STAT3 attenuates EGFR-mediated ERK activation and cell survival during oxidant stress in mouse proximal tubular cells.

We have shown that renal epithelial cell survival depends on the sustained activation of the extracellular signal-regulated protein kinase (ERK) and lack of this activation was associated with death during oxidative stress. ERK is activated via the canonical epidermal growth factor receptor (EGFR)-Ras-MEK pathway, which could be attenuated by oxidants. We now show that the failure to activate ERK in a sustained manner during severe oxidative stress is owing to the activation of the signal transducer and activator of transcription-3 (STAT3) rather than the failure to activate the EGFR. Tyrosine phosphorylation of the EGFR and STAT3 was studied in hydrogen peroxide (H(2)O(2))-treated mouse proximal tubule (TKPTS) cells or in mouse kidney after ischemia/reperfusion (I/R) injury by Western blotting. STAT3 activation was inhibited by either pharmacologically (AG490) through its upstream janus kinase (JAK2) or by a dominant-negative STAT3 adenovirus. EGFR was inhibited by AG1478. Survival was determined by fluorescence-activated cell sorter analysis and trypan blue exclusion. We found that the EGFR was phosphorylated on its major autophosphorylation site (Tyr1173) regardless of the H(2)O(2) dose. On the other hand, both I/R and severe oxidative stress - but not moderate stress - increased tyrosine phosphorylation of STAT3 in an EGFR and JAK2-dependent manner. Inhibition of JAK2 or STAT3 lead to increased ERK activation and survival of TKPTS cells during severe oxidative stress. Our data suggest a role of tyrosine-phosphorylated STAT3 in the suppression of ERK activation. These data suggest that the STAT3 pathway might represent a new target for improved survival of proximal tubule cells exposed to severe oxidant injury.

Metabolomic study of cisplatin-induced nephrotoxicity.

We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARalpha ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARalpha ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARalpha ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity.

Positive effect of the induction of p21WAF1/CIP1 on the course of ischemic acute renal failure.

BACKGROUND: The p21 protein is found in the nucleus of most cells where it modulates cell cycle activity. At low levels, p21 stabilizes interactions between D cyclins and their cyclin-dependent kinases (cdks), but at high levels after induction by several different stress pathways, it causes cell cycle arrest. The p21 mRNA is induced in murine kidney after several types of acute renal failure, including cisplatin administration, ischemia-reperfusion, and ureteral obstruction. We reported that after cisplatin injection, mice with a p21 gene deletion developed much more severe renal damage than wild-type mice. To dissociate the effects of cisplatin-induced DNA damage and subsequent initiation of DNA damage-dependent cell death pathways from effects of acute renal failure, we have now examined mice after ischemia-reperfusion, a model of renal failure not associated with genotoxin-induced DNA damage early after the injury. METHODS: Wild-type and p21(-/-) mice were made ischemic by clamping both renal hila for 30 or 50 minutes. At various times after reflow, mortality and parameters of renal function and morphology were quantified. Also, the nuclear proteins p21 and proliferating cell nuclear antigen (PCNA) were localized in kidney sections by immunohistochemistry. RESULTS: Kidney function was more impaired and mortality increased significantly in p21(-/-) mice as compared with p21(+/+) mice. We found more cell cycle activity, indicated by increased number of mitotic cells and nuclear PCNA-positive cells, in kidney of p21(-/-) mice. CONCLUSIONS: In this study, p21(-/-) mice were more susceptible to ischemia-induced acute renal failure, with similarly elevated levels of parameters of cell cycle activity. We propose that the increased and inappropriate cell cycle activity in kidney cells is responsible for the increased kidney impairment and mortality.

The lack of a functional p21(WAF1/CIP1) gene ameliorates progression to chronic renal failure.

Partial renal ablation leads to progressive renal insufficiency and is a model of chronic renal failure from diverse causes. We find that mice develop functional and morphologic characteristics of chronic renal failure after partial renal ablation, including glomerular sclerosis, systemic hypertension, and reduced glomerular filtration. However, we now report that littermates with a homozygous deletion of the gene for the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1), do not develop chronic renal failure after ablation. The markedly different reactions of the p21(+/+) and p21(-/-) animals was not because of differences in glomerular number or degree of renal growth but rather because of the presence or absence of a normal p21 gene. Although the reaction to the stress of renal ablation is both hyperplastic and hypertrophic in the presence of a functional p21 gene, it would appear that the absence of the p21 gene may induce a more hyperplastic reaction because proliferating-cell nuclear antigen expression, a marker of cell-cycle progression, in the renal epithelium of the remnant kidney was more than five times greater in the p21(-/-) mice than in the p21(+/+) animals. Because p21 is a potent inhibitor of the cell cycle, we speculate that p21 regulates the balance between hyperplasia and hypertrophy after renal ablation. We propose that this change in response inhibits the development of chronic renal failure. These studies suggest that controlling p21 function may ameliorate or even prevent progressive end-stage renal disease.

