Angiotensin‑converting enzyme‑2 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation

  • Authors:
    • Ziye Chen
    • Xinpan Chen
    • Yu Bai
    • Zongli Diao
    • Wenhu Liu
  • View Affiliations

  • Published online on: July 28, 2020     https://doi.org/10.3892/mmr.2020.11372
  • Pages: 3008-3016
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Abstract

Angiotensin‑converting enzyme 2 (ACE2), an important component of the renin‑angiotensin system, protects against renal tubulointerstitial fibrosis, but its level of involvement in the mechanism of diabetic nephropathy (DN) currently remains unclear. Herein, the effects of ACE2 in DN and the associated mechanisms were investigated using serum and renal biopsy specimens from patients with DN and control participants, and human renal proximal tubular epithelial cells (HRPTEpiCs). The present study determined that the circulating concentration of ACE2 was high, but renal ACE2 expression was markedly lower, and there was abundant expression of Arkadia, an E3 ubiquitin ligase, in patients with DN. In vitro, ACE2 attenuated high‑glucose‑induced tubular epithelial to mesenchymal cell transition (EMT), which was demonstrated by increased expression of α‑SMA and loss of E‑cadherin expression, as demonstrated by western blot analysis and reverse transcription‑quantitative PCR. Adenovirus‑mediated ACE2 overexpression was also revealed to significantly inhibit Arkadia expression and alleviated high‑glucose‑induced EMT, while ACE2 inhibition had the opposite effects. Furthermore, western blot analysis demonstrated that ACE2‑alleviated EMT was associated with downregulated Arkadia and increased SMAD family member 7 (Smad7) protein, followed by TGF‑β/Smad pathway inhibition in HRPTEpiCs. In conclusion, ACE2 is protective in DN, which may be due to the inhibition of Arkadia‑mediated Smad7 degradation, whereby TGF‑β/Smad‑mediated EMT is ameliorated in high‑glucose‑stimulated HRPTEpiCs.

Introduction

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide (1). One of the main mechanisms of DN is renin-angiotensin system (RAS) activation, and inhibition of the RAS can slow the development of diabetic kidney injury (2). Angiotensin-converting enzyme 2 (ACE2) is a component of the RAS that has counter-regulatory functions to the classic ACE/angiotensin II (Ang II)/Ang II receptor type 1 (AT1R) axis (3). In human renal tissues, ACE2 is highly expressed in renal tubular epithelial cells, and low tubular ACE2 expression has been revealed to be associated with the progression of renal fibrosis in DN (4). However, the mechanisms by which ACE2 protects against high-glucose-induced renal fibrosis remain unclear. Moreover, circulating ACE2 has not been well studied in patients with DN.

Tubulointerstitial fibrosis is essential in the development of end-stage renal disease in DN. The renal tubular epithelial to mesenchymal cell transition (EMT) plays a key role in this process. It has been demonstrated that the transforming growth factor-β (TGF-β)/SMAD family member (Smad) signaling pathway is a potent initiator and regulator of EMT (5). Previous studies have revealed that TGF-β-induced EMT is primarily activated by Smad3 phosphorylation and inhibited by Smad7, a negative feedback regulator of the TGF-β1/Smad signaling pathway that protects against fibrosis by inducing receptor degradation, which halts the recruitment and phosphorylation of Smad3 (5,6). A previous study by Liu et al (7) revealed that the level of Smad7 protein was dynamically modulated by the ubiquitin-proteasome degradation pathway, and this was mainly attributed to the E3 ubiquitin ligase Arkadia (also referred to as ring finger protein 111). However, whether this process occurs in DN and the mechanisms involved have yet to be determined.

The purpose of the present study was to investigate the protective mechanism of ACE2 in DN. To this end, renal tissue from patients with DN and primary human renal proximal tubular epithelial cells (HRPTEpiC) stimulated with a high glucose concentration were studied. In addition, the serum ACE2 expression was measured in DN patients with microalbuminuria.

