these differences in susceptibility may reflect intrinsic age-related differences in basal renal metabolism.

Mechanisms mediating cephaloridine inhibition of renal gluconeogenesis.

Incubation of renal cortical slices with cephaloridine (CPH) markedly inhibits pyruvate-supported gluconeogenesis, an effect which is independent of CPH-induced lipid peroxidation. CPH was found to inhibit pyruvate-supported gluconeogenesis in a time-and concentration-dependent manner. Pyruvate-supported gluconeogenesis was inhibited as early as 10 min following incubation of renal cortical slices with 5 mM CPH. Similarly, endogenous gluconeogenesis was impaired following CPH treatment. CPH depressed the renal cortical slice content of ATP by 50%, but only following 90 and 120 min of drug exposure, suggesting that mitochondrial dysfunction does not mediate the inhibition of gluconeogenesis by CPH. To identify the intracellular site(s) of CPH inhibition of gluconeogenesis, the effects of CPH on glucose production were evaluated using substrates catalyzed by rate-limiting reactions. CPH inhibited renal cortical slice gluconeogenesis when the following substrates were used: pyruvate (mitochondrial), oxaloacetate and fructose-1,6-diphosphate (FDP) (postmitochondrial), and glucose-6-phosphate (G6P, endoplasmic reticulum). Inhibition of G6P-supported gluconeogenesis occurred within 5 min of incubation with 5 mM CPH. Direct addition of CPH to microsomal suspensions inhibited G6Pase activity in a concentration-dependent fashion. By contrast, addition of CPH to cytosolic fractions did not affect FDPase activity. CPH increased the Km and decreased the Vmax of G6Pase, indicating mixed competitive and noncompetitive inhibition. These data indicate that the profound inhibition of renal cortical slice gluconeogenesis by CPH is mediated by inhibition of microsomal G6Pase activity.

Biochemical mechanisms of cephaloridine nephrotoxicity: time and concentration dependence of peroxidative injury.

These experiments were designed to elucidate the initiating biochemical events mediating cephaloridine (CPH) nephrotoxicity. Renal cortical slices from naive male Fischer-344 rats were incubated at 37 degrees C in a phosphate- or bicarbonate-buffered medium containing 0, 1, 5, or 10 mM CPH. Slices were incubated for 15, 30, 45, 60, 90, 120, and 180 min and evaluated for accumulation of organic ions [p-aminohippurate (PAH) and tetraethylammonium (TEA)], pyruvate-stimulated gluconeogenesis, malondialdehyde (MDA) production, and reduced glutathione (GSH) content. Renal cortical slice accumulation of PAH and TEA was decreased by 5 and 10 mM CPH as early as 120 and 90 min of incubation, respectively. CPH-induced MDA production by renal cortical slices preceded the effects of CPH on organic ion accumulation. Coincubation of CPH with the antioxidants promethazine and N,N'-diphenyl-p-phenylenediamine inhibited CPH-induced lipid peroxidation and changes in organic ion accumulation. In contrast, 5 or 10 mM CPH inhibited gluconeogenic capacity at all time points examined, an effect which was not influenced by antioxidant treatment. Depletion of renal cortical GSH by 5 or 10 mM CPH was evident following 30 min of incubation and was also unaffected by antioxidant treatment. These results support the hypothesis that lipid peroxidation mediates the effects of CPH on renal organic ion transport. The early and profound inhibition of gluconeogenesis by CPH suggests that the biochemical pathways of gluconeogenesis are either proximal to or represent a primary target for CPH nephrotoxicity.

Cephaloridine nephrotoxicity in aging male Fischer-344 rats.

Age-related differences in susceptibility to cephaloridine nephrotoxicity were evaluated in male Fischer-344 rats. Rats, 2.5, 4, 10-12 and 27-29 months old, were administered a single intraperitoneal dose of cephaloridine and renal function evaluated 24 h later. Susceptibility to cephaloridine-induced nephrotoxicity was age-related. Older rats (10-12 and 27-29 months) were the most susceptible to cephaloridine nephrotoxicity as indicated by a dose-related increase in relative kidney weight, elevation in blood urea nitrogen concentrations and a diminished capacity of renal cortical slices to accumulate the organic anion, p-aminohippurate (PAH) and the organic cation, tetraethylammonium (TEA). Impaired renal function following cephaloridine treatment was not detected in 2.5-month-old, apparent to a slight extent in 4-month-old, and most pronounced in 10-12- and 27-29-month-old rats. Serum and renal cortical concentrations of cephaloridine tended to be greater in older rats compared to that of young adults. Thus, the enhanced susceptibility of older rats to cephaloridine nephrotoxicity may be related in part to the increased renal cortical accumulation of cephaloridine.

