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Research ArticleNephrology
Open Access | 
10.1172/JCI180242
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
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1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
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1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Li, G. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Li, Y. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Fang, W. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Zhang, S. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Yu, R. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Jiang, S. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Gao, X. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Jiang, Y. in: JCI | PubMed | Google Scholar
1Nephrology Department, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou, China.
2Department of Endocrinology, The First Affiliated Hospital of Xinjiang Medical University, State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Urumqi, China.
3Department of Pediatrics, Jiangxi Children’s Medical Center, Nanchang, China.
4State Key Laboratory of Genetic Engineering, School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, China.
5The Key Laboratory of Experimental Teratology of the Ministry of Education and Department of Histology and Embryology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan, China.
6Department of Orthopedics, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China.
7Qingyuan People’s Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China.
Address correspondence to: Qi Wang, No. 35 Yinquan North Road, The Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan, China. Email: wq131415@qq.com. Or to: Yuhang Jiang, No. 3025, Shennan Middle Road, The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China. Email: xhaka2016@163.com. Or to: Xia Gao, No. 9 Jinsui Road, Guangzhou Women and Children’s Medical Center, Guangzhou, China. Email: gaoxiagz@vip.163.com.
Authorship note: DL and GA are co–first authors.
Find articles by Wang, Q. in: JCI | PubMed | Google Scholar
Authorship note: DL and GA are co–first authors.
Published February 6, 2025 - More info
Published in Volume 135, Issue 6 on March 17, 2025
AbstractFibrosis is the final common pathway leading to end-stage chronic kidney disease (CKD). However, the function of protein palmitoylation in renal fibrosis and the underlying mechanisms remain unclear. In this study, we observed that expression of the palmitoyltransferase ZDHHC18 was significantly elevated in unilateral ureteral obstruction (UUO) and folic acid–induced (FA-induced) renal fibrosis mouse models and was significantly upregulated in fibrotic kidneys of patients with CKD. Functionally, tubule-specific deletion of ZDHHC18 attenuated tubular epithelial cells’ partial epithelial-mesenchymal transition (EMT) and then reduced the production of profibrotic cytokines and alleviated tubulointerstitial fibrosis. In contrast, ZDHHC18 overexpression exacerbated progressive renal fibrosis. Mechanistically, ZDHHC18 catalyzed the palmitoylation of HRAS, which was pivotal for its translocation to the plasma membrane and subsequent activation. HRAS palmitoylation promoted downstream phosphorylation of MEK/ERK and further activated Ras-responsive element–binding protein 1 (RREB1), enhancing SMAD binding to the Snai1 cis-regulatory regions. Taken together, our findings suggest that ZDHHC18 plays a crucial role in renal fibrogenesis and represents a potential therapeutic target for combating kidney fibrosis.
Graphical Abstract
Introduction
Almost all forms of chronic kidney disease eventually progress to renal fibrosis (1, 2). Tubular epithelial cells (TECs) are the main component of the kidney. When kidney injury occurs, injured TECs undergo partial epithelial-mesenchymal transition (EMT) while still residing within the basement membrane of the tubules. They are characterized by their acquisition of mesenchymal features and coexpression of both epithelial and mesenchymal cell markers. TECs undergoing partial EMT release including proinflammatory and profibrotic factors into the renal interstitium, thereby remodeling the microenvironment to promote inflammation and fibrosis (3–5). Therefore, identifying key molecules involved in the partial EMT process in TECs may lead to the development of therapeutic approaches for preventing renal fibrosis.
Protein S-palmitoylation is a common posttranslational modification that increases the hydrophobicity of proteins and plays an important role in regulating protein transport, location, and functional activation (6, 7). S-palmitoylation links palmitate with specific cysteine residue (Cys) side chains of proteins through unstable thioester bonds. This modification is reversible (8, 9). Protein palmitoylation is catalyzed by a series of enzymes called zinc finger DHHC (ZDHHC) palmitoyltransferases, which contain the signature Asp-His-His-Cys (DHHC) motif (10). ZDHHC protein family members are involved in various physiological and pathological processes. ZDHHC13 has been reported to catalyze palmitoylation of the GPCR MC1R to inhibit the development of melanoma (11). ZDHHC8-KO mice exhibit prepulse inhibition defects, leading to behavioral abnormalities (12, 13). Previous studies have reported that polycystin 1 (PKD1) palmitoylation increases the protein level of PKD1 and promotes the occurrence of polycystic kidney disease (14). β-Catenin palmitoylation leads to protein degradation and inhibits the occurrence of renal fibrosis (15). However, as many as 23 ZDHHC enzymes can catalyze the s-palmitoylation of proteins. The role and function of the ZDHHC enzyme in renal fibrosis are not fully understood.