Materials and methods

Blood samples and renal tissue collection

Blood samples were collected from 10 patients with DN and 10 non-diabetic volunteers from December 2018 to March 2019. Diabetic renal tissue specimens were obtained from five patients with a pathological diagnosis of DN and control specimens were obtained from five patients with minimal change in nephropathy without renal interstitial fibrosis. None of the recruited participants had liver dysfunction, heart failure, malignancy and none were pregnant. The non-diabetic volunteers were those who visited the hospital for routine physical examination and had no history of diabetes or nephropathy with normal blood glucose and serum creatinine concentrations. The blood samples and renal tissues were obtained from Beijing Friendship Hospital, following a protocol approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University. Each participant provided their written informed consent before being included in the study.

Serum ACE2 measurements

Venous blood was placed in disposable sterile test tubes and centrifuged at 1,000 × g, 25°C for 10 min to isolate serum. The serum samples were then transferred to cryovials and frozen at −80°C until assayed.

The serum ACE2 expression levels were measured using a commercial ELISA kit (cat. no. E01A0499; Shanghai BlueGene Biotech Co., Ltd.), according to the manufacturer's instructions.

Histopathological examination

Renal sections were fixed in 4% neutral-buffered paraformaldehyde for 24 h at 25°C, embedded in paraffin, and cut into 4 µm-thick sections, which were then prepared for Periodic acid-Schiff (PAS), Periodic Schiff-Methenamine Silver (PASM) or Masson's modified trichrome histological staining. The stained renal tissue sections were scored as previously described (8). All the scoring was performed by one observer who was blinded to the group from which the tissue specimens originated. The data were recorded and used to compare the diabetic with the non-diabetic groups.

Immunohistochemistry

Paraffin-embedded sections of renal tissues were deparaffinized in xylene, rehydrated through an alcohol gradient from 99.5% to 70% alcohol, and underwent antigen retrieval in citrate buffer (cat. no. P0081; Beyotime Institute of Biotechnology, Inc.). Endogenous peroxidase activity was blocked with 3% H2O2, and then the sections were incubated with 5% normal goat serum at 37°C for 30 min. Sections were then incubated overnight at 4°C with primary antibodies targeting ACE2 (1:100; cat. no. ab87436; Abcam) or Arkadia (1:100; cat. no. OM290104; Omnimabs), followed by incubation with a biotinylated secondary antibody (1:2,000; cat. no. SP-9000; OriGene Technologies, Inc.) for 30 min at room temperature. After thorough rinsing, the antigens were detected by 3,3′-diaminobenzidine staining (OriGene Technologies, Inc.). Sections were counterstained with hematoxylin, dehydrated and cover-slipped. The stained renal tissue sections were scored as aforementioned.

Cell culture and treatments

The HRPTEpiCs were purchased from ScienCell Research Laboratories, Inc., (cat. no. 4100). Cells were cultured in epithelial cell medium (EpiCM) supplemented with 2% fetal bovine serum and epithelial cell growth supplement (EpiCGS; all from ScienCell Research Laboratories, Inc.), benzylpenicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere containing 5% CO2 as described in previous studies (9,10). Actively growing cells in their second to third passage were used for experiments. Cells were seeded at a density of 2.5×105 cells/well in 6-well plates. When cell confluency reached 70–80%, the medium was replaced with serum-free epithelial cell medium. After serum starvation for 12 h, cells were incubated in medium containing a normal (5.5 mmol/l D-glucose) or high glucose concentration (30 mmol/l D-glucose; Sigma-Aldrich; Merck KGaA) for 4, 12, 24, 48 or 72 h (9). Subsequently, the cells were stimulated with a high glucose concentration for 48 h in the presence or absence of adenoviruses overexpressing ACE2. For treatment with an ACE2 inhibitor, the ACE2 inhibitor DX600 (0.5 mmol/l; BioVision, Inc.) was added to high glucose medium for 48 h. Finally, the cells were treated with small interfering (si)RNA targeting Arkadia or scramble siRNA for 48 h while remaining in high glucose medium. The cells were then collected by trypsin digestion for subsequent analysis.

