Alcohol promotes renal fibrosis by activating Nox2/4-mediated DNA methylation of Smad7

Alcohol consumption causes renal injury and compromises kidney function. The underlying mechanism of the alcoholic kidney disease remains largely unknown. In this study, an alcoholic renal fibrosis animal model was firstly employed which mice received liquid diet containing alcohol for 4-12 weeks. The Masson’s Trichrome staining analysis showed that kidney fibrosis increased at week 8 and 12 in the animal model which was further confirmed by albumin assay, Western blot, immunostaining and real-time PCR of fibrotic indexes (collagen I and α-SMA). In vitro analysis also confirmed that alcohol significantly induced fibrotic response (collagen I and α-SMA) in HK2 tubular epithelial cells. Importantly, both in vivo and in vitro studies showed alcohol treatments decreased Smad7 and activated Smad3. We further determined how the alcohol affected the balance of Smad7 (inhibitory Smad) and Smad3 (regulatory Smad). Genome-wide methylation sequencing showed an increased DNA methylation of many genes and bisulfite sequencing analysis showed an increased DNA methylation of Smad7 after alcohol ingestion. We also found DNA methylation of Smad7 was mediated by DNMT1 in Ethyl alcohol (EtOH)-treated HK2 cells. Knockdown of Nox2 or Nox4 decreased DNMT1 and rebalanced Smad7/Smad3 axis, and thereby relieved EtOH-induced fibrotic response. The inhibition of reactive oxygen species by the intraperitoneal injection of apocynin attenuated renal fibrosis and restored renal function in the alcoholic mice. Collectively, we established novel in vivo and in vitro alcoholic kidney fibrosis models and found that alcohol induces renal fibrosis by activating oxidative stress-induced DNA methylation of Smad7. Suppression of Nox-mediated oxidative stress may be a potential therapy for long-term alcohol abuse-induced kidney fibrosis.


Introduction
Alcohol abuse is becoming a large social issue and its related diseases result in a heavy social-economic burden [1]. The long-term alcohol consumption is positively correlated with the risk of developing chronic kidney disease (CKD) [2,3], and associated with increased incidence of albuminuria, and is linked with a poor prognosis of CKD [4][5][6][7][8][9].
Accumulating evidence from basic research indicates alcohol abuse has detrimental effects on kidney. Although the liver is a primary organ most likely to be injured by alcohol, excessive alcohol intake destroys the structure and function of the kidneys, including tubular and glomerular dysfunction and electrolyte disorders [10]. Ethanol significantly increases reactive oxygen species (ROS) production, resulting in inflammatory response in kidney and renal cell apoptosis [11,12]. The chronic alcohol exposure also caused insulin resistance, β-cell dysfunction and hyperglycemia which potentially lead to glomerulosclerosis and interstitial fibrosis in kidney [13,14].
Hyperlipidemia induced by chronic alcohol consumption had the enhanced lipid oxidation, cytokine production and apoptosis in kidney [15,16]. Moreover, alcohol intoxication aggravated tubular abnormalities via oxidative stress and inflammation in the rhabdomyolysis-induced acute kidney injury (AKI) [17] and provoked renal fibrosis through myofibroblast infiltration and extracellular matrix (ECM) production in the ischemic reperfusion (IR)-induced AKI [18].
Given overwhelming data confirming the renal toxicity of alcohol abuse, there are significant gaps in the field of alcoholic renal disease. First, most studies have relied on animal models due to a dearth of in vitro models. Second, the mechanisms by which alcohol induces renal damage, especially at the late stage of renal fibrosis, are still obscure.
TGF-β1 plays a critical role in renal fibrosis by activating downstream regulatory Smads, including Smad2 and Smad3, via binding to TGF-β1 receptors. Activated Downloaded from https://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20191047/865195/cs-2019-1047.pdf by guest on 12 January 2020 Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20191047 Smad2/3 form a complex with Smad4 and then translocate into the nucleus, bind to target genes and initiate transcription [19,20]. Smad7 binds to Smurf2 to form an E3 ubiquitin ligase and degrades the TGF-β1 receptors, thereby negatively regulated TGF-β1/Smad signaling [21]. DNA methylation mediated by DNA methyltransferases, including DNMT1, DNMT3a and DNMT3b, may contribute to Smad7 reduction in liver fibrosis [22]. Additionally, previous studies showed that ROS could significantly induce DNA methyltransferases [23,24]. In the current study, we detected the suppression of Smad7 and activation of TGF-β/Smad3 in fibrotic kidney in response to alcohol, which was positively correlated with ROS production in kidneys, so we hypothesized that ROS-induced DNA methyltransferases may lead to DNA methylation and reduction of Smad7, thereby enhanced TGF-β/Smad3-mediated alcoholic renal injury.
Taken together, in the current study we aim to establish novel in vivo and in vitro alcohol-induced renal injury models and then studied the underlying mechanisms of alcoholic renal fibrotic response.

