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Acute kidney injury (AKI) is a major complication of cardiac bypass surgery. We examined whether levels of liver fatty acid-binding protein (L-FABP) can be an early biomarker for ischemic injury by measuring this protein in the urine of 40 pediatric patients prior to and following cardiopulmonary bypass surgery. AKI was defined as a 50% increase in the serum creatinine from baseline, which was normally not seen until 24-72 h after surgery. Enzyme-linked immunosorbent assay analysis showed increased L-FABP levels (factored for creatinine excretion) of about 94- and 45-fold at 4 and 12 h, respectively, following surgery in the 21 patients who developed AKI with western blot analysis, confirming L-FABP identity. Univariate logistic regression analyses showed that both bypass time and urinary L-FABP were significant independent risk indicators for AKI. After excluding bypass time from the model and using a stepwise multivariate logistic regression analysis, urinary L-FABP levels at 4 h after surgery were an independent risk indicator with the area under the receiver-operating characteristic curve 0.810, sensitivity 0.714, and specificity 0.684 for a 24-fold increase in urinary L-FABP. Our study shows that urinary L-FABP levels represent a sensitive and predictive early biomarker of AKI after cardiac surgery.

Previous studies from our laboratory showed that increased fatty acid oxidation by the kidney is cytoprotective during cisplatin (CP)-mediated nephrotoxicity. In this study, we determined the effects of CP and fibrates on peroxisome proliferation and the expression of liver fatty acid-binding protein (L-FABP) in normal mice, and in mice transgenically overexpressing human L-FABP (h-L-FABP). Labeling of peroxisomes demonstrated reduced peroxisomal staining in the proximal tubule of CP-treated mice compared with control mice. There was increased peroxisomal labeling in the proximal tubules of both control and CP-treated mice when either was treated with fibrate; a known peroxisome proliferator-activated receptor-alpha ligand. L-FABP protein expression, not detected in control or CP-treated mice, was significantly increased in the proximal tubules of fibrate-treated mice of either group. In the transgenic mice, CP increased the shedding of h-L-FABP in the urine, which was decreased by fibrate as was the acute renal failure. A cytosolic pattern of h-L-FABP expression was found in the proximal tubules of untreated transgenic mice with a nuclear presence in CP-treated mice. Fibrate pretreatment restored the cytosolic expression pattern in CP-treated mice. Our study shows that fibrate may improve CP-induced acute renal failure due to both peroxisome proliferation and increased L-FABP in the cytosol of the proximal tubule.

Copper 2 II ( 3 , 5 -ditertiarybutylsalicylate ) 4 ( ethanol ) 4 , Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 , was synthesized and characterized for evaluation as an anti-apoptotic superoxide dismutase (SOD)-mimetic in an in vitro 50 μM cis-diamminedichloroplatinum(II), [PtII(NH3)2(Cl)2]-treated kidney proximal tubule epithelial cell (LLC-PK) preparation. Synthesized Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 was characterized by elemental analysis, FTIR spectrophotometry, and X-ray crystallography. The IC50 for SOD-mimetic reactivity of Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 , determined with the xanthine/xanthine oxidase/nitroblue tetrazolium (NBT) system, was found to be 2.69 μM for the binuclear chelate. Pretreatment of LLC-PK cells with 20 μM Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 prevented 50 μM PtII(NH3)2(Cl)2-induced and superoxide-mediated apoptosis. This SOD-mimetic significantly suppressed PtII(NH3)2(Cl)2-induced translocation of pro-apoptotic Bax from the cytosol to the inner mitochondrial membrane, prevented PtII(NH3)2(Cl)2-induced release of cytochrome c from the inner mitochondrial membrane and the appearance of cytochrome c in the cytosol, and prevented conversion of procaspase-3 to active caspase-3. Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 treatment inhibited PtII(NH3)2(Cl)2-mediated tubular cell injury by preventing activation of cellular mechanisms that lead to proximal tubule kidney cell death. Based on these observations, PtII(NH3)2(Cl)2- induced O 2 - -mediated apoptosis can be mechanistically overcome with a small molecular mass SOD-mimetic, Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 . Prior treatment of patients who are to undergo treatment with PtII(NH3)2(Cl)2 for their neoplastic disease with Cu 2 II ( 3 , 5 -DTBS ) 4 ( Eth ) 4 may be beneficial to these patients.

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.