Adenovirus transduction and siRNA transfection

Adenoviruses overexpressing ACE2 (pHBAd-BHG; Hanbio Biotechnology Co., Ltd), siRNA targeting Arkadia (5′-TTCCATGCAGAAAGAGATTTGTAAA-3′) and a scrambled siRNA (5′-TTCTCCGAACGTGTCACGTAA-3′) were purchased from Hanbio Biotechnology Co., Ltd. HRPTEpiCs were either infected with adenoviruses using the polybrene method at an MOI of 100 or transfected with 100 nmol/l siRNA at 37°C and in 5% CO2 for 6 h. All transfections were carried out using Transfection Reagent (Hanbio Biotechnology Co., Ltd.,). The medium was then replaced with fresh epithelial cell medium after 6-h incubation. Empty adenovirus and scrambled siRNA were used as negative controls. HRPTEpiCs were used for experiments 48 h after transfection.

Western blot analysis

Western blotting was performed as previously described (9). Briefly, after blocking nonspecific binding with 5% BSA (Sigma-Aldrich; Merck KGaA), the membranes were incubated overnight at 4°C with primary antibodies. Primary antibodies against ACE2 (1:1,000; cat. no. 4355), Smad3 (1:1,000; cat. no. 9523), phosphorylated (p)-Smad3 (1:1,000; cat. no. 9520), E-cadherin (1:1,000; cat. no. 14472), α-smooth muscle actin (α-SMA; 1:1,000; cat. no. 19245) were purchased from Cell Signaling Technology, Inc. Antibodies targeting Arkadia (1:500; cat. no. ab174624), Smad7 (1:1,000; cat. no. ab190987), TGF-β (1:1,000; cat. no. ab92486) or β-actin (1:1,000; cat. no. ab8226) were purchased from Abcam. After the first incubation, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; cat. no. ZB-2301 or ZB-2305; ZSGB-BIO; OriGene Technologies, Inc.). Target proteins were detected and analyzed using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore). Densitometry analysis was performed using ImageJ software (v1.52; National Institutes of Health).

Reverse transcription-quantitative PCR (RT-qPCR)

RNA was extracted from cells with TRIzol® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), following the manufacturer's protocol. Complementary DNA (cDNA) was generated using a RevertAid cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using a SYBR1 Taq™ kit (Takara Bio, Inc.,) based on the ABI PRISM 7000 system. The thermocycling conditions consisted of an initial denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 20 sec, 55°C for 20 sec, and 72°C for 20 sec. β-actin was used as the reference gene. The results were analyzed using the 2−ΔΔCq method (9). The primer sequences were as follows: ACE2 forward, 5′-CATTGGAGCAAGTGTTGGATCTT-3′ and reverse 5′-GAGCTAATGCATGCCATTCTCA-3′; Arkadia forward, 5′-CCACATAGGATGCACCCAAAC-3′ and reverse, 5′-AATTCCCAGTTCCCAGGCA-3′; Smad7 forward, 5′-CCTTAGCCGACTCTGCGAACTA-3′ and reverse, 5′-CCAGATAATTCGTTCCCCCTGT-3′; E-cadherin forward, 5′-GAGAACGCATTGCCACATACAC-3′ and reverse, 5′-GCACCTTCCATGACAGACCC-3′; α-SMA forward, 5′-ACTGGGACGACATGGAAAAG-3′ and reverse, 5′-CATCTCCAGAGTCCAGCACA-3′; and β-actin forward, 5′-TGGGTCAGAAGGACTCCTATG-3′ and reverse, 5′-CAGGCAGCTCATAGCTCTTCT-3′.

Statistical analysis

Data were expressed as the means ± standard deviations (SDs). Comparisons between groups were performed using one-way analysis of variance, followed by the Student-Newman-Keuls test. Statistical analyses were performed using SPSS v17.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

Clinical characteristics of and serum ACE2 expression levels in diabetic and non-diabetic participants

In total, 20 participants were recruited to the present study (10 diabetic and 10 non-diabetic). The characteristics of the study participants are presented in Table I. The age of the diabetic patients (59.0±14.7 years) and non-diabetic volunteers (62.6±17.0 years) were similar (P=0.248). There were no significant differences in serum creatinine or systolic and diastolic blood pressure. However, the serum ACE2 level was significantly higher in patients with diabetes compared with non-diabetic volunteers (10.2±2.6 vs. 24.7±3.9 ng/ml, P=0.007).