Cephaloridine nephrotoxicity: strain and sex differences in mice.

Marked species and sex differences have been observed in the nephrotoxicity to the cephalosporin antibiotic cephaloridine (CPH). Preliminary studies have also indicated significant strain differences in mice to CPH nephrotoxicity. To investigate these findings further, male and female C57BL, BALB/c, CD-1, CFW, CBA/J, and DBA/2 mice were given either 4000 or 6000 mg/kg of CPH, sc. Renal function was assessed 48 hr later by the ability of renal cortical slices to accumulate the organic ions p-aminohippurate (PAH) and tetraethylammonium (TEA), changes in blood urea nitrogen (BUN) and kidney-to-body wt ratios. CPH produced dose-dependent nephrotoxicity in C57BL female mice. After 6000 mg/kg, PAH and TEA slice-to-medium (S/M) ratios were reduced by 70 and 49%, respectively; BUN was elevated 10-fold. The same dose given to CFW females had no effect. BALB/c, CD-1, CBA/J, and DBA/2 females showed intermediate signs of toxicity. Male mice of all strains tested exhibited no nephrotoxicity. CPH nephrotoxicity has been correlated with the concentration of CPH within the tubular cell; and C57BL female mice had relatively greater intracellular accumulation of CPH than C57BL male mice and CFW female mice in vitro and in vivo. Thus, differences in net renal cortical accumulation of CPH suggest possible differences in transport, binding, and/or metabolism of CPH may exist among strains and between sexes of mice.

Effects of gentamicin on renal function in isolated perfused kidneys from male and female rats.

The isolated perfused rat kidney was used to determine whether sex differences in gentamicin nephrotoxicity are related to intrinsic differences in renal response to gentamicin. Acute exposure to gentamicin decreased fractional reabsorption of water and electrolytes without changes in glomerular filtration rate in both sexes. Gentamicin decreased the tubular reabsorption of lysozyme but not glomerular permeability to lysozyme. No sex differences in renal responses were observed following in vitro exposures to gentamicin, suggesting that sex differences in susceptibility to gentamicin in vivo may be attributable to extrarenal factors, such as pharmacokinetics.

Acetaminophen nephrotoxicity in the rat: quantitation of renal metabolic activation in vivo.

Renal cortical necrosis induced by acetaminophen (APAP) may be related to generation of reactive intermediates by two mechanisms of metabolic activation, direct P-450 dependent metabolic activation (P-450) or metabolic activation subsequent to deacetylation of APAP to p-aminophenol (PAP). Generation of arylating intermediates by both pathways of metabolic activation was quantified in cyclohexamide (HEX)-pretreated or naive rats in vivo with specifically labeled [14C]APAP. The association of each type of metabolic activation with APAP-induced nephrotoxicity was determined in Fischer 344 (F344) and Sprague-Dawley (SD) rats, strains that are susceptible and resistant to APAP-induced nephrotoxicity, respectively. Covalent binding of [ring-14C]APAP to renal cortex was approximately four times greater than [acetyl-14C]APAP in HEX-pretreated F344 rats. In contrast, in SD rats pretreated with HEX covalent binding of [ring-14C]APAP and [acetyl-14C]APAP in the renal cortex was not different. Furthermore, covalent binding of [ring-14C]APAP to renal cortical protein was approximately four times greater in F344 rats than in SD rats. Arylation of hepatic protein by either [ring-14C]APAP or [acetyl-14C]APAP was similar regardless of strain or pretreatment regimen. These studies demonstrated arylation of renal macromolecules in vivo by reactive intermediates resulting from PAP in F344 but not SD rats. Since F344, but not SD, rats are susceptible to APAP-induced nephrotoxicity, it appears the formation of arylating intermediates by PAP is a requisite step in APAP-induced nephrotoxicity.

Metabolic heterogeneity of the proximal and distal kidney tubules.

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7-ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.