Fibrate prevents cisplatin-induced proximal tubule cell death. Background In previous studies we have shown that cisplatin inhibits peroxisome proliferator-activated receptor-α (PPAR-α) activity and consequently fatty acid oxidation, and these events precede proximal tubule cell death. In addition the use of fibrate class of PPAR-α ligands ameliorate renal function by preventing both inhibition of fatty acid oxidation and proximal tubule cell death. Methods LLC-PK1 cells were treated with cisplatin and apoptosis was established by the presence of nuclear fragmentation and by cell cycle analysis. Proximal tubular cells treated with cisplatin and bezafibrate were subjected to sub cellular fractionation and the presence of Bax, Bcl-2, cytochrome c, and active caspase-3 in the cytosolic and mitochondrial membrane fractions was determined by Western blot analysis. PPAR-α activity was measured by determining luciferase activity after transfection of LLC-PK1 cells with TK-Luc 3× PPAR response elements (PPRE), and the accumulation of nonesterified free fatty acids was measured in lysates obtained from cells treated with cisplatin and bezafibrate. Results Incubation of LLC-PK1 cells with 25 μmol/L cisplatin for 18 hours induced 41.5% apoptosis measured by cell cycle analysis. Cisplatin-induced apoptosis was significantly suppressed by bezafibrate, a fibrate class of PPAR-α ligand. Bezafibrate treatment of LLC-PK1 cells prevented cisplatin-induced translocation of proapoptotic Bax from the cytosol to the mitochondrial fraction, and increased the expression of antiapoptotic molecule Bcl-2. Cisplatin-induced inhibition of PPAR-α activity was accompanied by increased accumulation of nonesterified free fatty acids. Pretreatment with bezafibrate prevented both the inhibition of PPAR-α activity and the accumulation of nonesterified free fatty acids induced by cisplatin. Finally, bezafibrate prevented cisplatin-induced release of cytochrome c from the mitochondria to the cytosol, and the cleavage of procaspase-3 to active caspase-3. Conclusion Bezafibrate treatment inhibits cisplatin-mediated tubular injury by preventing the activation of various cellular mechanisms that lead to proximal tubule cell death. These findings support our previous observations where the use of fibrates represents a novel strategy to ameliorate proximal tubule cell death in cisplatin-induced acute renal failure.

Loss of heterozygosity (LOH) and/or inactivation of tumor suppressor genes are implicated in the initiation and progression of many malignancies, including colorectal cancer. Although accumulating evidence suggests a chemopreventive role for folate in colorectal cancer, regulatory mechanisms are poorly understood. The primary objective of the current investigation was to determine whether folic acid would prevent LOH of the three tumor suppressor genes, deleted in colorectal cancer (DCC), adenomatous polyposis coli (APC) and p53 in macroscopically normal appearing rectal mucosa of patients with adenomatous polyps. In addition, the effect of folic acid on rectal mucosal proliferation was determined. Twenty patients were randomized in a double-blind study to receive either folic acid 5mg once daily or identical placebo tablets for 1 year. Genomic DNA and total protein were extracted from the rectal mucosa at baseline and after 1 year of treatment and analyzed for LOH and protein levels of APC, DCC and p53 genes. In addition, paraffin-embedded mucosal specimens were analyzed for proliferating cell nuclear antigen (PCNA) immunoreactivity, as a measure of cellular proliferative activity. Folate supplementation prevented LOH of DCC gene in five out of five (100%) patients who demonstrated baseline heterozygosity, whereas two out of four (50%) placebo-treated patients with baseline heterozygosity demonstrated allelic loss. Mucosal protein levels of DCC were also reduced in 7 of 10 (70%) placebo-treated patients compared to only 2 of 10 (20%) of patients treated with folate. Levels increased, however, in eight and three patients in the folic acid and placebo groups, respectively (P0.02). Folic acid caused no change in allelic status of either APC or p53 gene. Folate supplementation caused a small, but not statistically significant, 16% reduction in mucosal proliferation, whereas placebo treatment resulted in a 88% (P0.05) increase in this parameter, when compared with the corresponding baseline values. Our results indicate that folic acid prevents an increase in proliferation and arrests LOH of DCC gene and also stabilizes its protein in normal appearing rectal mucosa of patients with colorectal adenomas. Taken together, our data suggest that one of the ways folate may exert its chemopreventive effect is by stabilizing certain tumor suppressor gene(s) and preventing further increases in proliferation.