Table I.

Clinical characteristics of study subjects.

Table I.

Clinical characteristics of study subjects.

CharacteristicsNon-diabetic (n=10)Diabetic (n=10)P-value
Age, years59.0±14.762.6±17.00.248
Sex, male/female5/55/5
SBP, mmHg135.2±15.4138.1±14.30.423
DBP, mmHg78.5±9.880.2±9.70.527
Glucose, mmol/l4.8±0.49.5±2.30.002
Serum creatinine, µmol/l56.6±7.557.1±10.70.454
UACR, mg/g112.5±11.8
ACE2, ng/ml10.2±2.624.7±3.90.007

[i] Data are presented as the mean ± SD. SBP, systolic blood pressure; DBP, diastolic blood pressure, UACR, urine albumin-to-creatinine ratio; ACE2, angiotensin-converting enzyme 2.

Expression of ACE2 is lower and Arkadia expression is higher in diabetic human kidneys

Since tubulointerstitial fibrosis is one of the major pathological alterations in DN (11), renal fibrosis was evaluated by Masson's trichrome staining in diabetic and non-diabetic human kidneys. Furthermore, immunohistochemistry was used to examine the expression of ACE2 and Arkadia in diabetic and non-diabetic human kidneys. Histologically, PAS and PASM staining demonstrated profound extracellular matrix deposition and Masson's trichrome staining revealed more severe renal fibrosis in diabetic than in non-diabetic human kidneys (Fig. 1A and B). Immunohistochemistry demonstrated significantly lower ACE2 expression in diabetic samples compared with the control kidneys, whereas the expression of Arkadia was higher (Fig. 1C and D).

High glucose reduces ACE2 and induces Arkadia expression and increases α-SMA and decreases E-cadherin expression in HRPTEpiCs

The present study subsequently investigated the effects of high glucose on the expression of ACE2, Arkadia and EMT in HRPTEpiCs. As revealed in Fig. 2, both the protein (Fig. 2A and B) and mRNA (Fig. 2C) expression levels of ACE2 decreased with time, while the mRNA and protein expression levels of Arkadia were increased with time in HRPTEpiCs incubated with a high glucose media. Furthermore, the expression of α-SMA, a myofibroblast marker, increased, whereas that of E-cadherin, an epithelial marker that plays a key role in maintaining the integrity of epithelial cells, decreased.

Smad7 mRNA increases while Smad7 protein expression level decreases in HRPTEpiCs stimulated with high glucose

Western blot analysis was used to determine the expression levels of Smad7 protein in HRPTEpiCs at different time-points during the stimulation of high glucose (Fig. 2A and D). High glucose gradually but significantly reduced the expression of Smad7 protein. To determine whether the decrease in Smad7 protein expression resulted from downregulation of Smad7 mRNA expression, mRNA expression levels were assessed by RT-qPCR. In contrast to the significant decrease in Smad7 protein, the expression levels of Smad7 mRNA increased with time in HRPTEpiCs stimulated with high glucose. These results indicated that Smad7 expression was downregulated at the protein level in the presence of a high glucose concentration.

ACE2 overexpression inhibits Arkadia expression and alleviates high-glucose-induced EMT in HRPTEpiCs

Next, ACE2 was overexpressed or inhibited in HRPTEpiCs stimulated with high glucose. Ad-ACE2 infection significantly increased the expression of ACE2 mRNA (Fig. S1A). The RT-qPCR and western blot analysis revealed that ACE2 overexpression significantly reduced both the mRNA and protein expression of Arkadia (Fig. 3A and B), whereas ACE2 inhibition increased Arkadia mRNA and protein levels (Fig. 4A and B) in HRPTEpiCs stimulated with high glucose. Accordingly, high-glucose-induced EMT, indicated by upregulation of α-SMA and downregulation of E-cadherin expression, was alleviated when ACE2 was overexpressed (Fig. 3A and C) and aggravated when ACE2 was inhibited (Fig. 4A and D).

