Loss of Klotho and activation of the renin-angiotensin system (RAS) are common pathological findings in chronic kidney diseases. However, whether these two events are intricately connected is poorly understood. We hypothesized that Klotho might protect kidneys by targeted inhibition of RAS activation in diseased kidneys. To test this hypothesis, mouse models of remnant kidney, as well as adriamycin nephropathy and unilateral ureteral obstruction, were utilized. At 6 weeks after 5/6 nephrectomy, kidney injury was evident, characterized by elevated albuminuria and serum creatinine levels, and excessive deposition of interstitial matrix proteins. These lesions were accompanied by loss of renal Klotho expression, up-regulation of RAS components, and development of hypertension. In vivo expression of exogenous Klotho through hydrodynamic-based gene delivery abolished the induction of multiple RAS proteins, including angiotensinogen, renin, angiotensin-converting enzyme, and angiotensin II type 1 receptor, and normalized blood pressure. Klotho also inhibited b-catenin activation and ameliorated renal fibrotic lesions. Similar results were obtained in mouse models of adriamycin and obstructive nephropathy. In cultured kidney tubular epithelial cells, Klotho dose-dependently blocked Wnt1-triggered RAS activation. Taken together, these results demonstrate that Klotho exerts its renal protection by targeted inhibition of RAS, a pathogenic pathway known to play a key role in the evolution and progression of hypertension and chronic kidney disorders. Chronic kidney disease (CKD) is increasingly recognized as a major public health problem, because it affects about 10% to 13% of the adult population worldwide.1e3 Among many risk factors for developing CKD, aging is an independent and strong predictor for progressive renal insufficiency. 4 The elderly population also tends to develop hypertension and cardiovascular disease, characteristic features consistent with the activation of the renin-angiotensin system (RAS). 5,6 These observations suggest that aging, CKD, and RAS activation might be intimately linked. However, the molecular details behind these connections are not fully understood.RAS consists of several key components, including angiotensinogen (AGT), renin, angiotensin-converting enzyme (ACE), and angiotensin II type I and type II receptors (AT1 and AT2, respectively).7e9 Two main enzymes in this system, renin and ACE, lead to the formation of angiotensin II, the principal active peptide of RAS, which mediates both blood pressureedependent and eindependent kidney damage in CKD. Several factors can induce RAS activation, such as reactive oxygen species, hyperglycemia, and albumin. 10,11 We recently found that all RAS genes contain T cell factor/ lymphoid enhancer-binding factor binding sites in their promoter regions and are directly regulated by canonical Wnt/ b-catenin signaling.12 These results have established that bcatenin is a master regulator controlling the expression of all RAS components in the kidneys.

AKI is a devastating condition with high morbidity and mortality. The pathologic features of AKI are characterized by tubular injury, inflammation, and vascular impairment. Whether fibroblasts in the renal interstitium have a role in the pathogenesis of AKI is unknown. In this study, we investigated the role of fibroblast-specific -catenin signaling in dictating the outcome of AKI, using conditional knockout mice in which-catenin was specifically ablated in fibroblasts (Gli1--cat-/-). After ischemia-reperfusion injury (IRI), Gli1--cat-/- mice had lower serum creatinine levels and less morphologic injury than Gli1--cat+/+ littermate controls. Moreover, we detected fewer apoptotic cells, as well as decreased cytochrome C release; reduced expression of Bax, FasL, and p53; and increased phosphorylation of Akt, in the Gli1--cat-/- kidneys. Gli1--cat-/- kidneys also exhibited upregulated expression of proliferating cell nuclear antigen and Ki-67, which are markers of cell proliferation. Furthermore, Gli1--cat-/- kidneys displayed suppressed NF-κB signaling and cytokine expression and reduced infiltration of inflammatory cells. Notably, loss of -catenin in fibroblasts induced renal expression of hepatocyte growth factor (HGF) and augmented the tyrosine phosphorylation of c-met receptor after IRI., treatment with Wnt ligands or ectopic expression of active -catenin inhibited HGF mRNA and protein expression and repressed HGF promoter activity. Collectively, these results suggest that fibroblast-specific-catenin signaling can control tubular injury and repair in AKI by modulating HGF expression. Our studies uncover a previously unrecognized role for interstitial fibroblasts in the pathogenesis of AKI.

In mouse model of adriamycin nephropathy (ADR), CXCR4 expression was significantly induced in podocytes as early as 3 days. This was accompanied by an increased upregulation of oxidative stress in podocyte, as demonstrated by malondialdehyde assay, nitrotyrosine staining and secretion of 8-hydroxy-2'-deoxyguanosine in urine, and induction of NOX2 and NOX4, major subunits of NADPH oxidase. CXCR4 was also induced in human kidney biopsies with proteinuric kidney diseases and colocalized with advanced oxidation protein products (AOPPs), an established oxidative stress trigger. Using cultured podocytes and mouse model, we found that AOPPs induced significant loss of podocyte marker Wilms tumor 1 (WT1), nephrin, and podocalyxin, accompanied by upregulation of desmin both in vitro and in vivo. Furthermore, AOPPs worsened proteinuria and aggravated glomerulosclerosis in ADR. These effects were associated with marked activation of SDF-1α/CXCR4 axis in podocytes. Administration of AMD3100, a specific inhibitor of CXCR4, reduced proteinuria and ameliorated podocyte dysfunction and renal fibrosis triggered by AOPPs in mice. In glomerular miniorgan culture, AOPPs also induced CXCR4 expression and downregulated nephrin and WT1. Innovation and Conclusion: These results suggest that chemokine receptor CXCR4 plays a crucial role in mediating oxidative stress-induced podocyte injury, proteinuria, and renal fibrosis. CXCR4 could be a new target for mitigating podocyte injury, proteinuria, and glomerular sclerosis in proteinuric chronic kidney disease. Antioxid. Redox Signal. 27, 345-362.