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Klotho is an anti-aging protein produced primarily by tubular epithelial cells (TECs). Down-regulated expression of Klotho in injured TECs plays a key pathogenic role in promoting renal fibrosis. Supplementation or overexpression of exogenous Klotho has shown good therapeutic efficacy in a variety of CKD mice models, however, high production costs, technical difficulties, potential biosafety risks, and low renal concentration greatly limit its clinical application. Recent studies have shown that nanoparticles are an ideal delivery system for drugs, which can achieve targeted delivery to organs and even cells, thereby improving efficacy and reducing side effects.
We designed and synthesized polydopamine-PEI 25K-l-serine nanoparticles carrying Klotho plasmids (PPSK), which could safely and targeted delivery of plasmid DNA to the injured TECs and sustained expression of Klotho protein. We characterized the physical and chemical properties of PPSK and evaluated its biocompatibility and distribution in vitro and in vivo. Hypoxia-reoxygenation (H/R) cell model and unilateral renal ischemia-reperfusion (UIRI) mouse model were used to assess the antifibrotic therapeutic effect of PPSK. Moreover, we performed transcriptome sequencing using the kidney cortex to reveal the molecular mechanism behind the pharmacological action of the nanomedicine-based Klotho gene therapy.
We demonstrated that the transfection ability of PPSK to TECs is better than that of commercial transfection reagents Lipo2000 and PEI 25K, and could effectively reduce H/R-induced fibrotic gene expression. In animals, we found that PPSK mainly accumulated in TECs of the injured kidney, a single-dose injection with PPSK preserved normal kidney architecture and arrested renal fibrosis. Mechanistically, PPSK upregulated the expression of peroxisome proliferator-activated receptor alpha (PPARα), a major regulator of lipid metabolism and mitochondrial homeostasis, thus maintaining fatty acid oxidation in TECs to protect against renal fibrosis. In addition, PPSK showed good biocompatibility and low toxicity.
In summary, this study has developed a novel nanomedicine-based Klotho gene therapy targeting the injured TECs, which could prevent CKD progression via sustaining fatty acid oxidation. PPSK may serve as a promising tubule-targeting therapeutic agent against renal fibrosis.