RAS is a well-known oncogene that regulates cell survival, growth, and differentiation (16, 17). RAS has 3 isoforms, HRAS, NRAS and KRAS. The sequences of these RAS isoforms share a high degree of sequence homology, but they have different biological effects (18). HRAS and the downstream MEK/ERK pathways are activated by unilateral ureteral obstruction (UUO) (19–21), and KO of HRAS in mice reduces UUO-induced renal fibrosis (22). The activation of RAS signaling depends on the subcellular localization of GTPase (23). HRAS is palmitoylated by ZDHHC9 in the Golgi apparatus. Palmitoylation greatly improves the affinity of HRAS for the plasma membrane (PM). HRAS is recruited to the PM and further activated by receptor-Grb2-SOS complexes. Activated HRAS proteins recruit RAF to the PM, where it becomes active and initiates the MEK/ERK signaling cascade (24). However, ZDHHC9 expression is significantly downregulated during renal fibrosis (15). So, how HRAS is activated during renal fibrosis and whether other ZDHHC family palmitoyltransferases modify its palmitoylation remain unknown.
Here, we found that the expression of ZDHHC18 was markedly upregulated during renal fibrosis. Knocking out ZDHHC18 in renal TECs inhibited the expression of partial EMT–related genes and alleviates renal fibrosis phenotypes in vivo. Mechanistically, ZDHHC18 catalyzes HRAS palmitoylation, facilitating its localization to the plasma membrane. HRAS palmitoylation activated Ras-responsive element–binding protein 1 (RREB1), promoting SMAD binding to the Snai1 and Has2 cis-regulatory regions. Collectively, our results demonstrate that ZDHHC18 may be an attractive therapeutic target for treating kidney fibrosis.
ResultsZDHHC18 is upregulated in fibrotic kidneys of patients with chronic kidney disease. We detected ZDHHC18 expression in microdissected kidney samples from patients with chronic kidney disease (CKD). The basic characteristics of the patients are summarized in Supplemental Table 1 (supplemental material available online with this article; https://doi.org/10.1172/JCI180242DS1). CKD samples showed significant interstitial fibrosis and tubular injury, as evidenced by Masson and H&E staining, compared with nonfibrotic kidney tissues (Figure 1, A, C, and D). IHC showed minimal ZDHHC18 expression in nonrenal fibrosis tissue but intense staining in fibrotic kidneys, predominantly in dilated proximal tubules lined by flat, thin epithelium lacking brush borders (Figure 1, A and B). Furthermore, ZDHHC18 levels showed significant positive correlations with the tubular injury score (Figure 1E), serum creatinine (sCr) levels (Figure 1F), and blood urea nitrogen (BUN) levels (Figure 1G). However, ZDHHC18 levels were negatively correlated with the estimated glomerular filtration rate (eGFR) (Figure 1H). The expression of α smooth muscle actin (α-SMA) and vimentin was markedly elevated in the kidney interstitium of fibrotic kidneys (Figure 1I). Linear regression analysis revealed a strong positive correlation between ZDHHC18 expression and the levels of both α-SMA and vimentin (Figure 1, J and K), indicating that ZDHHC18 played a significant role in kidney fibrosis.
Figure 1The expression of ZDHHC18 is markedly increased in the kidneys of patients with CKD. (A) Photomicrographs of ZDHHC18, H&E, and Masson staining in kidney sections from patients with nonrenal fibrosis (NRF) or renal fibrosis (RF). Red arrows indicate damaged tubules. Scale bars: 100 μm (enlarged insets: 20 μm). (B–D) Quantification of ZDHHC18 expression levels (B), kidney injury score (C), and fibrosis area (D) (n = 8 NRF, n = 15 RF). (E–H) Pearson’s correlation analysis showing the relationship between ZDHHC18 staining intensity and kidney injury score (E), sCr levels (F), BUN levels (G), and eGFR (H) in patients with RF (n = 15). (I) Photomicrographs of α-SMA and vimentin staining in kidney sections from the NRF and RF groups. Scale bar: 20 μm. (J) Quantitative analysis of α-SMA and vimentin staining in NRF (n = 8) and RF (n = 15) groups. (K) Pearson’s correlation analysis between ZDHHC18 levels and α-SMA and vimentin staining in the RF group (n = 15). Data are presented as the mean ± SD. ***P < 0.001, by 2-tailed Student’s t test (B–D and J).