ACE2 alleviates high-glucose-induced EMT in HRPTEpiCs by downregulating Arkadia-dependent ubiquitin degradation of Smad7 and inhibiting the TGF-β/Smad pathway

HRPTEpiCs were stimulated with high glucose in the presence of an ACE2 inhibitor and/or Arkadia siRNA to explore the mechanism by which ACE2 deactivation promotes EMT when exposed to a high glucose concentration. Transfection with siArkadia significantly decreased Arkadia expression (Fig. S1A). ACE2 inhibition was found to reduce Smad7 protein expression, but induced Smad7 mRNA expression, and substantially activated the TGF-β/Smad signaling pathway, as detected by p-Smad3, in high-glucose-stimulated HRPTEpiCs (Fig. 4A, C and E). To further confirm the effects of the ACE2/Arkadia/Smad7 axis on EMT, high-glucose-stimulated HRPTEpiCs were cotreated with an ACE2 inhibitor and Arkadia siRNA. All the effects of ACE2 deactivation observed were reversed following depletion of Arkadia (Fig. 5). Moreover, the expression of TGF-β, another crucial component of the TGF-β/Smad signaling pathway, was detected and exhibited a similar trend to that of p-Smad3 expression (Fig. S1B and C). Collectively, these findings indicated that ACE2 protected against high-glucose-induced renal fibrosis by inhibiting EMT, which may be at least in part due to inhibition of Arkadia-dependent ubiquitin degradation of Smad7 and the TGF-β/Smad signaling pathway.

Discussion

The present study has demonstrated the relationship between ACE2 and the ubiquitination pathway in DN. Inhibition of ACE2 appeared to suppress EMT in vitro and in vivo via an Arkadia-dependent mechanism. This process would lead to an increase in TGF-β/Smad signaling, the key regulatory pathway in EMT, by targeting Smad7 for ubiquitin-mediated degradation. Moreover, the present study also revealed that serum ACE2 was higher in patients with DN with microproteinuria.

It is widely accepted that the activation of RAS, particularly the intrarenal RAS, is important for the progression of DN (12). ACE2 is an ACE homolog that counter-regulates the RAS. It exists both as a tissue-bound enzyme, which is highly expressed in the proximal tubule brush border, and as a soluble protein that has enzymatic effects in the circulatory system (13,14). ACE2 expression is downregulated in the kidneys of patients with DN (15). More recently, ACE2 knockout was revealed to be associated with EMT and renal fibrosis in mouse models of obstructive, hypertensive and diabetic nephropathy, which feature α-SMA accumulation and E-cadherin deficiency (3,16,17). In contrast, ACE2 overexpression ameliorated renal fibrosis and albuminuria in diabetic mice (18). ACE2 is generally conceived as a renoprotective factor that works by converting Ang II to Ang(1–7), which has its cellular effects via the G protein-coupled receptor Mas (19,20). The cross talk between renal ACE and ACE2, as well as the balance between the ACE/Ang II/AT1R and ACE2/Ang(1–7)/Mas axes have also been emphasized in vivo and in vitro (2124). However, it appears that ACE2 has effects that are far more complex than RAS regulation (3,25). This led to the present investigation to decipher the detailed mechanism by which ACE2 exerts its renoprotective effects in patients with DN and HRPTEpiCs in a high glucose environment.