RNA extraction and real-time PCR
Total RNA was extracted by RNeasy Isolation Kit (Qiagen, Valencia, CA, USA) and converted into cDNA by the Reverse transcription kit (Bio-Rad, Hercules, CA, USA).
Real-time PCR was performed by using the Bio-Rad iQ SYBR Green supermix with Opticon2 (Bio-Rad, Hercules, CA) as previously described [28]. The sequences of primers are as follows: Human α-SMA, forward 5'-ATCAAGGAGAAACTGTGTTATGTAG-3', The ratio for the mRNA of interest was normalized to β-actin and presented as the mean ± S.D.

DCF Assay
Alcohol induced cellular oxidative stress were measured by the DCF (2',7' -dichlorofluorescein) Assay Kit (Beyotime, Jiangsu, China). DCF, the oxidized products of ROS, was measured by fluorescence microscopy with excitation of 488 HK2 cells were treated by ethanol (100mM) for 1 to 4 days, stained by DHE, and measured under fluorescence microscopy using an excitation wavelength between 480-520 nm and an emission wavelength between 570-600 nm. [26].

Determination of MDA and GSH
The levels of MDA and GSH in cell or in mouse tissues were detected by a commercial kit (Jiancheng Co., Nanjing, China) according to the manufacturer's instructions [26].  The resulting PCR products were purified using a MinElute Gel Extraction Kit (Generay, Shanghai, China) and cloned into pTG19-T vector (Generay). Individual clones were grown and plasmids were purified using a Plasmid DNA Purification Kit Downloaded from https://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20191047/865195/cs-2019-1047.pdf by guest on 12 January 2020 Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20191047 (Generay). For each condition, at least 10 clones of each sample were selected and sequenced by the Shanghai Generay Biotech Co., Ltd.

Determination of blood pressure and blood alcohol concentration
Blood pressure was measured using a tail-cuff method. Briefly, mice were gently restrained in a holder and warmed to 37 °C by a heating platform under the holder.
Animals were acclimated in the environment for 5-10 min after occlusion cuffs and pulse transducers (Softron, BP-2010 Series) were placed on the tail. The values of at least 10 readings for each mouse were used for blood pressure. The levels of blood alcohol were determined by headspace GC according to the manufacturer's instructions.

Statistical analyses
Data was analyzed by two sample t-test or one-way analysis of variance (ANOVA) followed by Tukey post hoc tests using GraphPad Prism 5 software. A p-value less than or equal to 0.05 (two tailed) was considered as the statistical significance. Figure 1A. We found although mice in Lieber-DeCarli ethanol liquid diet-feed group lose weight significantly in the first 4 weeks, their body weight then increased to comparable level to control group. ( Figure   1B). As the level of serum ethanol increased significantly compared with control group (supplementary Figure 1A), serum urea nitrogen and microalbumin analysis indicated alcohol treatment declined the renal function compared the control diet ( Figures 1C and 1D). We also found that ethanol treatment had no impact on blood

Alcohol induced aberrant methylation in kidney
The whole-genome methylation sequencing revealed significant changes of protein methylation in alcoholic kidneys (Figures 4A). We then examined the protein levels of the three major DNA methyltransferases, DNMT1, DNMT3a and DNMT3b in ethanol treated HK2 cells and in mice using Western blot analysis. Results showed that all three methyltransferases were significantly elevated in alcohol-stimulated tubular epithelial cells and alcoholic mice (Figures 4B and 4C, respectively).

Alcohol reduced Smad7 and activated TGF-β1/ Smad3 signaling-driven fibrosis in kidney
We previous reported that interrupting TGF-β1/Smad3 and Smad7 signaling leads to renal fibrosis in various renal disease models [28,29,32,33]. We further analyzed gene  Figure 5B). In addition, we tested the effects of other organic solvents, like dimethyl sulfoxide and isopropanol, on Smad7 expression to confirm the specificity of ethanol treatment, the results showed that ethanol, but not dimethyl sulfoxide or isopropanol, reduced the Smad7 level significantly (Supplementary Figure 3). Real-time PCR also indicated that TGF-β1 mRNA was significantly induced by the alcohol consumption ( Figure 5C). Furthermore, EtOH-induced fibrotic response in TβRII wild type (WT) cells was significantly suppressed in EtOH-treated TβRII dominant negative (DN) cells ( Figure 5D). Consistent findings from in vitro study also indicated the imbalance of Smad3 and Smad7 signaling in ethanol-treated HK2 cells ( Figure 5E).

