Introduction
Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD) and end-stage renal failure, constituting a major health concern worldwide (1). Damage to the glomer-ular filtration unit is one of the most important pathological characteristics of DN. The filtration unit comprises capillary endothelial cells, the glomerular basement membrane and podocytes, which are specialized epithelial cells. Once considered to be primarily quiescent and terminally differentiated cells, podocytes have been identified as a key factor in DN (2). Podocyte injury is characterized by the pathological loss of regularity in branching and widening of the foot processes, termed 'foot process effacement'. Severe insults lead to podo-cyte loss by apoptosis or detachment. Numerous studies have reported that disruption of multiple renal signaling pathways is crucial for the progression of such pathological damage (3,4). However, there are currently no effective therapeutic options for DN in the clinical setting. Indeed, the precise regulatory signaling pathways through which podocytes are injured in DN are largely unclear.
Autophagy is a major catabolic pathway through which mammalian cells degrade and recycle macromolecules and organelles. The major roles of autophagy are the removal of damaged organelles, degradation of proteins, and reconstitution of intracellular metabolism for the maintenance of intracellular homeostasis and cell health (5). Generally, autophagy is upregulated under conditions of oxidative stress and hypoxia in podocytes. Recently, convincing evidence indicates that, in the case of diabetes, autophagy plays a pivotal role in maintaining the homeostasis of lysosomes in podocytes. The impairment of autophagy, as it is implicated in the pathogenesis of podocyte loss, may lead to massive overflow proteinuria in DN (6-8). However, the exact role of autophagy in podocytes remains elusive.
Connexins (Cxs) are a multigenic family of transmem-brane proteins that form gap junctions and participate in the exchange of information between cells. Connexin 43 (Cx43) is considered to be the most abundant and widely expressed gap junction protein. Extensive studies have revealed that, in addition to its role in intercellular communication, Cx43 mediates gene transcription, cytoskeleton dynamics, ATP exocytosis, vesicle release and cell stress (9). Sawai et al (10) reported the presence of Cx43 in normal podocytes in a linear pattern, and demonstrated a shift in this linear distribution in patients with DN. Our previous studies also indicated that upregulation of Cx43 is involved in podocyte injury (11), suggesting that Cx43 may be a critical regulator in podo-cytes under DN conditions. Furthermore, Cx43 has recently been implicated in inflammation and fibrosis. Inhibiting Cx43 may alleviate kidney damage and maintain renal function. Therefore, new therapies targeting Cx43 blockade in ideal cell populations may be a viable option for effectively inhibiting the progression of CKD (12). Interestingly, Cx43 rapidly modulates autophagy response, playing a critical role in cell apoptosis (13). However, the effect of Cx43 on the regulation of podocyte autophagy under DN conditions remains unclear.
The aim of the present study was to determine the effect of Cx43 on impaired autophagic flux, and to determine whether the regulation of Cx43 can protect podocytes under DN conditions.
Materials and methods
Antibodies and reagents
Rapamycin (RP) and chloroquine (CQ) were purchased from Sigma-Aldrich; Merck KGaA. Antibodies against LC3, mammalian target of rapamycin (mTOR) and p-mTOR were acquired from Cell Signaling Technology, Inc. Anti-Cx43, anti-podocin, anti-nephrin and anti-p62 antibodies were obtained from Abcam. Anti-GAPDH was purchased from CWBio.
Animals
The study protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. A total of 24 male Sprague-Dawley rats (aged 5-6 weeks and weighing ~190 g) were housed under specific pathogen-free conditions at optimal temperature with a 12-h light/dark cycle, and were allowed free access to standard food and water. The rats were randomly divided into four groups: Group 1, PBS-infused rats (control, n=6); group 2, streptozocin (STZ; 60 mg/kg)-infused rats (n=6); group 3, STZ (60 mg/kg)-infused rats with scrambled siRNA (SCR, n=6); and group 4, STZ (60 mg/kg)-infused rats with Cx43 siRNA [oligodeoxynucleotide antisense (AS), n=6]. At the end of the 28-day infusion period, the rats were weighed and blood and urine samples were collected. The blood urea nitrogen and urine protein levels were analyzed according to the manufacturer's protocol (R&D Systems, Inc.). Tail capillary blood glucose levels were monitored with a glucometer (Accu-Chek Performa; Roche Diagnostics GmbH).
Cell culture
The immortalized mouse podocyte cell line MPC5 was kindly provided by Dr Junwei Yang (Nanjing Medical University) and the cells were cultured as previously described (14). Podocytes were differentiated without interferon-γ at 37°C for 14 days prior to the experiments. Differentiated podocytes were incubated in medium containing 0.1% fetal bovine serum for 24 h. The podocytes exposed to HG (30 mM) were then cultured for various time periods.