Zdhhc18 expression is upregulated in fibrotic kidneys of mice. RNA-Seq data showed Zdhhc18 upregulation in fibrotic kidneys of UUO mice or mice with folic acid–induced (FA-induced) renal fibrosis (Figure 2A). We confirmed these findings using UUO and FA mouse models (Supplemental Figure 1A). Quantitative real-time PCR (qRT-PCR) and Western blot (WB) analysis confirmed that the expression of ZDHHC18 was upregulated during renal fibrosis (Figure 2, B and C). Only a few ZDHHC family members (Zdhhc14, Zdhhc15, Zdhhc17, Zdhhc18, and Zdhhc24) were upregulated by both UUO and FA mice, and Zdhhc18 showed the highest upregulation (Supplemental Figure 1B). Apt1 and Apt2, thought to be responsible for depalmitoylation, were not upregulated during UUO- or FA-induced renal fibrosis (Figure 2A and Supplemental Figure 1C). Next, we examined the expression of fibrosis markers and their correlation with Zdhhc18 expression. Our findings revealed a significant increase in the mRNA expression of Col1a1, Col3a1, Fn1, and Acta2 in fibrotic kidneys compared with that in the control group (Supplemental Figure 2A). Among all Zdhhc family members, Zdhhc18 exhibited the strongest positive correlation with these fibrosis markers (Supplemental Figure 2, B and C). Single-cell combinatorial indexing RNA-Seq (sci-RNA-Seq) analysis revealed that Zdhhc18 had highest cumulative expression in the failed repair of the proximal tubule (PT-FR) subtype during UUO progression (Figure 2D) and displayed the most significant upregulation in PT-FR and descending limb–thin ascending limb of the loop of Henle (DTL-ATL) subtypes at day 10 after UUO compared with healthy kidneys (Figure 2E). Both PT-FR, a proximal tubule subtype, and DTL-ATL, part of the loop of Henle, belong to TECs. Notably, PT-FR became the largest TEC subpopulation in late-stage UUO (from day 6 to day 10) (Figure 2F). sci-RNA-Seq data showed that ZDHHC18 expression was also slightly upregulated in endothelial cells. Immunofluorescence experiments showed that ZDHHC18 was markedly increased in the VCAM1+ PT-FR cells of UUO and FA mice (Figure 2G), but no obvious changes were observed in CD31+ endothelial cells (Supplemental Figure 1D). These findings suggest that Zdhhc18 abundance was predominantly upregulated in TECs from fibrotic mouse kidneys.
Figure 2Zdhhc18 is elevated in mouse fibrotic kidneys. (A) Heatmap of ZDHHCs and APTs gene expression in mouse kidneys after UUO (GSE125015) and FA injection (GSE65267). (B) Zdhhc18 mRNA levels in mouse UUO kidneys (0, 3, 7, and 10 days) and at FA kidneys (0, 7, 14, 21, and 28 days) (n = 4). (C) WB analysis of ZDHHC18 expression in kidneys after 10 days of UUO and 28 days of FA, with ZDHHC18 levels quantified using ImageJ software (NIH) (n = 3). (D) Distribution and relative expression of Zdhhc18 in different types of renal cells from mouse kidneys after UUO (GSE190887). (E) Relative expression of Zdhhc18 on different days and cell subpopulations in UUO mouse kidneys. (F) Connected bar plots displaying the proportional abundance of subpopulations of TECs in different days of UUO. (G) Confocal microscopy images show staining for ZDHHC18 (green), VCAM1 (red), and DAPI (blue) in UUO (left) and FA (right) kidneys. Scale bars: 20 μm. Data are presented as the mean ± SD. **P < 0.01 and ***P < 0.001, by 2-tailed Student’s t test. PT, proximal tubule; PT-AcInj, acute injury PT; PT-Inj, injured PT; PT-R, repairing PT; DCT, distal convoluted tubule; CNT, connecting tubule; PC, principal cell of collecting duct; DTL, descending limb of loop of Henle (LoH); ATL, thin ascending limb of the LoH; TAL, thick ascending limb of the LoH; ICA, type A intercalated cell of the collecting duct; ICB, type B intercalated cell of the collecting duct; EC, endothelial cell; Pod, podocyte; Fib, fibroblast; Myofib, myofibroblast; Uro, urothelium; avg. exp., average expression; pct. exp., percentage of expression.
