Kidney Week

Abstract: TH-OR017

Ubiquitin-Proteasome System Actively Maintains Homeostasis of Proximal Tubules

Session Information

• AKI: Mechanisms - Injury and Repair
  November 07, 2019 | Location: Salon A/B, Walter E. Washington Convention Center
  Abstract Time: 05:42 PM - 05:54 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Hirano, Keita, Department of Nephrology, Kyoto University Graduate School of Medicine, Kyoto, Japan

Keita Hirano, MD



• Tashiro, Yoshitaka, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan

Yoshitaka Tashiro,

• Yanagita, Motoko, Department of Nephrology, Kyoto University Graduate School of Medicine, Kyoto, Japan

Motoko Yanagita,

Background

Autophagy and ubiquitin-proteasome system (UPS) are two major protein degradation pathways essential for cellular protein homeostasis. While the role of autophagy in the kidney has been extensively analyzed in the proximal tubules of the kidney, the impact of UPS function in the maintenance the proximal tubules has been unclear.

Methods

In order to analyze the role of UPS function in the proximal tubule, we crossed conditional knockout mice of the proteasome subunit Rpt3 with proximal tubule specific inducible Cre (Ndrg1-Cre^ERT2), to generate Rpt3fl/fl:Ndrg1-Cre^ERT2(PT-Rpt3-CKO) mice, in which the expression of Rpt3 can be deleted in proximal tubules at desired time points by tamoxifen administration. Utilizing PT-Rpt3-CKO mice, we investigated how UPS regulates the maintenance of proximal tubules.

Results

As early as one day after tamoxifen administration for 5 consecutive days, proximal tubules of PT-Rpt3-CKO mice showed mild injury in light microscopy, whose mitochondria were fragmented in electron microscopy. Proximal tubule injury at this time point was also confirmed by the upregulation of Kim1, cleaved caspase 3, and gH2AX in immunostaining. Immunoblotting also revealed the upregulation of ubiquitin and p62, and downregulation of LC3 1/2ratio, indicating autophagy insufficiency. Four days after tamoxifen administration, serum creatinine and blood urea nitrogen (BUN) were elevated and proximal tubules were detached from the basement membrane. In addition to further upregulation of Kim1, cleaved caspase 3, and gH2AX, and p62 compared to those in day 1, the expression of p21was upregulated and cyclinD1 was downregulated indicating possible G1 cell cycle arrest. 8 days after tamoxifen administration, all PT-Rpt3-CKO mice died possibly due to renal insufficiency. The administration of the proteasome inhibitor MG-132 to a proximal tubule cell line NRK52E resulted in decreased mitochondrial membrane potential and oxygen consumption rate.

Conclusion

Our results provide strong evidence showing that the dysfunction of UPS rapidly triggers mitochondrial dysfunction, autophagy insufficiency, cell cycle arrest and apoptosis of proximal tubules, leading to renal insufficiency and death. Compared to mild phenotypes of conditional knockout mice of autophagy related molecules in the proximal tubules, UPS plays a crucial role in the active maintenance of proximal tubules.