MAPK activation determines renal epithelial cell survival during oxidative injury.

Ischemia/reperfusion (I/R) injury induces both functional and morphological changes in the kidney. Necrosis, predominantly of the proximal tubule (PT), is the hallmark of this model of renal injury, whereas cells of the distal nephron survive, apparently intact. We examined whether differences in cellular outcome of the various regions of the nephron may be due to segmental variation in the activation of the mitogen-activated protein kinases (MAPKs) in response to I/R injury. Whereas c-Jun N-terminal kinase (JNK) is activated in both the cortex and inner stripe of the outer medulla, the extracellular regulated kinase (ERK) pathway is activated only in the inner stripe in which thick ascending limb (TAL) cells predominate. These studies are consistent with the notion that ERK activation is essential for survival. To test this hypothesis directly, we studied an in vitro system in which manipulation of these pathways and their effects on cellular survival could be examined. Oxidant injury was induced in mouse PT and TAL cells in culture by the catabolism of hypoxanthine by xanthine oxidase. PT cells were found to be more sensitive than TAL cells to oxidative stress as assessed by cell counting, light microscopy, propidium iodide uptake, and fluorescence-activated cell sorting (FACS) analysis. Immunoprecipitation/kinase analysis revealed that JNK activation occurred in both cell types, whereas ERK activation occurred only in TAL cells. We then examined the effect of PD-098059, a MAP kinase kinase (MEK)-1 inhibitor of the ERK pathway, on PT and TAL survival. In TAL cells, ERK inhibition reduced cell survival nearly fourfold (P < 0.001) after oxidant exposure. In PT cells, activation of the ERK pathway by insulin-like growth factor I (IGF-I) increased survival by threefold (P < 0.001), and this IGF-I-enhanced cell survival was inhibited by PD-098059. These results indicate that cell survival in the kidney after ischemia may be dependent on ERK activation, suggesting that this pathway may be a target for therapeutic treatment in I/R injury.

Lessons learned from ischemic and cisplatin-induced nephrotoxicity in animals.

Following ischemic or nephrotoxic injury, the regenerating kidney assumes an earlier developmental stage and a less mature phenotype. Recovery involves the activation of a group of genes, including protooncogenes and growth factor genes that initiate and sustain cell growth. Inflammation also plays an important role in the recovery process as several of the changes in gene expression implicate the participation of the inflammatory cascade. Many of the changes in gene expression may eventually be reflected in the urine of the damaged kidney. By exploiting these changes in urine composition as a consequence of injury it should be possible to detect evidence of biologic effects of exposure and may yield predictions of eventual risk of serious damage to kidney.

Cell cycle events during renal injury.

Recovery from ischemic and nephrotoxic acute renal failure requires replacement of damaged tubule cells in order to restore the morphological and functional integrity of the renal epithelium. A more thorough understanding of the renal stress response and its molecular interactions with the cell cycle machinery will be important areas of biomarker research in nephrotoxicity. Many of these molecular targets should be detectable hopefully without resorting the invasive techniques.

Expression of the polymeric immunoglobulin receptor and excretion of secretory IgA in the postischemic kidney.

The humoral mucosal immune response of the kidney involves the transport of secretory IgA (S-IgA) through renal epithelial cells by the polymeric immunoglobulin receptor (pIgR). The pIgR is cleaved and released as free secretory component (FSC) or attached to IgA (S-IgA). We examined the effects of an ischemic model of acute renal failure (ARF) on the expression of pIgR and the secretion of FSC and S-IgA in the urine. Kidney pIgR mRNA levels decreased in ischemic animals by 55% at 4 h and by 85% at 72 h compared with controls. pIgR protein expression in the medullary thick ascending limb (TAL) decreased within 24 h and was nearly undetectable by 72 h. Urinary S-IgA and FSC concentrations decreased by 60% between days 3 and 6. pIgR mRNA and pIgR protein in the kidney returned to approximately 90% of control levels and urinary FSC and S-IgA concentrations returned to approximately 55% of control levels by day 7. We demonstrate that ischemic ARF decreases renal mucosal S-IgA transport in vivo and may contribute to the increased incidence of urinary tract infections.

