LONP1 targets HMGCS2 to protect mitochondrial function and attenuate chronic kidney disease

Article11 January 2023Open Access Source DataTransparent process LONP1 targets HMGCS2 to protect mitochondrial function and attenuate chronic kidney disease Mi Bai Mi Bai Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Data curation, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Mengqiu Wu Mengqiu Wu orcid.org/0000-0001-9488-5572 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Data curation, Formal analysis, Visualization, Methodology, Writing - original draft Search for more papers by this author Mingzhu Jiang Mingzhu Jiang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Data curation, ​Investigation, Visualization Search for more papers by this author Jia He Jia He orcid.org/0000-0001-7849-2322 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Data curation, ​Investigation, Methodology Search for more papers by this author Xu Deng Xu Deng Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Data curation, Funding acquisition, ​Investigation, Methodology Search for more papers by this author Shuang Xu Shuang Xu Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Jiaojiao Fan Jiaojiao Fan Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Mengqiu Miao Mengqiu Miao Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Ting Wang Ting Wang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Yuting Li Yuting Li Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Methodology Search for more papers by this author Xiaowen Yu Xiaowen Yu Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Funding acquisition, Methodology Search for more papers by this author Lin Wang Lin Wang orcid.org/0000-0002-6677-7061 Key Laboratory of Molecular Pharmacology and Drug Evaluation, Yantai University, Yantai, China Contribution: Methodology Search for more papers by this author Yue Zhang Yue Zhang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Validation, ​Investigation Search for more papers by this author Songming Huang Songming Huang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Funding acquisition, Validation, ​Investigation Search for more papers by this author Li Yang Li Yang orcid.org/0000-0002-2528-5087 Renal Division, Peking University First Hospital, Beijing, China Contribution: Validation, ​Investigation Search for more papers by this author Zhanjun Jia Corresponding Author Zhanjun Jia jiazj72@hotmail.com orcid.org/0000-0002-3107-0429 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Conceptualization, Formal analysis, Funding acquisition, Validation, ​Investigation, Writing - review & editing Search for more papers by this author Aihua Zhang Corresponding Author Aihua Zhang zhaihua@njmu.edu.cn orcid.org/0000-0001-7438-4404 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Conceptualization, Formal analysis, Funding acquisition, Validation, ​Investigation, Writing - review & editing Search for more papers by this author Mi Bai Mi Bai Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Data curation, Funding acquisition, Validation, ​Investigation, Visualization, Methodology, Writing - original draft Search for more papers by this author Mengqiu Wu Mengqiu Wu orcid.org/0000-0001-9488-5572 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Data curation, Formal analysis, Visualization, Methodology, Writing - original draft Search for more papers by this author Mingzhu Jiang Mingzhu Jiang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Data curation, ​Investigation, Visualization Search for more papers by this author Jia He Jia He orcid.org/0000-0001-7849-2322 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Data curation, ​Investigation, Methodology Search for more papers by this author Xu Deng Xu Deng Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Data curation, Funding acquisition, ​Investigation, Methodology Search for more papers by this author Shuang Xu Shuang Xu Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Jiaojiao Fan Jiaojiao Fan Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Mengqiu Miao Mengqiu Miao Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Ting Wang Ting Wang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: ​Investigation, Methodology Search for more papers by this author Yuting Li Yuting Li Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Methodology Search for more papers by this author Xiaowen Yu Xiaowen