#2303 PDE5 inhibitor LW1646 improves kidney fibrosis by alleviating ER stress and mitochondrial calcium overload

Abstract Background and Aims Renal fibrosis is the terminal pathological manifestation of most chronic kidney diseases (CKDs). Inhibitors targeting phosphodiesterase 5 (PDE5) have been used to treat erectile dysfunction and pulmonary arterial hypertension by increasing cGMP levels and activating the cGMP/PKG signaling pathway. Studies have shown that PDE5 inhibitors can attenuate the progression of CKD, although the underlying mechanisms remain unclear. LW1646 is a newly identified inhibitor with higher specificity and potency against PDE5. This study aims to investigate the therapeutic effects of LW1646 on renal fibrosis and its underlying mechanisms. Method In vitro, pro-fibrotic response in HK2 cells was induced by TGF-β1 and intervened with LW1646. In vivo, renal interstitial fibrosis model was established by unilateral ureteral obstruction for 7 days (7UUO) in Pde5a-/- mice. mRNA expression level of Pde5a was analyzed by qPCR. Western blot was used to assess changes in fibrosis markers, endoplasmic reticulum stress indicators, mitochondrial homeostasis metrics, and components of mitochondrial-associated membranes (MAM). Mitochondrial membrane potential (MMP) and reactive oxygen species were evaluated using flow cytometry, fluorescent imaging was employed to visualize mitochondrial networks, accumulation of mitochondrial calcium (mtCa2+) and formation of MAM. Histological assessments of renal fibrosis were performed using H&E staining and Masson's trichrome staining. Morphology of mitochondria and ER and mitochondria-ER contact in kidney cortex were investigated by transmission electron microscope (TEM). Results mRNA expression level of Pde5a was elevated in renal cortex in 7UUO-induced kidney fibrosis mice. Expression of fibrosis markers and endoplasmic reticulum stress indicators were markedly upregulated both in vitro and in vivo. Meanwhile, mitochondrial biogenesis was inhibited, which was accompanied by increased oxidative stress, decreased MMP and augmented mitochondrial fission. MAMs formation and mtCa2+ level were enhanced. LW1646 significantly prevented development of fibrosis in mice with 7UUO and exhibited a more pronounced effect than sildenafil. Pde5a knockout was associated with alleviated fibrosis in mice. LW1646 or Pde5a knockout attenuated ER stress and altered mitochondrial homeostasis in fibrotic kidneys and HK2 cells exhibiting pro-fibrotic responses. Conclusion Our data demonstrated that LW1646 ameliorated renal fibrosis likely through inhibiting ER stress and maintaining mitochondrial homeostasis. Mechanistically, activation of cGPM/PKG pathway by LW1646 or Pde5a knockout conferred mitochondrial protection by inhibiting the formation of MAMs, thereby reducing mtCa2+ overload and alleviating renal fibrosis.