Regulation of the polymeric immunoglobulin receptor by water intake and vasopressin in the rat kidney.

The polymeric immunoglobulin receptor (pIgR) transports polymeric immunoglobulins (IgA) from the basolateral to the apical surface of epithelial cells. At the apical surface, its amino-terminal domain, termed secretory component (SC), is proteolytically cleaved and released either unbound (free SC) or bound to IgA. We examined the effects of changes in water balance and vasopressin on the production and secretion of the pIgR in the rat kidney in vivo. Water deprivation induced a 2.7-fold increase in the pIgR mRNA and a 2.2-fold increase in intracellular pIgR protein compared with water-loaded animals. Physiological doses of desmopressin reproduced the effects of water deprivation on mRNA and intracellular protein levels, suggesting that pIgR expression may be regulated by a vasopressin-coupled mechanism. Secretion of free SC and secretory IgA in the urine, however, correlated directly with water intake and urine flow. These results suggest that hydration status and vasopressin may affect the mucosal immunity of the kidney by regulating at different steps the epithelial cell production and secretion of the polymeric immunoglobulin transporter/ secretory component.

Induction of p21WAF1/CIP1/SDI1 in kidney tubule cells affects the course of cisplatin-induced acute renal failure.

The p21 protein is found in the nucleus of most cells at low levels and is induced to elevated levels after DNA damage, causing cell-cycle arrest. We have reported that p21 mRNA is rapidly induced to high levels in murine kidney after acute renal failure. The function(s) in the kidney of p21 induction in cisplatin-induced acute renal failure was studied with mice that are homozygous for a p21 gene deletion. After drug administration, as compared with their wild-type littermates, p21(-/-) mice display a more rapid onset of the physiologic signs of acute renal failure, develop more severe morphologic damage, and have a higher mortality. Therefore, the induction of p21 after cisplatin administration is a protective event for kidney cells. Using both bromodeoxyuridine incorporation and nuclear proliferating cell nuclear antigen detection, we found that cisplatin administration caused kidney cells to start entering the cell-cycle. However, cell-cycle progression is inhibited in wild-type mice, whereas kidney cells in the p21(-/-) mice progress into S-phase. We propose that p21 protects kidneys damaged by cisplatin by preventing DNA-damaged cells from entering the cell-cycle, which would otherwise result in death from either apoptosis or necrosis.

The p53-independent activation of transcription of p21 WAF1/CIP1/SDI1 after acute renal failure.

In three different models of acute renal failure (ischemia, ureteral obstruction, and cisplatin administration), the p21WAF1/CIP1/SDI1 gene, the protein product of which is associated with cell-cycle interruption, terminal differentiation, and cellular senescence, was activated in murine kidney cells. This transcription was localized in kidney only to cells of thick ascending limbs and distal convoluted tubules. Although the tumor suppressor protein, p53, can trans-activate the p21 gene in some cells, increased levels of nuclear p53 protein could be demonstrated only in the cisplatin model of acute renal failure. High levels of p21 mRNA were induced in kidney of p53 "null" mice, demonstrating that p21 gene activation was through a p53-independent pathway. We also present evidence that, in the cisplatin model, both p53-independent and p53-dependent induction of p21 mRNA occur simultaneously. We conclude that p21 gene activation is a general response to renal injury and could be a key determinant of cell fate in the cell in which it is expressed.

Regulation of transcription by the rat EGF gene promoter in normal and ischemic murine kidney cells.

Epidermal growth factor (EGF) is a small polypeptide belonging to a class of molecules that can mediate cell growth, differentiation, and acute phase responses. EGF mRNA is transcribed primarily in cells of the salivary gland and the kidney. We have found that the tissue and cellular specificities of EGF gene expression are controlled by a promoter region located upstream from the start of mRNA transcription. In a variety of experimentally induced forms of acute renal failure, the mRNA and protein levels for kidney EGF fall markedly and remain low for a prolonged period. This decrease was determined by nuclear runoff transcription to be a consequence of diminished transcription of the EGF gene rather than increased instability of the mRNA. Using transgenic mice, we found the effect of renal ischemia on EGF mRNA transcription to be a result of a disease-mediated interruption of the function of this upstream promoter region, presumably from alterations in the activity of one or more cellular trans-acting factors.

Effect of aspirin on experimental diabetic nephropathy.