ZDHHC18 enhances the TGF-β1–induced partial EMT in TECs. To explore the role of ZDHHC18 upregulation in TECs, we next established an in vitro cell model by culturing human tubular epithelial HK-2 cells in the presence of TGF-β1. In response to TGF-β1 stimulation, ZDHHC18 expression was upregulated at both the mRNA (Supplemental Figure 3A) and protein levels (Supplemental Figure 3B). ZDHHC18 knockdown in HK-2 cells (Supplemental Figure 3, C and D) resulted in upregulation of E-cadherin (CDH1), an epithelial cell marker, when treatment with TGF-β1 (Supplemental Figure 3E). The expression levels of TGF-β1–induced mesenchymal markers (SNAI1, SNAI2, and VIM) and fibrosis markers (COL1A1, COL3A1, FN1, and ACTA2) were significantly downregulated in response to ZDHHC18 knockdown (Supplemental Figure 3, E and F). In contrast, overexpression of ZDHHC18 (Supplemental Figure 3, G and H) increased the expression of TGF-β1–induced mesenchymal markers and fibrosis markers (Supplemental Figure 3, I and J).
Next, we generated mice with TEC-specific deletion of Zdhhc18 using a conditional gene-targeting approach based on Cre/loxP recombination (Supplemental Figure 4A). Mice that were homozygous for the Zdhhc18-loxP–targeted allele (Zdhhc18fl/fl) were bred with TEC-specific Cdh16 Cre lines, which was confirmed by tail genotyping (Supplemental Figure 4B). WB analysis confirmed the reduction of ZDHHC18 protein levels specifically in renal tubules of Zdhhc18 conditional-KO (Zdhhc18-CKO) mice, with no detectable changes in glomeruli or endothelial cells (Supplemental Figure 4C). We also established an in vitro cell model of partial EMT by culturing primary TECs (PTECs) from Zdhhc18-CKO and WT mice. Following TGF-β1 treatment, PTECs from WT mice exhibited increased expression of mesenchymal markers (Snai1, Snai2, and Vim) concomitant with reduced expression of the epithelial marker Cdh1. Whereas Zdhhc18-CKO PTECs showed lower expression of TGF-β1–induced mesenchymal markers and higher expression of the epithelial marker Cdh1, these data for PTECs together with the data for HK-2 cells suggest that Zdhhc18 promotes TGF-β1–induced partial EMT in vitro. In the process of renal fibrosis, TECs undergoing partial EMT contribute to fibroblast activation and inflammatory niche formation through TGF-β1 and proinflammatory cytokines secretion (25, 26). Our results indicate that Zdhhc18 deficiency attenuated TGF-β1–induced expression of Tgfb1, a key cytokine for fibroblast activation (Supplemental Figure 4E). In addition, Zdhhc18 KO suppressed the expression of proinflammatory cytokines and chemokines (Il1b, Il6, Tnfa, Ccl2, and Ccl5) (Supplemental Figure 4F).
TEC-specific Zdhhc18 deletion inhibits renal fibrosis. The Zdhhc18-CKO mice were born without any apparent abnormalities. At 2 months of age, there were no significant differences in terms of body weight (Supplemental Figure 5A), kidney weight (Supplemental Figure 5B), sCr levels (Supplemental Figure 5C), or BUN levels (Supplemental Figure 5D) between the Zdhhc18-CKO mice and Zdhhc18fl/fl littermates without Cre (WT). Furthermore, under normal conditions, we observed no apparent alterations in kidney structure (Figure 3B), indicating that specific deletion of Zdhhc18 in TECs did not lead to phenotypic changes in mice.
