Early onset of cisplatin-induced nephrotoxicity in streptozotocin-diabetic rats treated with insulin.

Mechanism of protection against cisplatin nephrotoxicity in streptozotocin-diabetic rats is unclear but is associated with decreased renal platinum accumulation. This study was designed to determine whether normalization of hyperglycaemia by insulin treatment to six week streptozotocin-diabetic rats reversed protection against cisplatin nephrotoxicity. Male Sprague-Dawley rats divided into 3 groups (n=10/group) (1) non-diabetic (2) untreated streptozotocin-diabetic and (3) insulin-treated streptozotocin-diabetic groups were rendered diabetic using streptozotocin (65 mg/kg body weight, intravenous). At the end of 6 weeks, Group 3 animals were treated with insulin (subcutaneously) for 21 days to normalize glucose. After 21 days of insulin treatment, the mean +/- S.D. plasma glucose (mg%) in Group 3 animals at 144.8 +/- 22.03, was significantly lower than Group 2 animals (412 +/- 24.69) and comparable to age-matched non-diabetic (Group 1) animals. Blood urea nitrogen at 24 hr after intraperitoneal administration of cisplatin (5 mg/kg body weight) increased by a factor 2.5 in Group 3 compared to 1.1 and 1.3 in Group 1 and Group 2 animals respectively. In the same animals, at 96 hr the blood area nitrogen increased by a factor of 3.2 and 2.9 in Group 1 and Group 3 respectively compared to 1.14 for Group 2 animals. Renal platinum levels in Group 1, Group 2 and Group 3 after 96 hr after cisplatin administration were 6.92 +/- 0.83, 3.46 +/- 0.77 & 6.20 +/- 0.64 (microg/g wet weight of tissue) respectively. Results indicate that 21 day insulin treatment to streptozotocin-diabetic animal reverses protection against cisplatin toxicity. Moreover, insulin treatment increased the susceptibility of streptozotocin-diabetic rats to cisplatin-induced renal toxicity.

Reduced expression of organic cation transporters rOCT1 and rOCT2 in experimental diabetes.

Recent reports have documented a functional deficit of organic cation transport in diabetic rats by an unknown mechanism. This study was designed to test the hypothesis that experimental diabetes decreases expression of organic cation transporters at the basolateral membrane. Streptozotocin-induced diabetic rats were maintained for varying durations after induction of diabetes. A second group of age-matched control rats was maintained in a parallel manner. Kinetic analysis of tetraethylammonium accumulation in freshly isolated proximal tubular cells indicated a significantly lower V(max) value for the diabetics versus controls with no statistical difference in K(m) values between the two groups. Cortex sections were processed by standard procedures for Northern and immunoblot analysis. Protein expression of the organic cation transporters rOCT1 and rOCT2 progressively decreased with increasing duration of diabetes. After 21 days of diabetes, rOCT1 and rOCT2 were maximally reduced by 50 and 70%, respectively. Quantification of mRNA expression revealed that the roct1 transcript remained unchanged, whereas the roct2 transcript was decreased by 50% after 14 days of diabetes. Treatment with insulin prevented the reductions in transporter levels. These results support the hypothesis by demonstrating that experimental diabetes decreased expression of both rOCT1 and rOCT2 protein and also of roct2 mRNA accumulation. On the other hand, roct1 mRNA levels were unaffected by the diabetic state. This suggests that differences in rOCT2 protein may result from transcriptional and/or translational changes, whereas rOCT1 deficits may be due to posttranscriptional alterations.

Cellular and molecular studies on cisplatin-induced apoptotic cell death in rat kidney.

Using morphological and molecular approaches, we characterized cisplatin-induced cell necrosis and apoptosis in rat kidney. Male Sprague-Dawley rats ( n=5 per group) received a single intraperitoneal injection of either cisplatin (5 mg/kg) or saline, and were killed on day 5. Functionally, cisplatin-treated rats developed polyuric acute renal failure. Morphologically, kidneys of cisplatin-treated rats showed overt tubular necrosis associated with apoptosis in the corticomedullary junction. Cell necrosis was segment-specific and was distributed in radial fashion at the corticomedullary junction. The apoptosis was limited to discrete cells in apparently intact tubules in the vicinity of the necrosed tubules. The apoptotic changes were confirmed by TUNEL (TdT-mediated deoxyuridine triphosphate nick-end labeling) and staining for cleaved caspase-3. Analysis of outer medullary tissue for apoptosis-related molecules by RNase protection assay revealed a significant increase in the expression of pro-apoptotic mRNAs (caspases 1, 2, and 8, and Bax) in cisplatin-treated rats. On the other hand, the expression of mRNA for the anti-apoptotic Bcl-2 did not change, resulting in a decrease in relative ratio of Bcl-2/Bax, and thus favoring apoptosis. The above changes were paralleled by a marked increase in caspase-3 precursor, the executioner protease. Furthermore, these pro-apoptotic molecular changes were associated with a 3-fold increase in the activity of JNK1 in the outer medulla, but not in the cortex, of cisplatin-treated rat kidneys, localizing to the site of maximal apoptosis. Upregulation of JNK1 activity in the outer medulla was not accompanied by changes in the activities of ERK or p38 kinase. In conclusion, these data suggest that cisplatin-induced apoptotic cell death in native kidney may be mediated by cooperative activation of the JNK1 pathway and Bax in the outer medulla.

Functional impairment of renal organic cation transport in experimental diabetes.

This study was designed to determine the effect of diabetes on the function of the renal organic cation transport system that mediates the excretion of a wide variety of toxicants and drugs. The experiments compared the ability of renal cortex slices from streptozotocin-induced diabetic and non-diabetic rats to accumulate the model cation, 14C-tetraethylammonium under controlled conditions. Initial experiments demonstrated a progressive decline in tetraethylammonium accumulation with increasing duration of diabetes. The maximal decrease was observed at 21 days after streptozotocin injection. Time-dependent incubations revealed that tetraethylammonium uptake from both diabetic and non-diabetic rats followed a curvilinear pattern expected of an active process. However, at steady state the diabetic-derived slices accumulated a significant 38% less tetraethylammonium versus slices from non-diabetics. Concentration-dependent incubations of tetraethylammonium (0.01-10 mM, 60 min.) demonstrated saturable transport in both diabetic and non-diabetic slices with a significantly decreased capacity of diabetic-derived slices to accumulate tetraethylammonium. Cellular respiration rates in the two groups were not different. Insulin treatment of the diabetic rats prevented the transport decline. While the causative factor of the transport impairment in diabetes is unresolved, this study documents an aspect of diabetic nephropathy that has not been previously reported but which may have important implications for renal excretion of cationic drugs and toxicants. The results also provide a mechanism for the well-documented "protection phenomenon" by which the kidneys of diabetic rats are resistant to nephrotoxicity induced by the chemotherapeutic agent cisplatin.