Aging and gastrointestinal malignancies, including that of the stomach are associated with increased activation of EGF-receptor (EGFR). Although the intracellular events that regulate this process are poorly understood, we hypothesize that loss of ERRP (EGFR-related protein; GenBank accession number AF187818), a recently identified negative regulator of EGFR, that possesses a substantial homology to the ligand binding extracellular domain of EGFR, may contribute to this event. In support of our hypothesis, we have observed that in Fischer-344 rats, whereas aging is associated with increased activation of EGFR in the gastric mucosa, expression of ERRP decreases in this tissue during this period. The latter is accompanied by a concomitant reduction in the amount of TGF-α bound to ERRP. In contrast, the amount of TGF-α bound to EGFR is found to be higher in the gastric mucosa of aged than in young rats. This is accompanied by a concomitant rise in EGFR levels. In the gastric mucosa, EGFR and ERRP are found to be colocalized. Gastric adenocarcinoma in humans, which has been shown to be associated with increased activation of EGFR, shows a substantial reduction in ERRP expression, when compared with benign tissues. We conclude that increased activation of EGFR in the gastric mucosa during aging and carcinogenesis may partly be due to the loss of ERRP.

Introduction and Aims: Epidermal growth factor (EGF) and its receptor (EGFR) play crucial roles in cellular signaling in many malignancies, including pancreatic neoplasia. Attenuation of EGFR signaling has been considered novel strategy for the management of human malignancies in several ongoing clinical trials. We recently isolated a novel negative regulator of EGFR, termed EGF receptor related protein (ERRP), whose expression appears to attenuate EGFR activation. In the current study, the expression of ERRP in normal and neoplastic pancreas was investigated and correlated with the clinicopathologic parameters in pancreatic ductal adenocarcinoma (DA). Methodology: Using rabbit polyclonal antibody that specifically interacts with ERRP, immunohistochemical staining was performed on 45 benign pancreata and 106 cases of DA. The intensity and percentage of cells with cytoplasmic and membranous staining were scored as 0, 1, 2, or 3. A combined score was calculated as intensity x percent/3, and for comparative analysis, the data were arbitrarily divided into three groups

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.

Our previous studies suggest that peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a critical role in regulating fatty acid beta-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARalpha in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARalpha expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia-reperfusion-induced acute kidney injury. Ischemia-reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARalpha activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARalpha ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury.

Copper(2)(II)(3,5-ditertiarybutylsalicylate)(4)(ethanol)(4), Cu(2)(II)(3,5-DTBS)(4)(Eth)(4), was synthesized and characterized for evaluation as an anti-apoptotic superoxide dismutase (SOD)-mimetic in an in vitro 50 microM cis-diamminedichloroplatinum(II), [Pt(II)(NH(3))(2)(Cl)(2)]-treated kidney proximal tubule epithelial cell (LLC-PK) preparation. Synthesized Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) was characterized by elemental analysis, FTIR spectrophotometry, and X-ray crystallography. The IC(50) for SOD-mimetic reactivity of Cu(2)(II)(3,5-DTBS)(4)(Eth)(4), determined with the xanthine/xanthine oxidase/nitroblue tetrazolium (NBT) system, was found to be 2.69 microM for the binuclear chelate. Pretreatment of LLC-PK cells with 20 microM Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) prevented 50 microM Pt(II)(NH(3))(2)(Cl)(2)-induced and superoxide-mediated apoptosis. This SOD-mimetic significantly suppressed Pt(II)(NH(3))(2)(Cl)(2)-induced translocation of pro-apoptotic Bax from the cytosol to the inner mitochondrial membrane, prevented Pt(II)(NH(3))(2)(Cl)(2)-induced release of cytochrome c from the inner mitochondrial membrane and the appearance of cytochrome c in the cytosol, and prevented conversion of procaspase-3 to active caspase-3. Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) treatment inhibited Pt(II)(NH(3))(2)(Cl)(2)-mediated tubular cell injury by preventing activation of cellular mechanisms that lead to proximal tubule kidney cell death. Based on these observations, Pt(II)(NH(3))(2)(Cl)(2)- induced O(2)(-)-mediated apoptosis can be mechanistically overcome with a small molecular mass SOD-mimetic, Cu(2)(II)(3,5-DTBS)(4)(Eth)(4). Prior treatment of patients who are to undergo treatment with Pt(II)(NH(3))(2)(Cl)(2) for their neoplastic disease with Cu(2)(II)(3,5-DTBS)(4)(Eth)(4) may be beneficial to these patients.