Yu Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Funding acquisition, Methodology Search for more papers by this author Lin Wang Lin Wang orcid.org/0000-0002-6677-7061 Key Laboratory of Molecular Pharmacology and Drug Evaluation, Yantai University, Yantai, China Contribution: Methodology Search for more papers by this author Yue Zhang Yue Zhang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Validation, ​Investigation Search for more papers by this author Songming Huang Songming Huang Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Contribution: Funding acquisition, Validation, ​Investigation Search for more papers by this author Li Yang Li Yang orcid.org/0000-0002-2528-5087 Renal Division, Peking University First Hospital, Beijing, China Contribution: Validation, ​Investigation Search for more papers by this author Zhanjun Jia Corresponding Author Zhanjun Jia jiazj72@hotmail.com orcid.org/0000-0002-3107-0429 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Conceptualization, Formal analysis, Funding acquisition, Validation, ​Investigation, Writing - review & editing Search for more papers by this author Aihua Zhang Corresponding Author Aihua Zhang zhaihua@njmu.edu.cn orcid.org/0000-0001-7438-4404 Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China Contribution: Conceptualization, Formal analysis, Funding acquisition, Validation, ​Investigation, Writing - review & editing Search for more papers by this author Author Information Mi Bai1,2,3,†, Mengqiu Wu1,2,3,†, Mingzhu Jiang1,2,†, Jia He1,2, Xu Deng1,2, Shuang Xu1,2, Jiaojiao Fan1,2, Mengqiu Miao1,2, Ting Wang1,2, Yuting Li1,2, Xiaowen Yu1,2,3, Lin Wang4, Yue Zhang1,2, Songming Huang1,2, Li Yang5, Zhanjun Jia *,1,2,3 and Aihua Zhang *,1,2,3 1Department of Nephrology, State Key Laboratory of Reproductive Medicine, Children's Hospital of Nanjing Medical University, Nanjing, China 2Jiangsu Key Laboratory of Pediatrics, Nanjing Medical University, Nanjing, China 3Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China 4Key Laboratory of Molecular Pharmacology and Drug Evaluation, Yantai University, Yantai, China 5Renal Division, Peking University First Hospital, Beijing, China † These authors contributed equally to this work *Corresponding author. Tel: +86 18951769656; E-mail: jiazj72@hotmail.comCorresponding author. Tel: +86 18951769017; E-mail: zhaihua@njmu.edu.cn EMBO Mol Med (2023)15:e16581https://doi.org/10.15252/emmm.202216581 AbstractSynopsis The paper explained Introduction Results Discussion Materials and Methods Data availability Acknowledgements Author contributions Disclosure and competing interest statement For more informationSupporting InformationReferencesPDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. MetricsMetricsTotal downloads901Last 6 Months901View all metrics ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondria comprise the central metabolic hub of cells and their imbalance plays a pathogenic role in chronic kidney disease (CKD). Here, we studied Lon protease 1 (LONP1), a major mitochondrial protease, as its role in CKD pathogenesis is unclear. LONP1 expression was decreased in human patients and mice with CKD, and tubular-specific Lonp1 overexpression mitigated renal injury and mitochondrial dysfunction in two different models of CKD, but these outcomes were aggravated by Lonp1 deletion. These results were confirmed in renal tubular epithelial cells in vitro. Mechanistically, LONP1 downregulation caused mitochondrial accumulation of the LONP1 substrate, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), which disrupted mitochondrial function and further accelerated CKD progression. Finally, computer-aided virtual screening was performed, which identified a novel LONP1 activator. Pharmacologically, the LONP1 activator attenuated renal fibrosis and mitochondrial dysfunction. Collectively, these results imply that LONP1 is a promising therapeutic target for treating CKD. Synopsis Activation of the mitochondrial protease Lon protease 1 (LONP1) attenuates mitochondrial dysfunction and renal fibrosis in chronic kidney disease (CKD) mice, suggesting that targeting LONP1 might represent a novel therapeutic strategy for CKD. CKD patients and mice have decreased LONP1 expression. The substrate of LONP1, HMGCS2, accumulates in mitochondria causing mitochondrial dysfunction and accelerating CKD progression. Activation of LONP1 by the newly identified activator 84-B10 improves mitochondrial function and reduces renal fibrosis in