Chronic kidney disease (CKD) is characterized by renal structure disorder and dysfunction, including glomerulosclerosis, tubule-interstitial fibrosis and glomerular filtration rate (GFR) decrease caused by many initial diseases. Because of the excretory role, kidney injury induced by metabolic disease is also a common cause of kidney diseases, including hyperhomocysteinemia (HHcy). Also, as an antineoplastic drug, the most common side effect of cisplatin is kidney injury. Epithelial-mesenchymal transition (EMT) is an essential mechanism of fibrosis. Zinc finger E-box binding homeobox 1 (ZEB1) has been identified as a directly transcriptional inhibitor of EMT, repressing CDH1 (gene of E-cadherin) transcription by binding to two E-box sequences in the promoter. cAMP-response element binding protein (CREB) is a crucial transcription factor involved in the development of fibrosis in multiple organs, including heart, liver, lung and kidney. However, the underlying mechanism involved in CREB and EMT in kidney injury remains unknown. Recent years, Hydrogen sulfide (H2S) has been identified as a protective gas among various pathological progresses. It was mainly generated by cystathionine β synthase (CBS), cystathionine γ lyase (CSE) in kidney. Protein post-translational modifications (PTMs) have been implicated as vital mechanisms of regulating structure, location, activity and function of protein. Studies have confirmed that H2S plays a part in protein PTMs through S-sulfhydration, which is cystetine (-SH) of protein combines with HS- from H2S to form -SSH. In addition, S-sulfhydration acts important roles in inflammation, fibrosis, lipid metabolism and cell death. Therefore, it is worth exploring the precise mechanism of H2S in alleviating kidney injury. In the present study, our findings focused on investigating how H2S prevented kidney fibrosis via regulating EMT.

2.1 Animal models C57BL/6 mice aged 8 weeks were used to carry out animal experiments, which obtained from Capital Medical University. All of generation according to with the Guide for the Care and Use of Laboratory Animals. The HHcy mice were fed with chow contained 2.5 methionine for 32 weeks meanwhile the Control mice were fed with normal diet. For inducing classic kidney injury, cisplatin (TargetMol, USA) was injected 5 mg/kg a week and last for 4 weeks in total of 20 mg/kg. NaHS was injected 5.6 mg/kg per day and last for a month. Adeno-associated virus serotype 9 (AAV9) was constructed to express wild-type CREB (CREB-WT) or CREB S-sulfhydration site-mutated CREB (CREB-C286A, Cys286 mutated to alanine). The AAV-null group was established by empty vector, as a negative control. 2×1012 VG/ml AAV was in situ injected into mouse kidney cortex (40 μl per mouse). 2.2 Detection of H2S concentration The serum and kidney H2S concentration were detected by Methylene blue spectrophotometric method using H2S Content Assay kit (Solarbio, BC2050) according to its instruction. H2S level in cells was also measured by a selective fluorescent probe 4-Methyl-7-azidocoumarin (AzMC, Sigma Aldrich, 802409, 50 μM at 37 ◦C for 1h). The images were collected by STED (TCS SP8 STED, Leica). 2.3 Protein S-sulfhydration detection The modified biotin switch assay was used to measure the S-sulfhydration level of CREB in tissues or HK2 cells. Briefly, cells or tissues were lysed in HEN buffer and then centrifuged at 12500 rpm for 15 min at 4 ◦C. The protein blocked by blocking buffer was added to the lysates at 50 ◦C for 30 min with frequent shaking (600 r/min). The precipitations were resuspended in 4 mM Biotin-HPDP (Abcam, ab145614) labeling buffer (20 mM Biotin-HPDP diluted in dimethylformamide) for 4 h at room temperature (at least 25 ◦C). The streptavidin-agarose beads (Sigma Aldrich, S1638) incubated with protein overnight at 4 ◦C and subjected to western blot with an anti-CREB antibody. 2.4 Statistical analysis All statistical analyses were performed using GraphPad Prism 8.0 software. Data were represented as the means ± SD. Unpaired Student’ s t-test was used to analyze data with only two groups. One-way analysis of variance (ANOVA) was used to compare multiple groups. P-value <0.05 was considered as statistically significant.