In the present study, ACE2 expression was downregulated, whereas the expression of Arkadia, an E3 ubiquitin ligase critical for the TGF-β/Smad signaling pathway during EMT (26,27), was upregulated in both diabetic human kidneys and HRPTEpiCs stimulated with high glucose. These results indicated that ACE2 affected EMT by modulating Arkadia expression. Accumulating evidence suggests that Smad7 is a major regulator of the intensity and duration of Smad signals (2831). In the present study, Smad7 protein expression level was decreased while its mRNA expression level was increased. Therefore, it was hypothesized that the decrease in Smad7 protein expression was caused by greater degradation rather than lower expression. Arkadia has been reported to aggravate organ damage by inducing Smad signals via the degradation of Smad7 in pulmonary fibrosis, atrial fibrillation and aristolochic acid nephropathy (3234). In fact, inhibition or overexpression of ACE2 caused upregulation and downregulation of Arkadia expression, respectively, which resulted in changes in Smad7 protein and mRNA expression levels in high-glucose-stimulated HRPTEpiCs. Consistently, Arkadia knockdown increased Smad7 protein expression levels and attenuated high-glucose-induced EMT when ACE2 was inhibited. Therefore, it was hypothesized that ACE2 was able to suppress EMT by preventing Arkadia-dependent ubiquitination, which degrades Smad7 protein in high-glucose-stimulated HRPTEpiCs. This process is essential for renal fibrosis in DN and had not been reported in previous studies.

Another finding of the present study was that patients with DN with microalbuminuria displayed a significantly higher serum ACE2 expression level compared with the controls. Few human studies have previously measured circulating ACE2 in the context of DN. In experimental studies, circulating ACE2 activity has been reported to be higher in mouse models of diabetes (3538). To date, only one study of patients with type I diabetes with vascular complications demonstrated higher ACE2 activity in the circulation (39). In concordance, the present study revealed a high serum ACE2 level in patients with DN with microalbuminuria. Numerous previous studies have linked high circulating ACE2 expression levels with an increased risk of cardiovascular disease, which is common in patients with DN (38,4042). The increased circulating ACE2 expression level in these patients may stem from cardiovascular injury or occur to compensate for the downregulated renal ACE2. However, this result was difficult to confirm due to the small sample size. Nevertheless, the present findings still suggest a possible diagnostic role for the measurement of circulating ACE2 in DN, but further larger-scale clinical studies are still required to confirm this finding.

In conclusion, the present study has demonstrated that intrarenal ACE2 had a protective effect in DN. ACE2 suppressed Arkadia-dependent ubiquitin degradation of Smad7, which may be an essential mechanism by which TGF-β/Smad-mediated EMT was ameliorated in high-glucose-stimulated HRPTEpiCs.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

This study was supported by the Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support, Grant/Award no. ZYLX201824 and the National Natural Science Foundation of China, Grant/Award no. 81570660.

Availability of data and materials

The datasets used and analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

WL and ZC provided the concept and designed the study. ZC and XC performed the experiments, and the results were then interpreted by ZC, ZD and YB. ZC prepared the figures and drafted the manuscript. ZC, ZD and WL edited and revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All procedures were approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University. Each participant provided their written informed consent before being included in the study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

ACE2

angiotensin-converting enzyme 2

DN

diabetic nephropathy

HRPTEpiCs

Human renal proximal tubular epithelial cells

EMT

epithelial to mesenchymal cell transition

RAS

renin-angiotensin system

TGF-β

transforming growth factor-β

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October-2020
Volume 22 Issue 4

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Online ISSN:1791-3004

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Spandidos Publications style
Chen Z, Chen X, Bai Y, Diao Z and Liu W: Angiotensin‑converting enzyme‑2 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation. Mol Med Rep 22: 3008-3016, 2020
APA
Chen, Z., Chen, X., Bai, Y., Diao, Z., & Liu, W. (2020). Angiotensin‑converting enzyme‑2 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation. Molecular Medicine Reports, 22, 3008-3016. https://doi.org/10.3892/mmr.2020.11372
MLA
Chen, Z., Chen, X., Bai, Y., Diao, Z., Liu, W."Angiotensin‑converting enzyme‑2 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation". Molecular Medicine Reports 22.4 (2020): 3008-3016.
Chicago
Chen, Z., Chen, X., Bai, Y., Diao, Z., Liu, W."Angiotensin‑converting enzyme‑2 improves diabetic nephropathy by targeting Smad7 for ubiquitin degradation". Molecular Medicine Reports 22, no. 4 (2020): 3008-3016. https://doi.org/10.3892/mmr.2020.11372