Although accumulating evidence suggests a chemopreventive role for folic acid (FA) in colorectal carcinogenesis, the underlying mechanisms are largely unknown. Previously, we reported that supplemental FA inhibits the expression and activation of epidermal growth factor receptor (EGFR) in colon cancer cell lines. To determine the mechanism(s) by which FA affects EGFR function, we have examined whether and to what extent supplemental FA or its metabolites 5-methyltetrahydrofolate (MTF), dihydrofolate (DF), and tetrahydrofolate (TF) will modulate basal and serum-induced activation of the EGFR promoter in the HCT-116 colon cancer cell line. HCT-116 cells were preincubated with or without (control) FA or one of its metabolites (10 μg/ml) for 48 h, transfected with the EGFR promoter luciferase reporter construct, and incubated for 48 h with FA, DF, TF, or 5-MTF in the absence or presence of 10% FBS. Supplemental FA as well as its metabolites markedly inhibited EGFR promoter activity and its methylation status. Exposure of the cells to 10% FBS caused a marked stimulation of EGFR promoter activity and its expression, both of which were greatly abrogated by supplemental FA and 5-MTF. In contrast, serum-induced activation of c- fos promoter activity was unaffected by 5-MTF. The 5-MTF-induced inhibition of serum-mediated stimulation of EGFR promoter activity and EGFR expression was reversed when methylation was inhibited by 5-aza-2′-deoxycytidine. Our data suggest that FA and its metabolite 5-MTF inhibit EGFR promoter activity in colon cancer cells by enhancing methylation. This could partly be responsible for FA-mediated inhibition of growth-related processes in colorectal neoplasia.

Aging and gastrointestinal malignancies, including that of the stomach are associated with increased activation of EGF-receptor (EGFR). Although the intracellular events that regulate this process are poorly understood, we hypothesize that loss of ERRP (EGFR-related protein; GenBank accession number AF187818), a recently identified negative regulator of EGFR, that possesses a substantial homology to the ligand binding extracellular domain of EGFR, may contribute to this event. In support of our hypothesis, we have observed that in Fischer-344 rats, whereas aging is associated with increased activation of EGFR in the gastric mucosa, expression of ERRP decreases inthis tissue during this period. The latter is accompanied by a concomitant reduction in the amount of TGF-alpha bound to ERRP. In contrast, the amount of TGF-alpha bound to EGFR is found to be higher in the gastric mucosa of aged than in young rats. This is accompanied by a concomitant rise in EGFR levels. In the gastric mucosa, EGFR and ERRP are found to be colocalized. Gastric adenocarcinoma in humans, which has been shown to be associated with increased activation of EGFR, shows a substantial reduction in ERRP expression, when compared with benign tissues. We conclude that increased activation of EGFR in the gastric mucosa during aging and carcinogenesis may partly be due to the loss of ERRP.

Background & Aims: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor–related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor-related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor–related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor-related protein expression is altered in colorectal cancer. Methods: A 55-kilodalton epidermal growth factor receptor–related protein fusion protein with V5 and His tags was generated in a drosophila expression system and subsequently purified by a His antibody affinity column. Rabbit polyclonal antibodies against epidermal growth factor receptor-related protein were used to examine the expression of epidermal growth factor receptor–related protein. Results: Epidermal growth factor receptor-related protein expression was found to be high in benign human colonic epithelium but low in adenocarcinoma. Exposure of the colon cancer cell lines HCT-116 and Caco-2 to purified recombinant epidermal growth factor receptor–related protein caused a marked inhibition of proliferation, as well as attenuation of basal and ligand-induced stimulation of epidermal growth factor receptor phosphorylation. Epidermal growth factor receptor-related protein-induced inhibition of proliferation of colon cancer cells was prevented by epidermal growth factor receptor–related protein antibodies. Reduced epidermal growth factor receptor phosphorylation was partly due to sequestration of epidermal growth factor receptor ligands by epidermal growth factor receptor-related protein, resulting in the formation of inactive heterodimers with epidermal growth factor receptor. Intratumoral or subcutaneous (away from the tumor site) injections of purified epidermal growth factor receptor–related protein caused regression of palpable colon cancer xenograft tumors in some severely compromised immunodeficient mice and arrested tumor growth in others. Conclusions: We propose that epidermal growth factor receptor-related protein inhibits cellular growth by attenuating epidermal growth factor receptor signaling processes and is an effective therapeutic agent for colorectal cancer.