DNMT1 promoted the loss of Smad7 in alcohol-treated mTECs
To determine which DNA methyltransferases played the critical role in alternating Smad7 methylation, we successfully knocked down the methyltransferases of DNMT1, DNMT3a, and DNMT3b in mTECs ( Figure 6A, B). Among the EtOH-treated mTECs with knockdown of DNMT1, DNMT3a, and DNMT3b respectively, inhibition of DNMT1 significantly restored Smad7 levels compared with alcohol-treated empty vector cells ( Figure 6C).

Alcohol induced reactive oxygen species production in vivo and in vitro
We next investigated the underlying mechanisms by which alcohol induced DNMT1 and resulted in the imbalance of Smad3/Smad7. We found that MDA levels were significantly increased in kidneys, while GSH, an antioxidant index, decreased in vivo ( Figures 7A and 7B), suggesting the significant increase of ROS levels by alcohol. In addition, DCF fluorescence data showed that alcohol increased ROS in HK2 cells, which was further confirmed by DHE staining (Figures 7D, E). Nox2 and Nox4 are two important Nox family members that mediate ROS production [34]. We found that Nox2 and Nox4 levels increased after alcohol ingestion in mice ( Figure 7C). These results were consistent with the findings that Nox2 and Nox4 were highly induced in EtOH-treated HK2 cells in a time-dependent manner ( Figure 7F).

Alcohol promoted renal fibrosis through Nox2/4-dependent mechanisms
To determine the function of Nox2 and Nox4 in EtOH-treated tubular epithelial cells, we silenced Nox2 and Nox4 respectively (Figure 8A and 9A). Western blot and real-time PCR results show knockdowns of either Nox2 or Nox4 suppressed EtOH-induced fibrosis (Collagen I and α-SMA) in cells (Figures 8B&C and 9B&C).
Moreover, Western blot results showed that silencing Nox2 or Nox4 alleviated DNMT1 protein level and restored the balance of Smad7/Smad3 ratio ( Figure 8D and 9D). These results suggested that both Nox2 and Nox4 affected DNA methylation and caused imbalance of Smad7/Smad3 in EtOH-induced fibrosis.
Apocynin prevented alcohol-induced renal fibrosis by suppressing DNA methyltransferase and rebalancing Smad3/Smad7 signaling Next, we determined if apocynin, a NADPH oxidase inhibitor, prevented alcohol-induce renal fibrosis. We found that alcohol induced Nox2 and Nox4 were significantly reduced in mice receiving apocynin twice per week for 8 weeks ( Figure   10A). Consistently, apocynin decreased levels of ROS and improved renal function in mice as demonstrated by GSH, MDA and BUN assays analysis ( Figures 10B, 10C and 10D). Treatment of apocynin attenuated total collagen deposition ( Figure 11A) as well as other fibrosis indexes (Collagen I and α-SMA) in kidneys. Importantly, apocynin reduced DNMT1 level and thereby restored the balance of Smad7 and Smad3 signaling in alcoholic kidneys ( Figure 11B, 11C and 11D). The findings were further confirmed by in vitro study that apocynin attenuated EtOH-induced fibrotic response in tubular epithelial cells (Figures 11Eand 11F). Additionally, treatment of apocynin didn't alter the level of aldehyde dehydrogenases (ALDHs) Figure 4).