Transfection of small interference RNA
Podocytes were transfected with Cx43 siRNA (50 nM) (sense, 5′-AAAGUUGCUGCUGGACAU GAATT-3′ and antisense, 5′-UUCAUGUCCAGCAGCAACUUUTT-3′) or negative control siRNA (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense, 5′-ACGUGACACGUUCGGAGAATT-3′) for 24 h using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Thereafter, the level of targeting protein with knockdown of Cx43 was detected by western blotting.
Western blotting
The cells were harvested after treatment with the different compounds for the indicated times. Protein levels were detected by western blotting according to established protocols (15). Primary antibodies against Cx43 (1:1,000), LC3 (1:1,000), p62 (1:2,000), podocin (1:1,000), synaptopodin (1:1,000), mTOR (1:1,000), p-mTOR (1:1,000) and GAPDH (1:2,000) were used.
Annexin V-fluorescein isothiocyanate conjugated with propidium iodide (PI) staining
Podocyte injury was quantified by Annexin V/PI staining (BD Biosciences) following the manufacturer's protocol. Briefly, cells were harvested and washed twice with PBS. Subsequently, the cells were resuspended in 100 µl binding buffer, then incubated with 5 μl Annexin V and 10 μl PI for 15 min at 25°C in the dark. After mixing gently in 400 μl of binding buffer, the cells were observed under a fluorescence microscope (DS-Ri1; Nikon Corporation).
Immunofluorescence staining
Paraffin sections (3-μm) were prepared and incubated with primary antibodies against synaptopodin, Wilms' tumor-1 (WT-1) and p-mTOR in PBS containing 1% BSA overnight at 4°C after blocking with 5% BSA for 1 h. Subsequently, the sections were incubated with secondary antibody in PBS containing 1% BSA in the dark. After washing with PBS, cell nuclei were stained with DAPI. Immunostained samples were visualized under a fluorescence microscope (Nikon Corporation).
Immunohistochemistry
Paraffin-embedded sections were used for immunohistochemistry. Briefly, the sections were incubated with primary antibodies against Cx43 and podocin overnight at 4°C. The sections were then analyzed using a streptavidin peroxidase detection system (Maixin) according to the manufacturer's protocol. Reactions were conducted using a DAB substrate kit (Maixin) and counterstaining was performed using hematoxylin. The sections were visualized under a microscope (Nikon Corporation).
Statistical analysis
Data are presented as the mean ± the standard error of the mean. When more than two groups were compared, one-way analysis of variance followed by Bonferroni's correction was employed to analyze the differences using SPSS 22.0 statistical software (IBM Corp.). A two-sided P-value of <0.05 was considered to indicate statistically significant differences.
Results
Cx43 silencing attenuates HG-induced podocyte injury
It was observed that podocyte injury increased significantly in the HG group, as did the expression of Cx43, suggesting that abnormal activation of Cx43 may be involved in HG-induced podocyte injury. To examine this hypothesis, Cx43 function was inhibited with Cx43 siRNA in the podocytes. siRNA-control served as a negative control. Interestingly, following transfection with Cx43 siRNA, we observed that, compared with the siRNA-control group, podocyte injury was markedly attenuated, as evidenced by the number of Annexin V/PI-positive cells (green) (Fig. 1A). In addition, increased levels of podocin and synaptopodin (surrogate markers for podocytes) were observed in podocytes transfected with Cx43 siRNA via western blot analysis (Fig. 1B). These findings indicate that the inhibition of Cx43 plays a protective role against HG-induced podocyte injury.
Cx43 negatively regulates autophagic flux by enhancing mTOR signaling in HG-induced podocytes
In addition, impaired autophagic flux was also observed in HG-induced podocytes, as demonstrated by the increased expression levels of p62 and LC3II/LC3I. Convincing evidence has demonstrated that connexins play a key role in autophago-some formation (16). To examine whether Cx43 regulates autophagic flux in HG-induced podocytes, Cx43 siRNA was also used to inhibit Cx43 expression. Of note, compared with podocytes transfected with NC-siRNA, the LC3II/LC3I ratio and the level of p62 expression were markedly decreased in Cx43 siRNA-treated podocytes, as detected by western blotting, indicating that Cx43 also plays a key role in regulating autophagic flux. Subsequently, the exact mechanism of the regulatory role of Cx43 on autophagic flux was investigated. Accumulating evidence has demonstrated that mTOR signaling is a pharmacological target for autophagy regulation (17). Therefore, attention was focused on mTOR signaling. The results revealed that the p-mTOR/mTOR protein ratio decreased significantly in the Cx43 siRNA group (P<0.05; Fig. 2), confirming that Cx43 inhibited autophagy by activating the mTOR signaling pathway in HG-induced podocytes.
Impaired autophagic flux blocks the degradation of Cx43 in HG-induced podocytes
Accumulating evidence demonstrates that Cx43 is also regulated by autophagy under certain pathological conditions (18,19), suggesting that autophagy may regulate Cx43 expression in HG-induced podocytes. To examine this hypothesis, CQ and RP, acting as an autophagy inhibitor and an autophagy inducer, respectively (20,21), were used to regulate autophagic flux. Interestingly, compared with the HG group, the level of Cx43 increased significantly in the HG + CQ group (P<0.05). By contrast, the level of Cx43 decreased significantly in the HG + RP group compared with the HG group (P<0.05; Fig. 3). These results indicated that Cx43 activation is also regulated by impaired autophagic flux in HG-induced podocytes.