3.1 H2S alleviated EMT through inhibiting CREB transcriptional activity in HHcy induced kidney fibrosis To investigate the role of H2S on kidney function, HHcy induced kidney fibrosis model was constructed by feeding chow containing 2.5% methionine for 32 weeks. First, we detected serum Hcy and H2S levels. The result showed that the serum Hcy level was increased while both serum and renal H2S levels were decreased. In addition, we have also confirmed that H2S levels were down-regulated in other HHcy models. To further verify the role of H2S, we also established diabetes mice model (HFD diet for 32 weeks and STZ 40 mg/kg for 3 days, intraperitoneally). The renal H2S level was decreased in diabetes mellitus (DM) mice compared with Control. We found that intravenous insulin tolerance tests (ITT) and oral glucose tolerance tests (OGTT) were alleviated by supplement of NaHS (exogenous of H2S), which indicated that H2S could reversed both glucose homeostasis and insulin resistance. In HHcy model, the result showed that CBS was the dominant H2S generation enzyme in kidney tissue. The H2S fluorescent probe 7-Amino-4-methylcoumarin was used to detect the H2S level of HK2 cells incubation with Hcy, which indicated that Hcy could also decreased H2S level in vitro. Hematoxylin and eosin (H&E) and Masson staining were indicated that kidney fibrosis of HHcy model. Also, the protein expression and mRNA expression showed that collagens levels were increased in kidney of HHcy model. However, there were no difference of renal functions, such as serum creatinine (Scr), blood urea nitrogen (BUN), and uric acid (UA) in HHcy model compared with Control model. Moreover, there is no evidence showed that HHcy induced apoptosis. As we all know, EMT was the essential pathway involved in kidney fibrosis, we further confirmed that HHcy induced EMT in kidney fibrosis model, while H2S supplement reversed the progress. These results showed that HHcy induced EMT and kidney fibrosis, which could be suppressed by H2S. It is well known that CREB is an important transcription factor of fibrosis, also promotes EMT in kidney injury. The transcriptional activity of CREB is presented by its nuclear translocation and regulation of target genes. The results showed that Hcy up-regulated the CREB protein level of nuclear and promoted CREB nuclear translocation in HK2 cells. We also measured CREB fibrosis related target genes transforming growth factor (Tgfb2), α-smooth muscle actin (Acta2), Collagen type I alpha 2 chain (Col1a2) and Fibronectin (Fn1) in HK2 cells and confirmed that H2S donor lessened the expressions induced by Hcy. Additionally, the protein and mRNA level of CREB were up-regulated in HHcy induced kidney fibrosis model, and down-regulation of CREB could reverse EMT induced by Hcy. Also, the regulation of CREB could affect the kidney injury factors including inflammation and fibrosis induced by Hcy. All of above suggest that kidney fibrosis and EMT were induced by HHcy, while administration of H2S suppressed the progress by inhibiting CREB transcriptional activity. 3.2 H2S S-sulfhydrated CREB at Cys286 to inhibit its transcriptional activity Recent years, S-Sulfhydration has been identified as an important post-translational modification mediated by H2S, which is considered to be the vital way in regulating protein structure, location and function etc. To determine the protective role of H2S in EMT of kidney, we confirmed that H2S S-sulfhydrated CREB by treated with NaHS and dithiothreitol (DTT, a reducing agent). The result showed that NaHS up-regulated the CREB S-sulfhydration level and DTT abolished the effect. To identify the S-sulfhydrated cysteine site, CREB-encoding plasmids in which Cys286, Cys296, or Cys316 were mutated to alanine (C286A, C296A, or C316A) or WT plasmids were transfected into HK2 cells. We confirmed that the effective site was Cys286, which exactly locates in nuclear localization sequence of CREB. Moreover, the cysteine sites were highly conserved among different species (human, mouse, rat). Then we observed that Hcy down-regulated the S-sulfhydration of CREB and H2S supplement reversed it. While the effect of H2S on up-regulating S-sulfhydration was abolished after Cys286 mutation of CREB, and subsequent reversed its transcriptional activity, including nuclear translocation and target genes. These results confirmed that inhibition of CREB transcriptional activity by H2S was dependent on S-sulfhydration of Cys286. 3.3 H2S suppressed EMT by inhibiting CREB transcriptional activity in cisplatin- induced kidney injury To further explore the broad-spectrum role of CREB in kidney injury, we also constructed cisplatin induced kidney injury models. First, we analyzed the renal function of cisplatin treated mice. The data showed that the kidney/body weight ratio, Scr and BUN levels were increased after cisplatin injection, while treatment with NaHS reversed Scr and BUN levels but not kidney/body weight ratio. As we expected, both serum and kidney H2S concentration were down-regulated in cisplatin mice model. Interestingly, the serum H2S level was decreased and homocysteine level was increased on the first day after cisplatin injection, which were earlier than renal function indicators such as Scr and BUN. H2S supplement abolished cisplatin induced cell inflammation and apoptosis both in vivo and in vitro. The histological results showed the kidney damage including tubular cells died and fell off to block the lumen, tubular interstitial fibrosis, and significant podocyte injury (mainly manifested as exacerbated diffuse foot processes) was showed by transmission electron micrography in cisplatin mice, while mice with administration of NaHS showed a lesser extent of kidney injuries. Moreover, kidney injury