CKD mouse model. The paper explained Problem Chronic kidney disease (CKD) is a chronic renal structural and functional disorder caused by various diseases and conditions. The incidence of CKD worldwide is more than 10%, and there is a lack of effective treatment. A considerable proportion of patients progress to end-stage renal disease (ESRD). Renal fibrosis is a common pathology leading to ESRD and it is directly related to the prognosis of renal function. Kidney fibrosis progresses gradually until it is necessary to rely on renal replacement therapy to maintain life. Therefore, it is urgent to elucidate the molecular mechanism of the occurrence and development of renal fibrosis and to find effective intervention targets to delay its progression. Results Here, we identified LONP1/HMGCS2 signaling in proximal tubular cells as a therapeutic target for retarding kidney fibrosis. Tubular-specific Lonp1 overexpression mitigated renal injury and mitochondrial dysfunction in two different models of CKD, and these outcomes were aggravated by Lonp1 deletion. Mechanistically, LONP1 downregulation caused mitochondrial accumulation of the LONP1 substrate, HMGCS2, which disrupted mitochondrial function and further accelerated CKD progression. More importantly, we identified a novel LONP1 activator, which may be a promising therapeutic for treating CKD. Impact Mitochondrial dysfunction in proximal tubular cells contributes to CKD progression, while the specific therapeutic targets to improve mitochondrial function in proximal tubular cells are still limited. These results not only demonstrate the important role of the LONP1/HMGCS2 signaling pathway in delaying renal fibrosis, but also suggest that the pathway may be a therapeutic target for CKD by activating LONP1. Introduction Chronic kidney disease (CKD), the end stage of various kidney diseases, has become a worldwide health problem with a rapidly increasing prevalence, high morbidity, and economic burden (Zhang et al, 2012; Webster et al, 2017). Renal tubular-interstitial fibrosis is a common pathophysiological pathway involved in the development of multiple CKDs that lead to end-stage renal disease (ESRD). Proximal tubular cells, which are rich in mitochondria and require high energy to meet the tremendous energy demands of tubular reabsorption and secretion, are particularly sensitive to various insults such as hypoxia, oxidative stress, and toxins (Chevalier, 2016). Several reports have shown that the impairment of proximal tubular cells can initiate and promote an inflammatory and profibrogenic response, activate mesenchymal fibroblasts, and drive the production of many extracellular matrix proteins, which ultimately results in tubulointerstitial fibrosis and a continuous loss of renal function (Liu, 2011; Leung et al, 2013; Meng et al, 2014; Liu et al, 2020). Hence, identifying novel endogenous kidney protectants and specific mechanisms by which to mitigate proximal tubular cell injury can provide insights into new treatment strategies for CKD. Proximal tubular cells are highly dependent on mitochondria to maintain their cellular functions and viability. As the energy factory of cells, mitochondria maintain cellular homeostasis through a complex and sophisticated monitoring system, which includes the timely degradation of damaged misfolded proteins and the elimination of mutant mitochondrial DNA (mtDNA) and free radicals (Jiang et al, 2020). Data from previous studies showed that mitochondrial dysfunction is not only an early event in kidney injury, but also contributes to CKD development and progression, and maintaining normal mitochondrial function can effectively alleviate kidney injury (Yuan et al, 2012; Bai et al, 2019; Miguel et al, 2021). Thus, looking for intervention targets to maintain the normal functions of mitochondria and restoring the renal tubular mitochondrial function in CKD may be an important research direction for preventing and treating CKD. The Lon protease 1 (LONP1) is a highly conserved ATP-dependent protease that ensures mitochondrial proteostasis and regulates adaptive responses to cellular stress (Bota & Davies, 2001; Lu et al, 2003; Bahat et al, 2015). Previous findings have shown that LONP1 upregulation helps lung fibroblast cells adapt to acute stress and is important for preserving normal cell viability (Ngo et al, 2011) and overcoming the hypoxic, metabolic, and proteotoxic stress associated with the oncogenic transformation