The decline in glomerular filtration rate (GFR) in long-term diabetes in humans and animals in preceded by a period of hyperfiltration that may be responsible for it. The mediators of the increase in glomerular filtration are unknown, but recent studies suggest a prominent role for prostaglandins. To test the hypothesis that prostaglandins mediate early hyperfiltration and contribute to the progression of diabetic nephropathy, we examined the effects of long-term aspirin (ASA) treatment on whole kidney GFR and renal prostaglandin E2 (PGE2) synthesis in control and diabetic rats 8 days and 16 weeks after streptozocin administration. The rats were divided into four groups, control, control with ASA (C/ASA), diabetic, and diabetic with ASA (D/ASA). We found that 8 days after streptozocin treatment, PGE2 synthesis and GFR were increased in diabetic rats. ASA treatment inhibited renal prostaglandin synthesis and prevented the GFR increase. ASA given to control rats reduced PGE2 synthesis without changing GFR. In the 16-week study diabetic rats had lower GFR and increased renal PGE2 synthesis. Diabetic rats also had thickened glomerular basement membrane compared with control rats. By contrast GFR did not fall and thickening of the glomerular basement membrane did not occur in diabetic rats receiving ASA. ASA had no effect on GFR or glomerular basement membrane in normal rats but decreased renal PGE2 synthesis. The data demonstrate that aspirin prevents early hyperfiltration and prevents the fall in GFR and glomerular basement membrane thickening that occurs over time in diabetic rats. Inhibition of PGE2 synthesis by aspirin, or some other effect of aspirin, may be responsible for the protection observed.

The role of prostaglandins in early polyuria induced by cisplatin in the rat.

To assess a possible role of prostaglandins in the early phase of cisplatin-induced abnormalities in renal concentrating ability, three groups of rats were studied. In a first group we measured prostaglandin production from renal medullary microsomes isolated from rats sacrificed at different time periods after cisplatin, 5 mg/kg alone (PB/CP) or cisplatin plus aspirin, 300 mg/kg p.o., 1 h before cisplatin and daily (ASA/CP). In a second group of rats, balance studies were performed in PB/CP and ASA/CP animals for 4 days after cisplatin to determine the effect of such treatment on the renal excretion of solute and water. In another group of rats inulin clearance was measured in PB/CP and ASA/CP animals 4 days after such treatment. The rats received aspirin or phosphate buffer alone (50 mg/ml sodium phosphate, pH 8) to determine the effect of such treatment on prostaglandin production and renal function. In PB/CP Uosm fell and prostaglandin synthesis increased on days 1-3. Prostaglandin synthesis returned to baseline values by day 4, but Uosm remained low. Inulin clearance was low 4 days after cisplatin. In ASA/CP rats prostaglandin synthesis did not increase and the early polyuria was ameliorated. Aspirin did not prevent the later polyuria. Inulin clearance in the ASA/CP group was markedly reduced to levels below those observed with cisplatin alone. These data demonstrate that elevated rates of prostaglandin synthesis occur early in the course of cisplatin-induced renal failure and suggest that prostaglandins may play a role in the early cisplatin-induced concentrating defect.

Inhibition of amino acid transport by cis-diamminedichloroplatinum(II) derivatives in L1210 murine leukemia cells.

The uptake of cis-diamminedichloroplatinum(II) (cisplatin) has been studied in the L1210 murine lymphoid leukemia cell line. Labeled cisplatin and its aquated derivatives were resolved by high-performance liquid chromatography on a strong cationic exchange column. After 10 min of incubation of cisplatin with the cells, the major portion of the non-protein-bound platinum was in the form of cisplatin. However, a portion of this platinum was converted with time to a derivative which coeluted with the monoaquo derivative of cisplatin. With the appearance of this derivative, there was a concomitant inhibition of sodium-dependent amino acid transport as measured by the uptake of aminoisobutyric acid and methionine. Furthermore, the exposure of L1210 cells to a preparation of predominantly aquated product(s) of cisplatin inhibited amino acid uptake following a brief (2-min) incubation, whereas measurable inhibition of amino acid uptake by cisplatin required a longer preincubation period. This inhibition of aminoisobutyric acid and methionine was dependent on the concentration of platinum. Aminoisobutyric acid and methionine were shown to be concentrated in L1210 cells in the presence of sodium ions, and competition experiments suggest similar uptake systems. Since L1210 cells are methionine-auxotrophic leukemic cells, inhibition of essential amino acid transport by cisplatin may be a mechanism of cytotoxic action.