molecule-1(KIM1) and neutrophil gelatinase associated lipocalin (NGAL) as biomarkers indicated early kidney injury, were remarkably decreased by H2S donor in cisplatin induced kidney dysfunction model. Mice treated with NaHS also showed lower expression of fibrosis related proteins and higher expression of epithelial marker in cisplatin model. The fluorescent density data showed NaHS inhibited cisplatin induced EMT in HK2 cells. These results confirmed that H2S could protect kidney injury in cisplatin mice. Next, we detected H2S generation enzyme in kidney tissues of cisplatin model, the result showed that both CBS and CSE were down-regulated after cisplatin injection. The H2S fluorescence intensity of HK2 cells also confirmed that the lower H2S level with administration of cisplatin compared with PBS. Then we analyzed the CREB expression in nuclear through fluorescence intensity and protein expression. Mechanistically, H2S donor attenuated the CREB nuclear translocation induced by cisplatin. Also, the expression levels of target genes of CREB were reversed by H2S donor in cisplatin treated HK2 cells. The above results illustrated that supplement of H2S also had a prospectively therapeutic effect in kidney injury induced by cisplatin. 3.4 Inhibition of CREB S-sulfhydration by Cys286 mutation abolished the suppression of CREB transcriptional activity of H2S in cisplatin treated HK2 cells In order to explore the protective role of H2S in cisplatin model, we measured the S-sulfhydration level of CREB, the result showed that lower expression in cisplatin model compared with PBS treatment likewise. Correspondingly, H2S donor failed to increase S-sulfhydration level of CREB after Cys286 mutation in treatment with cisplatin HK2 cells. The prevention roles of H2S in down-regulating genes including death of epithelial cells, inflammation and fibrosis, which were also abolished by Cys286 mutation of CREB in HK2 cells with cisplatin. Furthermore, after CREB mutation at Cys286, H2S failed to promote CREB nuclear translocation. In summary, the above results demonstrated that inhibition of CREB S-sulfhydration by Cys286 mutation prevented H2S protective role in cisplatin induced CREB transcriptional activation. 3.5 Restraint of CREB S-sulfhydration by Cys286 mutation abolished the effect of H2S on EMT in cisplatin treated mice To further determinate the role of S-sulfhydration of CREB in cisplatin induced kidney injury, we constructed AAV injection mice model to express CREB-WT or CREB C286A. In vivo three-dimensional kidney imaging had proved that the injection efficiency of AAV was basically consistent. Two weeks after AAV injection, we treated mice with NaHS (5.6 mg/kg, daily) for 4 weeks and then cisplatin for 4 weeks (5 mg/kg, 1 time per week). After 2 times injection of cisplatin, the body weight of mice declined dramatically among all groups, while there was little difference between groups before sacrificed. First, we evaluated the renal functions, cisplatin induced kidney dysfunction was still obviously when CREB overexpression. Meanwhile, the results showed no difference in kidney/body weight ratio, Scr and BUN between AAV-WT and AAV-C286A groups although the early renal function indicators showed extent trend. The mRNA levels of kidney injury, as indicated by inflammation, fibrosis and apoptosis factors, showed that CREB S-sulfhydration by Cys286 mutation might affect kidney injury mainly from fibrosis in vivo. Renal histological observations indicated that H2S donor could alleviate kidney fibrosis partially while S-sulfhydration of CREB mutation at Cys286 reversed the effect. These results illustrated that inhibition of CREB S-sulfhydration by Cys286 mutation abolished the role of H2S in kidney fibrosis in vivo. Mechanistically, we further confirmed that CREB S-sulfhydration affect fibrosis through EMT. Moreover, NaHS could inhibit the nuclear translocation of CREB in CREB-WT-treated and cisplatin injection mice but not in CREB-C286A-treated and cisplatin injection mice. These results revealed that S-sulfhydration of CREB could regulate EMT and fibrosis by modulating its transcriptional activity. 3.6 NaHS suppressed CREB’s binding to ZEB1 and inhibited EMT To further explore the promotion role of CREB in EMT, dual luciferase reporter assay was used to verified whether CREB suppressed EMT directly. The data showed that CREB could not reduce CDH (epithelial gene) directly. To uncover CREB effect, an affinity-based mass spectrometry was performed to identify CREB binding partners in HEK293T cells and ZEB1 was among binding partners. It is well known that ZEB1 is an essential and direct transcription factor in EMT. We then used STRING interaction network to predict the relation between CREB and ZEB1. The prediction showed that there might be gene neighborhood (green line) and co-expression (black line). Then we confirmed the binding between CREB and ZEB1 using CoIP. After downregulating CREB expression by siRNA, the result showed that the interaction between CREB and ZEB1 was decreased. Moreover, the interaction was enhanced after Hcy treatment while H2S donor shorten the bind. These results indicated that the interaction between CREB and ZEB1 might play an important role in EMT.

In conclusion, this study identified that H2S could alleviated kidney injury through S-sulfhydrated CREB at Cys286. Furthermore, the effect of S-sulfhydration CREB at Cys286 in EMT and fibrosis were confirmed by AAV-CREB injection to mice. Mechanistically, our results showed that CREB could bind to ZEB1 thus indirectly promoted EMT. These findings not only provided evidences of relationship between S-sulfhydration and kidney fibrosis but also proposed a more precisely perspective target in preventation and treatment of kidney diseases.