of tumor cells (Lu, 2017). In addition, LONP1 can protect cardiomyocytes from injury due to ischemia/reperfusion (Venkatesh et al, 2019). Previously, we reported that reduced LONP1 expression in podocytes contributed to the pathogenesis of podocytopathy (Gong et al, 2021). However, no reports have demonstrated the involvement of LONP1 in renal fibrosis. LONP1 dysfunction directly leads to the failure of mitochondrial protein degradation, and the accumulation of abnormal mitochondrial proteins leads to a series of cellular and tissue injuries. In this study, we found that hydroxymethyl glutaryl coenzyme A synthase (HMGCS2) might be an LONP1 substrate. HMGCS2 (which belongs to the HMG-CoA synthase family) is a mitochondrial enzyme that catalyzes the first reaction of ketogenesis, a metabolic pathway that provides lipid-derived energy for various organs during times of carbohydrate deprivation, such as fasting (Geisler et al, 2019). HMGCS2 has been suggested to play important roles in diabetes, tumor, Alzheimer's disease, and intestinal cell differentiation (Wan et al, 2018, 2019; Cheng et al, 2019; Kim et al, 2019; Zou et al, 2019; Wang et al, 2019b). Recent data indicated that high HMGCS2 expression caused reactive oxygen species (ROS) accumulation and loss of the mitochondrial membrane potential (MMP), which then induced diabetic cardiomyopathy (Wang et al, 2020a). However, the exact role of HMGCS2 in renal fibrosis remains unclear. Here, we identified LONP1 as an endogenous mitochondrial regulator in renal tubular cells under CKD conditions, in both rodents and humans. Tubule-specific Lonp1 overexpression mitigated mitochondrial dysfunction and markedly increased tubular injury and renal fibrosis in two mouse models of CKD (one involving unilateral ureteral obstruction [UUO] and one involving 5/6 nephrectomy [5/6Nx]), but these outcomes were aggravated by tubule-specific Lonp1 abrogation. These results were confirmed in vitro, using transforming growth factor (TGF)-β1-treated renal tubular epithelial cells. Mechanically, we found that HMGCS2 is a possible substrate for LONP1 and that mitochondrial HMGCS2 accumulation disrupted mitochondrial function and further aggravated CKD. We validated these results by demonstrating that the pharmacological activator of LONP1 could attenuate renal fibrosis and mitochondrial function. Therefore, our findings provide a rationale for designing targeted LONP1 activators as therapeutic agents against CKD. Results LONP1 expression in fibrotic kidneys of CKD patients and UUO mice To examine the association of LONP1 with CKD, we performed immunohistochemical (IHC) staining with biopsied kidney tissues from 30 patients with CKD. Compared with the richness of signal in control human kidneys, significant downregulation of LONP1 was observed in patient kidneys, primarily in the renal tubular cells (Fig 1A and B). The mRNA levels of CKD patients also decreased in an online dataset (Kang et al, 2015) (Fig EV1A). More importantly, the level of LONP1 expression negatively correlated with the degree of kidney fibrosis, as assessed by the atrophy and fibrosis score (AFS; r = −0.716, P < 0.001; Fig 1C), as well as negatively correlated with BUN and Scr in CKD patients (Fig EV1E and F). Then, we studied UUO models at different time points and found that LONP1 expression decreased in a time-dependent manner (Figs 1D and E, and EV1C). We verified the reduction of Lonp1 mRNA levels in UUO models using an online database (Conway et al, 2020) (Fig EV1B and D). Figure 1. LONP1 is down-regulated in the kidneys of CKD patients and mice and proximal tubular-specific overexpression of Lonp1 alleviates renal injury and mitochondrial dysfunction in UUO model A. Immunohistochemical analysis of LONP1 expression in CKD children with mild (n = 11), moderate (n = 10), or severe fibrosis (n = 9). N = 4 in Normal group. Scale bar: 50 μm. B. Immunohistochemical semi-quantitative IOD analysis of LONP1. C. Pearson correlation analysis of LONP1 and atrophy and fibrosis score (AFS) in renal biopsy specimens. D. Western blot analysis for the expression of LONP1 in UUO models at different time points. E. Densitometric analysis for the expression of LONP1 (n = 3, biological replicates). F. Deposition of total fibrosis in kidney tissues was determined by Masson's trichrome staining. Scale bar: 50 μm. G. Sirius red staining in WT and cKI mice after UUO. Scale bar: 50 μm. H. Fibrotic area statistics of Sirius red