DISCUSSION
In the present study, we established and validated novel models of alcohol-induced renal fibrosis both in vivo and in vitro. We provided experimental evidence showing alcohol impaired renal function and increased ECM deposition mice as well as promoted fibrotic response in tubular epithelial cells. The models established by us may be used to elucidate mechanisms of alcoholic kidney injury and serve as a useful platform to screen drugs for the injury. We also demonstrated that alcohol promoted renal fibrosis by activating Nox2/4-mediated DNA methylation of Smad7, which enhanced Smad3-mediated renal fibrosis. Importantly, inhibition of ROS by apocynin protected against alcoholic renal fibrosis.
We measured the detrimental effects of chronic, heavy alcohol intake on kidneys in a novel murine model modified from the NIAAA model [25]. Results show alcohol aggravated renal injury and fibrotic response in a time-dependent manner; deposition of collagen and accumulation of α-SMA+ myofibroblasts were key characteristics.
These findings were consistent with a series of well-designed studies by Dr.
McIntyre's group, which showed that chronic ethanol ingestion induced oxidative kidney injury and fibrosis [35][36][37]. Of note, the renal fibrotic response may be led by the abnormal repair from the ethanol-induced acute kidney injury, which need to be further determined [38]. Additionally, it is important to note that limited in vitro data has been collected due to lack of in vitro models for evaluating alcohol-induced kidney injury; this is clearly an obstacle for understanding the mechanism more deeply. Given this, we established a new model by treating tubular epithelial cells with ethanol at various time points. We found that ethanol stimulation decreased the E-cadherin while increasing protein level of collagen and α-SMA in a time-dependent manner.
We also found that DNMT1-mediated DNA methylation of Smad7 is the central mechanism for alcohol-induced renal fibrosis. We, and others, identified that activation of TGF-β1/Smad3 signaling and loss of Smad7 enhanced renal fibrosis in Downloaded from https://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20191047/865195/cs-2019-1047.pdf by guest on 12 January 2020 various kidney disease models [39,28,29,32,19,33,40]. In the present study, genome-wide methylation sequencing and bisulfite sequencing showed that alcohol induced DNA methylation of Smad7 led to downregulation of Smad7 and overactivation of TGF-β/Smad3 signaling. By knocking down three major DNA methyltransferases (DNMT1, 3a and 3b), we showed that DNA methylation of Smad7 was mediated by DNMT1. Previous results showed that DNMT1 is induced in TGF-β-treated mouse kidney fibroblasts and regulates the expression of genes involved in fibroblast activation [41]. In addition, silencing the DNMT1 gene decreased Smad7 expression in response to TGF-β1 in liver fibrosis [22]. All these lines of evidence indicate DNMT1-mediated imbalance of Smad7/Smad3 signaling mediates alcohol-induced renal fibrosis.
Interestingly, we found that Nox-mediated ROS production plays a role in the induction of DNA methyltransferases in alcohol-stimulated kidneys. The increased expression of DNA methyltransferases caused by oxidative stress is gaining attention [42][43][44][45]. Kidneys are rich in alcohol-oxidizing enzyme [46], which may be associated with a complex interaction between alcoholic kidney injury and EtOH-induced oxidative stress [47]. Oxidative stress is a key trigger in kidney injury caused by alcohol, directly or indirectly. NADPH, specifically Nox2 and Nox4, are important enzymes in mediating oxidative stress-induced renal injury [48,36,47,34]. Indeed, Nox4 contributes to several types of renal diseases, including diabetic nephropathy, hypertensive nephropathy and obstructive nephropathy [34]. In the current study, we found alcohol induced Nox2 and Nox4 production both in vivo and in vitro. And, blocking these enzymes reduced ECM production in response to alcohol, indicating these enzymes are involved in alcohol-induced renal fibrosis. It is worth noting that the alcohol-induced fibrotic response peaked at week 8 and reduced at week 12, consistent with the finding that acute ethanol administration causes a dose-dependent injury of the antioxidant system [47]. It is possible that prolonged alcohol stimulation may activate antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, thereby protecting against renal fibrosis in a negative feedback loop Finally, we evaluated the therapeutic effect of ROS cleavage on alcoholic renal fibrosis. Apocynin, inhibition NADPH oxidases activity and ROS generation and scavenging, was intraperitoneally injected to inhibit ROS production in alcohol-fed mice [48,27,53,54]. Apocynin restored renal function and suppressed alcohol-induced renal fibrotic response, which was further confirmed in EtOH-stimulated HK2 cells.
These results indicate that targeting Nox-mediated ROS production may be a viable therapy for treating alcohol-induced renal injury.
Of note, in our model, we found that ethanol treatment caused hepatic injury and steatosis, but no significant fibrosis in liver. However, we found evidence showing that ethanol diet induced fibrotic response in kidney, we presumed that liver may be an organ with better metabolic and regenerative capacities compared with kidney.
In conclusion, based on our novel in vivo and in vitro alcoholic renal injury models, we found that Nox2/Nox4-mediated ROS production induced DNA methylation of Smad7 via DNMT1-dependant mechanism (Figure 12). Suppression of Smad7 and activation of Smad3 appears to promote alcohol-induced renal fibrosis. We further showed that inhibition of Nox-mediated oxidative stress by apocynin protected against alcoholic renal injury. Taken together, these data underscore a potential therapy to treat long-term alcohol abuse-induced kidney fibrosis by targeting ROS production.

Clinical perspectives
(i) Alcohol consumption causes renal injury but the underlying mechanism of the alcoholic kidney disease remains largely unknown.
(ii) Established novel in vivo and in vitro alcoholic kidney fibrosis models and found that alcohol induces renal fibrosis by activating oxidative stress-induced DNA