Figure 3TEC-specific Zdhhc18 deficiency inhibits renal fibrosis induced by UUO in mice. (A) Experimental design. Kidneys from WT and Zdhhc18-CKO mice were harvested after sham or UUO surgery for 10 days. (B) Gross appearance of the kidneys (scale bar: 2 mm) as well as images of H&E, Masson, and α-SMA staining of WT and Zdhhc18-CKO mouse kidneys after UUO. Scale bar: 20 μm. (C–E) Quantification of the tubular injury score (C), Masson staining of interstitial collagen (D), and α-SMA+ area (E) (n = 8). (F) Immunofluorescence images of staining. Square frames highlight digital enlargement of the tubule; white arrows indicate costaining for vimentin and E-cadherin. Scale bars: 20 μm. (G) Statistical analysis showing the percentage of vimentin- and E-cadherin–stained areas (n = 8). (H) Immunofluorescence images of ZDHHC18 (red), TGF-β1 (green), and α-SMA (white) expression in the kidneys of Zdhhc18-CKO mice after UUO. Scale bar: 20 μm. (I) p-P65, F4/80, and CD3 staining of kidneys from WT and Zdhhc18-CKO mice after UUO. Blue arrow indicates pP65+ cells in the renal tubules. Scale bars: 20 μm and 10 μm (enlarged insets). (J–L) Quantification of the proportion of pP65+ cells (J), F4/80+ area (K), and proportion of CD3+ cells (L) (n = 8). (M) mRNA levels of fibrotic markers in kidneys of Zdhhc18 CKO and WT mice (n = 8). Data are presented as the mean ± SD. **P < 0.01 and ***P < 0.001, by 2-way ANOVA with Tukey’s multiple-comparison test (C–E, G, and J–M).
In response to UUO (Figure 3A), Zdhhc18 KO significantly improved kidney morphology and attenuated tubular injury, as shown by H&E staining (Figure 3, B and C). Compared with those in the sham control group, mice subjected to UUO displayed significant extracellular matrix (ECM) accumulation, but tubule-specific deletion of Zdhhc18 decreased the extent of renal tubulointerstitial fibrosis, as demonstrated by Masson and Picrosirius red staining (Figure 3, B and D, and Supplemental Figure 5, E and F). Periodic acid–Schiff (PAS) staining revealed significant tubular dilatation and atrophy in the obstructed kidneys. However, these changes were much milder in Zdhhc18-CKO mice than in WT mice (Supplemental Figure 5E). Moreover, the interstitial accumulation of α-SMA+ myofibroblasts was upregulated by UUO, but the upregulation of α-SMA+ myofibroblasts was significantly reduced by Zdhhc18 KO (Figure 3, B and E). Immunofluorescence analysis revealed that UUO induced partial EMT, as indicated by the presence of remaining TECs on the basement membrane and coexpression of the epithelial cell marker E-cadherin and the mesenchymal cell marker vimentin. However, this partial EMT progression was inhibited by Zdhhc18 KO (Figure 3, F and G). Injured epithelial cells produce TGF-β1, which promotes the proliferation and activation of interstitial fibroblasts (25). Inhibition of partial EMT in TECs downregulates TGF-β1 expression and consequently attenuates fibroblast activation (26). Consistent with this, we found that Zdhhc18 KO reduced TGF-β1 expression in renal tubules after UUO and decreased the number of interstitial α-SMA+ myofibroblasts (Figure 3H). We further found that Zdhhc18 deficiency mitigated inflammatory reactions by decreasing the levels of proinflammatory mediators, such as Il1b, Il6, Il18, and Tnfa (Supplemental Figure 5G), and chemokines, such as Ccl1, Ccl2, Ccl3, Ccl4, and Ccl5 (Supplemental Figure 5H), suppressed nuclear phosphorylated NF-κB (p-P65) levels in tubular cells and F4/80+ macrophages and CD3+ T cell infiltration (Figure 3, I–L). Attenuated inflammation and decreased fibroblast activation collectively resulted in mitigated fibrosis in the kidneys of Zdhhc18-KO mice. qRT-PCR further confirmed that the expression of the fibrosis markers Col1a1, Col3a1, Fn1, and Acta2 and of Tgfb1 was significantly suppressed in the UUO model following Zdhhc18 KO (Figure 3M).
