Mechanisms of progression of chronic kidney disease (CKD), a major health care burden, are poorly understood. EGFR stimulates CKD progression, but the molecular networks that mediate its biological effects remain unknown. We recently showed that the severity of renal lesions after nephron reduction varied substantially among mouse strains and required activation of EGFR. Here, we utilized two mouse strains that react differently to nephron reduction -FVB/N mice, which develop severe renal lesions, and B6D2F1 mice, which are resistant to early deterioration -coupled with genome-wide expression to elucidate the molecular nature of CKD progression. Our results showed that lipocalin 2 (Lcn2, also known as neutrophil gelatinase-associated lipocalin [NGAL]), the most highly upregulated gene in the FVB/N strain, was not simply a marker of renal lesions, but an active player in disease progression. In fact, the severity of renal lesions was dramatically reduced in Lcn2 -/-mice. We discovered that Lcn2 expression increased upon EGFR activation and that Lcn2 mediated its mitogenic effect during renal deterioration. EGFR inhibition prevented Lcn2 upregulation and lesion development in mice expressing a dominant negative EGFR isoform, and hypoxia-inducible factor 1α (Hif-1α) was crucially required for EGFR-induced Lcn2 overexpression. Consistent with this, cell proliferation was dramatically reduced in Lcn2 -/-mice. These data are relevant to human CKD, as we found that LCN2 was increased particularly in patients who rapidly progressed to end-stage renal failure. Together our results uncover what we believe to be a novel function for Lcn2 and a critical pathway leading to progressive renal failure and cystogenesis.

Tubular function is altered in chronic renal failure (CRF). Whether drug secretion by renal tubules is modified in CRF is questioned because of frequent accumulation of various toxins in CRF. This function mainly involves ATP-dependent drug transporters, particularly P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) 2, both present in apical membrane of epithelial cells. The present study was aimed at determining the changes in P-gp and MRP2 expression induced by experimental CRF in kidney and liver. The relationship between MRP2 and glutathione metabolism changes was examined because MRP2 transports GSSG and glutathione conjugates. Rats underwent either 80% subtotal nephrectomy (Nx) or sham operation, and determinations were performed 3 and 6 wk later. CRF induced a 70--200% rise in protein and mRNA expression of MRP2 after 3 and 6 wk post-Nx in remnant kidney and after 6 wk in liver. However, P-gp expression was unchanged by CRF. Relative to whole kidney mass, total MRP2 levels decreased by only 27% in Nx rats whereas total P-gp levels were reduced by 60%. Renal GSSG and total glutathione levels were increased by 30% in Nx rats, but glutathione-S-transferase (GST) activity was normal; liver GSSG levels and GST activity were reduced in Nx rats. In conclusion, CRF resulted in specific overexpression of MRP2 in kidney and liver. This could be an adaptative response to some elevated circulating toxins. The later MRP2 induction and different glutathione changes in liver compared with kidney suggest different mechanisms for MRP2 induction and/or action in these two tissues.

Abstract-Extracellular adenosine production by the GPI-anchored Ecto-5Ј-Nucleotidase (Ecto-5Ј-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5ЈNu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5Ј-Nu stimulation by HRIs, Ecto-5Ј-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5Ј-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5Ј-Nu and decreased endocytosis of Ecto-5Ј-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5Ј-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5Ј-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5Ј-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5Ј-Nu endocytosis through a decrease of Rho-GTPases isoprenylation. This benefit is already apparent at the early step of endothelial dysfunction, which precedes the development of atherosclerosis. 4,5 Some of the effects of these cholesterol-lowering drugs, particularly those observed in short-term exposure, could be supported by mechanisms independent of lowering of plasma LDL concentration. 1,6 Induction of adenosine production in the extracellular space by the GPI-anchored Ecto-5Ј-Nu could be one of these mechanisms. Indeed, the production of adenosine by Ecto-5Ј-Nu is implicated in the regulation of endothelial functions 7-9 and in defense against hypoxia. 9 -12 Ecto-5Ј-Nu activity is increased by tissular hypoxia, notably in cardiomyocytes and endothelial cells. 13,14 The enzyme has been implicated in myocardial preconditioning, 13,15 and it was recently shown that HRIs potentiate the stimulation of Ecto-5Ј-Nu activity induced by ischemia in cardiomyocytes. 16 However, it is not known whether HRIs also enhance Ecto-5Ј-Nu activity in endothelium, a main site of adenosine production, 17 and the mechanisms whereby HRIs enhance Ecto-5Ј-Nu have not been studied. Some hypotheses can be put forward. First, it has been shown that HRIs can modulate the activity of enzymes, such as endothelial nitric oxide synthase, at the transcriptional level. 6 On the other hand, HRIs can induce posttranslational events, which influence cell surface expression of proteins....

The mechanisms of progression of chronic kidney disease (CKD) are poorly understood. Epidemiologic studies suggest a strong genetic component, but the genes that contribute to the onset and progression of CKD are largely unknown. Here, we applied an experimental model of CKD (75% excision of total renal mass) to six different strains of mice and found that only the FVB/N strain developed renal lesions. We performed a genome-scan analysis in mice generated by back-crossing resistant and sensitive strains; we identified a major susceptibility locus (Ckdp1) on chromosome 6, which corresponds to regions on human chromosome 2 and 3 that link with CKD progression. In silico analysis revealed that the locus includes the gene encoding the EGF receptor (EGFR) ligand TGF-␣. TGF-␣ protein levels markedly increased after nephron reduction exclusively in FVB/N mice, and this increase preceded the development of renal lesions. Furthermore, pharmacologic inhibition of EGFR prevented the development of renal lesions in the sensitive FVB/N strain. These data suggest that variable TGF-␣ expression may explain, in part, the genetic susceptibility to CKD progression. EGFR inhibition may be a therapeutic strategy to counteract the genetic predisposition to CKD. Human chronic kidney diseases (CKD), regardless of their etiology, are characterized by progressive destruction of the renal parenchyma and loss of functional nephrons, leading to ESRD. Approximately 13% of adults suffer from CKD in industrialized countries and the incidence of ESRD increases by 6% to 8% per year. Therefore, understanding the pathophysiology of CKD is a key challenge for public health.The mechanisms of CKD progression are poorly understood. Although clinical studies point to the important role of environmental factors in the biologic processes leading to renal deterioration, epidemiologic studies have underscored the importance of genetic components. Indeed, it has been observed that the evolution of CKD varies considerably among individual patients exposed to the same risk factors. Only a proportion of patients with diabetes or hypertension develop renal failure, and this occurs independently of glycemic control or hypertension. 1,2 However, the propensity to develop ESRD differs among ethnic groups 3-7 and it shows familial clustering. [7][8][9][10] Similarly, the rate of progression of primary hereditary kidney diseases can vary among members of the same family, [11][12][13] suggesting that genes unrelated to the disease