Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded
proteins retained in the ER, is implicated in various diseases including
chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial
fibrosis (RIF). Methods: Following establishment of a mouse RIF model by unilateral
ureteral obstruction (UUO), a specific ERAD inhibitor,
Eeyarestatin I (EerI), was administered to experimental animals by
intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by
enzyme-linked
immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1,
glucose-regulated
protein 78 (
GRP78),
activating transcription factor 4 (ATF4) and
protein disulfide isomerase (PDI). HK-2 cells were exposed to
transforming growth factor (TGF)-β1 with or without EerI, and expressions of related
proteins including Klotho, Derlin-1,
GRP78, ATF4 and PDI were determined by western blotting analyses. Results: UUO induced severe kidney
injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho
protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of
GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-β1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded
proteins, subsequently contributing to the additional production of mature Klotho
protein. Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.