staining in WT and cKI mice after UUO (n = 6 in WT+Sham group, n = 8 in cKI+Sham or WT+UUO group, n = 7 in cKI+UUO group, biological replicates). I. qRT-PCR analysis of FN1, Collagen I and Collagen III in WT and cKI mice after UUO (n = 7 in WT+Sham group, n = 8 in the other three groups, biological replicates). J, K. Western blot and densitometric analysis for the expression of FN1, Collagen I, Collagen III and TGF-β1 (monomer) in WT and cKI mice after UUO (n = 3 or 4, biological replicates). L. qRT-PCR analysis of mtDNA expression in WT and cKI mice after UUO (n = 6 or 7, biological replicates). M. Succinate dehydrogenase (SDH) staining in WT and cKI mice after UUO. Scale bar: 50 μm. N. Transmission electron microscopy images of the mitochondria in tubular cells in WT and cKI mice after UUO. Scale bar: 500 nm. Data information: In (B, E), data are presented as mean ± SEM. Student's t-test. In (H–L), data are presented as mean ± SEM. One-way ANOVA. Source data are available online for this figure. Source Data for Figure 1 [emmm202216581-sup-0005-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. LONP1 was down-regulated in CKD kidneys based on the analysis of online datasets and proximal tubular-specific overexpression of Lonp1 alleviated the reduction of mitochondrial genes expression and protected mitochondrial morphology in UUO model A. Lonp1 expression from online human RNA sequencing data (Data ref: Yi-An, 2014). N = 20 in Ctl group an n = 19 in CKD group (biological replicates). B. Lonp1 expression from online mouse UUO model RNA sequencing data (Data ref: Denby et al, 2020). N = 4 in each group (biological replicates). C. The mRNA expression of Lonp1 in our UUO models (n = 5–7, biological replicates). D. Lonp1 expression from kidney single cell datasets Gene Atlas of Reversible Unilateral Ureteric Obstruction Model (http://www.ruuo-kidney-gene-atlas.com/). E, F. Pearson correlation analysis of LONP1 and BUN and Serum Creatinine in CKD patients (n = 30). G. Western blot and densitometric analysis for the expression of LONP1 of proximal tubular cells isolated from WT and cKI mice (n = 3, biological replicates). H, I. qRT-PCR analysis of mitochondrial genes in WT and cKI mice after UUO (n = 8 in each group, biological replicates). J. Transmission electron microscopy images of intact renal tubule cells and mitochondria in WT and cKI mice after UUO. Scale bar: 5 μm, 2 μm and 500 nm. Data information: Data are presented as mean ± SEM. Student's t-test. In the boxplot of B, the central band represents the median line; the boxes represent range between 25 and 75%; the whiskers represent range within the 1.5 IQR (Inter-Quartile Range). Source data are available online for this figure. Download figure Download PowerPoint Tubule-specific Lonp1 overexpression or deletion attenuated or aggravated CKD induced by UUO To ascertain the role of LONP1 in CKD, we generated proximal tubule Lonp1 conditional knock-in mice (cKI). Compared with wild-type (WT) mice, the expression of LONP1 in renal tubule cells of cKI mice was approximately doubled (Fig EV1G). With the UUO model, Lonp1-specific overexpression in renal proximal tubules significantly attenuated tubular brush border loss, tubule atrophy, cellular infiltration, and tubulointerstitial fibrosis in the kidneys determined by Masson's trichrome and Sirius red staining (Fig 1F–H). The production of extracellular matrix components (Collagen I, Collagen III, and fibronectin 1 [FN1]) was lower in cKI mice (Fig 1I–K). The profibrotic factor TGF-β1 also decreased after Lonp1 overexpression (Fig 1J and K). In addition, mitochondrial dysfunction was ameliorated significantly, as evidenced by restored mitochondrial genes expression (Fig EV1H and I), mtDNA copy numbers (Fig 1L), succinate dehydrogenase (SDH) activity (Fig 1M) and mitochondrial morphology (including mitochondrial swelling and disorganized fragmented cristae in the renal tubular cells of the UUO model) (Figs 1N and EV1J). Next, we generated proximal tubule Lonp1 conditional knockout (cKO) mice. Compared with WT mice, LONP1 was almost not expressed in primary proximal tubule cells extracted and cultured from cKO mice (Fig EV2A and B). In contrast to our previous report showing that podocyte-specific Lonp1 knockout was lethal, mice with tubule-specific Lonp1 knockout grew and developed normally. Even in elderly mice (18 months), we observed normal body weights, blood pressures (BPs), serum creatinine (Scr) l