We also used the FA model to investigate the role of Zdhhc18 in kidney fibrosis (Figure 4A). The results of sCr and BUN measurements indicated that the severity of acute renal failure was not different between WT and Zdhhc18-KO mice on day 2 following FA administration (Supplemental Figure 6, A and B). On day 28 after FA administration, Zdhhc18-KO mice exhibited significantly improved renal function compared with WT mice, with lower sCr and BUN levels (Supplemental Figure 6, A–C). H&E, PAS, Masson, and Picrosirius red staining revealed that tubule-specific deletion of Zdhhc18 ameliorated tubular atrophy and tubulointerstitial fibrosis in mice on day 28 after FA administration (Figure 4, B–D, and Supplemental Figure 6, D and E). Moreover, the number of interstitial α-SMA+ myofibroblasts was upregulated by FA treatment, but the upregulation of α-SMA was significantly reduced by Zdhhc18 KO (Figure 4, B and E). Immunofluorescence analysis showed that Zdhhc18 KO inhibited the FA-induced partial EMT process in renal TECs (Figure 4, F and G). Similar to the UUO model, Zdhhc18 KO reduced TGF-β1 expression in renal tubules after FA and decreased the number of interstitial α-SMA+ myofibroblasts (Figure 4H). At day 28 after FA injection, Zdhhc18-CKO mice exhibited significantly reduced renal inflammation compared with WT mice, as evidenced by decreased mRNA levels of proinflammatory cytokines (Supplemental Figure 6F) and chemokines (Supplemental Figure 6G), along with attenuated nuclear phosphorylated NF-κB (pP65) in tubular cells, and reduced F4/80+ macrophages and CD3+ T cell infiltration (Figure 4, I–L). Ultimately, the expression of renal fibrosis markers in FA-induced Zdhhc18-KO mice was reduced (Figure 4M). In sum, data from the UUO and FA models demonstrate that TEC-specific KO Zdhhc18 reduced renal fibrosis and inflammation in mouse CKD.
Figure 4TEC-specific Zdhhc18 deficiency inhibits renal fibrosis induced by FA in mice. (A) Experimental design. Harvesting kidneys of WT and Zdhhc18-CKO mice after saline or FA injection for 28 days. (B) Gross appearance of the kidneys (scale bar: 2 mm) as well as images of H&E, Masson, and α-SMA staining of WT and Zdhhc18-CKO mouse kidneys after FA. Scale bar: 20 μm. (C–E) Quantification of the tubular injury score (C), Masson staining of interstitial collagen (D), and α-SMA+ area (E) (n = 8). (F) Immunofluorescence images of staining. Square frame highlight digital enlargement of the tubule; white arrows indicate costaining of vimentin and E-cadherin. Scale bars: 20 μm. (G) Analysis of the percentage of vimentin- and E-cadherin–stained areas (n = 8). (H) Immunofluorescence images of ZDHHC18 (red), TGF-β1 (green), and α-SMA (white) expression in the kidneys of Zdhhc18-CKO mice after FA. Scale bar: 20 μm. (I) pP65, F4/80, and CD3 staining of WT and Zdhhc18-CKO mouse kidneys after FA. Blue arrow indicates pP65+ cells in the renal tubules. Scale bars: 20 μm (Enlarged: 10 μm). (J–L) Quantification of the proportion of pP65+ cells (J), F4/80+ area (K) and the proportion of CD3+ cells (L) (n = 8). (M) mRNA levels of fibrotic markers in kidneys of Zdhhc18 CKO and WT mice (n = 8). Data are presented as the mean ± SD. **P < 0.01 and ***P < 0.001, by 2-way ANOVA with Tukey’s multiple-comparison test (C–E, G, and J–M).
Zdhhc18 overexpression exacerbates renal fibrosis. We used an adeno-associated virus (AAV) serotype 9 carrying the Cdh16 (a kidney-specific cadherin exclusively expressed in TECs) promoter to overexpress GFP-tagged Zdhhc18 in TECs. Four weeks after AAV injection, we found that GFP was expressed in the renal tubules but not in the glomeruli (Supplemental Figure 7A). IHC and qRT-PCR results showed that ZDHHC18 expression in the renal tubules was significantly increased (Supplemental Figure 7, B and C). Next, we analyzed AAV9-Ctrl and AAV9-Zdhhc18 mice in the UUO kidney disease model (Figure 5A). Zdhhc18 overexpression significantly exacerbated renal tubular inj
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