The role of Cullin 3- mediated protein ubiquitination and degradation in the initiation of kidney fibrosis

• Applicant: Privatdozent Dr. Turgay Saritas
• Subject Area: Nephrology
• Term: from 2016 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 332853055

Chronic kidney disease (CKD) is characterized by gradual loss of kidney function over time, and may eventually lead to kidney failure, which requires dialysis or a kidney transplant to maintain life. CKD affects one in ten adults in the general population worldwide, and is primarily caused by hypertension or diabetes. Fibrosis is the final pathway of virtually all chronic injury to the kidney. Inhibiting fibrosis therefore represents a logical strategy to inhibit progression of CKD to its conclusion. However, the initiating causes are poorly understood and importantly, no specific therapy exists.Cullins play critical roles in regulating a variety of biological processes, including the cell cycle, tumorigenesis, and fibrosis. Cullin 3 (Cul3) belongs to the Cullin-RING ligase (CRL) family, which adds ubiquitin moieties to proteins, thus controlling expression level of proteins by signaling it for degradation. David Ellison and Jim McCormick recently reported that kidney tubule-specific Cul3 deletion in adult mice (KS-Cul3-/-) led to progressive loss of kidney function within weeks, implying an essential physiological role of Cul3 in the kidney. On a collaborative basis, I observed in these mice well-accepted features of progressive tubulointerstitial fibrosis, such as increased extracellular matrix accumulation, apoptosis, cell proliferation and T-cell infiltration. However, it is unclear how Cul3 mediates its anti-fibrotic effects. So far, we only know that Cul3 disruption is associated with increased expression of the major cell cycle regulator cyclin E. The aim of this proposal is to investigate the role of Cul3 in the pathogenesis of renal fibrosis and CKD. In the labs of David Ellison (M.D., Professor of Medicine) and Jim McCormick (Ph.D., Assistant Professor of Medicine), both principal investigators at Oregon Health and Science University, USA, I will characterize the time-course of kidney fibrosis in KS-Cul3-/- mice. I will test if KS-Cul3-/- mice could serve as new tubulointerstitial fibrosis model since other mouse models are imperfect. In order to determine the maladaptive processes in vivo, this proposal will focus on the cell cycle, release of cytokines and reactive oxygen species, activation of myofibroblasts, and production of tubulointerstitial collagen upon renal Cul3 deletion. In addition to the KS-Cul3-/- model, I will also analyze the Cul3 ubiquitination pathway in three different mouse models of kidney fibrosis and in kidney tissue of patients with CKD of various etiologies. In vitro, I will test the hypothesis that Cul3 inactivation by CRISPR/Cas9 system in human primary renal proximal tubule epithelial cells causes cell-cycle arrest and paracrine myofibroblast activation. Understanding the role of Cul3 in fibrosis could open new therapeutic opportunities for targeting pro-fibrotic mechanisms in patients with CKD. In addition, the results of the planned studies may not only apply to kidney, but also to other organs.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Dr. Jim McCormick, Ph.D.