Fig 2: HMGCS2 may be the degradation substrate of LONP1 The proximal renal tubules of Lonp1 cKO mice were extracted and detected by mass spectrometry (n = 3, biological replicates).Subcellular localization of up-regulated proteins.Western blot validation of the up-regulated proteins in mitochondria (n = 2, biological replicates).CoIP analysis of interaction between LONP1 and HMGCS2.GST pull down analysis of interaction between LONP1 and HMGCS2.Enzymatic hydrolysis of LONP1 to HMGCS2.Immunofluorescence co-localization analysis of LONP1, HMGCS2 and mitochondria in mPTC (up, scale bar: 20 µm) and human kidney tissue (down, scale bar: 200 µm). Source data are available online for this figure.

Fig 3: Effect of HMGCS2 on mitochondrial function and renal fibrosis AImmunohistochemical analysis of HMGCS2 expression in CKD children. Scale bar: 50 µm.BImmunohistochemical semi-quantitative IOD analysis of HMGCS2 (n = 8 in Control group, n = 15 in CKD group).CPearson correlation analysis of HMGCS2 and atrophy and fibrosis score (AFS) in renal biopsy specimens.DWestern blot analysis for the expression of HMGCS2 in UUO model.EDensitometric analysis for the expression of HMGCS2 in UUO model (n = 3, biological replicates).F, GWestern blot and densitometric analysis of HMGCS2 in WT and cKI mice of UUO model (n = 3 or 4, biological replicates).H, IqRT-PCR and western blotting analysis of Hmgcs2 expression in WT and Hmgcs2+/- mice (n = 3, biological replicates).JFibrotic area statistics of Sirius red staining in WT and Hmgcs2+/- mice of UUO model (n = 12 in WT groups, n = 8 in Hmgcs2+/- groups, biological replicates).KDeposition of total fibrosis in kidney tissues in WT and Hmgcs2+/- mice after UUO was determined by Masson's trichrome staining. Scale bar: 50 µm.LSirius red staining of WT and Hmgcs2+/- mice of UUO model. Scale bar: 50 µm.M, NWestern blot and densitometric analysis for the expression of FN1, a-SMA and Collagen III in WT and Hmgcs2+/- mice after UUO (n = 6 in control groups, n = 8 in UUO groups, biological replicates).OqRT-PCR analysis of FN1, a-SMA and Collagen III in WT and Hmgcs2+/- mice after UUO (n = 10 or 12 in WT groups, n = 8 in Hmgcs2+/- groups, biological replicates).PSuccinate dehydrogenase (SDH) staining in WT and Hmgcs2+/- mice after UUO. Scale bar: 100 µm. Data information: In (B, E, H, I), data are presented as mean ± SEM. Student's t-test. In (G, J, N, O), data are presented as mean ± SEM. One-way ANOVA. Source data are available online for this figure.

Fig 4: The anti-fibrotic response effect of a novel LONP1 activator in mPTC cells A10 high docking score molecules showed LONP1 activating effect in cell free LONP1 protease activity assay system, of which 84-B10 showed stronger activity. Bortezomib (Bor), a reported LONP1 inhibitor, is involved as a contrast.B84-B10 dose dependently induced the peptidase activity of LONP1 as estimated by detecting the fluorogenic di-peptide substrate (Z-AA)2-Rh110 (n = 6 or 3, biological replicates).CChemical structure of 84-B10.DProposed binding model of 84-B10 binding to the human LONP1 catalytic domain. Three and two-dimensional illustrations of the interaction between 84-B10 and human LONP1 catalytic domain.EPhysical interaction between 84-B10 and LONP1 performed by Surface Plasmon Resonance (SPR).FProtein levels of HMGCS2 and LONP1 were estimated in mPTCs treated with different concentration of 84-B10 or vehicle (0.1% DMSO) for 24 h.G, HRepresentative immunoblot and densitometric analysis of HMGCS2, FN1, Collagen III, a-SMA and Vimentin in mPTCs treated as indicated. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n = 3 in each group).IqRT-PCR analysis of Lonp1, Collagen I, Collagen III, a-SMA, Vimentin and Periostin transcript levels in mPTCs treated as indicated (n = 4 in each group, biological replicates).J–NRepresentative Western blot and densitometric analysis of LONP1, FN1, a-SMA and Vimentin of each group was shown. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n = 3 in each group). mPTCs were transfected with siNC or siLONP1, and then pre-treated with 84-B10 (10 µM) 2 h before TGF-ß1 (10 ng/ml) treatment for another 24 h. Data information: Data are presented as mean ± SEM. Student's t-test. Source data are available online for this figure.

Fig 5: HMGCS2 aggravated TGF-ß1-induced mitochondrial dysfunction and fibrotic response in mPTCs and primary proximal tubular cells AThe expression of known substrates of LONP1 and HMGCS2 in our proteomics (n = 3, biological replicates). Compared with Hmgcs2, there was little difference in their expression in WT and Lonp1 cKO groups.BWestern blot showing the intramitochondrial localization of HMGCS2 in mPTC cells after transfected with Vector or Hmgcs2 plasmids (n = 2, biological replicates).CQuantification of mitochondrial ROS production in mPTC cells after transfected with Vector or Hmgcs2 plasmids following TGF-ß1 treatment (n = 4 in each group, biological replicates).DQuantitation of mitochondrial membrane potential (??m) by JC-1 staining in mPTC cells after transfected with Vector or Hmgcs2 plasmids following TGF-ß1 treatment (n = 4 in each group, biological replicates).EqRT-PCR analysis of FN1, Collagen III and a-SMA in mPTC cells after transfected with Vector or Hmgcs2 plasmids following TGF-ß1 treatment (n = 4 or 5, biological replicates).F, GWestern blot and densitometric analysis for the expression of FN1 and Collagen III in mPTC cells after transfected with Vector or Hmgcs2 plasmids following TGF-ß1 treatment (n = 3 in each group, biological replicates).HqRT-PCR analysis of FN1 and Collagen I in mPTC cells after transfected with shNC or shHmgcs2 plasmids following TGF-ß1 treatment (n = 6 in each group, biological replicates).I, JWestern blot and densitometric analysis for the expression of FN1 and Collagen III in mPTC cells after transfected with shNC or shHmgcs2 plasmids following TGF-ß1 treatment (n = 3 in each group, biological replicates).KThe proximal tubular cells were isolated from C57BL/6J mice and infected with HMGCS2-lentivirus (pLVX-Puro-mHmgcs2-HA) or control lentivirus (vector). Western blot and densitometric analysis for the expression of HA (n = 3 in each group, biological replicates).L, MThe infected primary cells were treated with TGF-ß1 for 24 h after infected with HMGCS2-lentivirus or control lentivirus. Western blot and densitometric analysis for the expression of FN1 and Collagen III. Three independent experiments were carried out and quantification of the abundance of these proteins is shown in panel (n = 3 in each group).NThe infected primary cells were treated with TGF-ß1 for 4 h. Quantification of mitochondrial ROS production (n = 4 in each group, biological replicates). Data information: In (K), data are presented as mean ± SEM. Student's t-test. In (C–E, G, H, J, M, N), data are presented as mean ± SEM. One-way ANOVA. Source data are available online for this figure.