Cx43 silencing improves autophagic flux in the glomeruli of DN rats
To determine the effect of Cx43 on autophagic flux in the glomeruli in a DN model in vivo, Cx43 siRNA or scrambled negative control siRNA was administered to the rats via the tail vein. After induction of diabetes, the DN rats and Cx43 siRNA-treated DN rats were compared in terms of body weight, blood urea nitrogen (BUN), urine protein and blood glucose levels. Of note, body weight was higher while blood glucose, urine protein and BUN levels were lower in Cx43 siRNA-treated diabetic rats compared with their control diabetic littermates (Table I). Immunohistochemical analysis revealed that Cx43 siRNA treatment significantly reduced Cx43 expression in the glomeruli (Fig. 4A). Interestingly, compared with scrambled negative control-injected mice, Cx43 siRNA administration efficiently reversed the enhanced autophagic flux, as detected by western blotting (Fig. 4B). Thus, the inhibition of Cx43 attenuated the enhanced autophagic flux in the glomeruli of rats with DN.
Cx43 negatively regulates autophagic flux by enhancing mTOR signaling in the glomeruli of DN rats
To examine whether mTOR signaling is involved in the regulatory effect of Cx43 on autophagic flux in vivo, mTOR levels were measured. Consistently with the results in vitro, immunofluorescence analysis revealed that the number of p-mTOR-positive cells decreased in the glomeruli of DN rats following treatment with Cx43 siRNA (Fig. 5A). Furthermore, the p-mTOR/mTOR protein ratio was markedly decreased, as confirmed by western blotting (Fig. 5B). Therefore, activation of the mTOR pathway may negatively contribute to Cx43-regulated autophagic flux.
Cx43 silencing attenuates podocyte injury in DN rats
To determine the role of Cx43 in podocyte injury in vivo, immunostaining for WT-1 and synaptopodin was performed. The results revealed that the number of podocytes was significantly increased following treatment with Cx43 siRNA (P<0.05; Fig. 6A and B). Similarly, compared with the NC-siRNA-treated DN rats, podocin (a marker for podocytes) also increased after knockdown of Cx43, as assessed by immunohistochemical staining analysis (Fig. 6C). Thus, Cx43 activation appears to promote podocyte injury.
Discussion
The critical role of podocyte injury, the characteristic pathological change of DN, in the initiation and progression of DN has been recognized for several years, and impaired autophagy appears to play a key role in podocyte injury (22,23). It was recently reported that Cx43 is involved in the autophagic process (24). However, whether Cx43 plays a critical role in podocyte autophagy in DN remains largely unknown. The present study demonstrated the involvement of increases in Cx43 expression and impaired autophagic flux in HG-induced podocyte injury. The silencing of Cx43 expression ameliorated impaired autophagic flux and reduced podocyte injury by suppressing the mTOR pathway. In vivo studies indicated that the inhibition of Cx43 improved impaired autophagic flux in STZ-induced DN animal models. Furthermore, the pathogenic effect of Cx43 on podocyte injury was also confirmed. These findings may facilitate the identification of novel therapeutic targets for the treatment of podocyte injury in DN.
Autophagy ('self-eating') is a tightly regulated process that delivers senescent intracellular constituents to lysosomes for degradation in order to maintain intracellular homeostasis. An emerging body of research demonstrated that autophagy regulation is closely linked to human health (25,26). Consistently with previous studies on aldosterone-induced podocyte injury (27), we herein observed that impaired autophagic flux was involved in HG-induced podocyte injury. Furthermore, increased Cx43 expression was also observed in association with this pathogenic process. However, whether Cx43 plays a critical role in impaired autophagic flux remains elusive.
Accumulating evidence has demonstrated that Cx43 may be involved in the process of podocyte injury in various kidney diseases (10,11,28). Our in vitro and in vivo data demonstrated that the expression of Cx43 was also elevated by treatment with HG, which may facilitate podocyte injury. Interestingly, after silencing Cx43 expression, HG-induced injury in cultured podocytes was partially attenuated by improving impaired autophagic flux, suggesting that Cx43 was partially involved in HG-induced podocyte injury and that the amelioration of impaired autophagic flux represents a novel mechanism mediating the effect of Cx43 on podocyte injury. However, the exact regulatory mechanisms of Cx43 on impaired autophagic flux have yet to be fully elucidated.
mTOR is a well-known master regulator of cellular metabolism, growth and proliferation in response to a wide range of triggers. The deregulation of mTOR signaling, which plays a key role in the regulation of autophagy, has been implicated in a number of human diseases, including diabetes, neurodegenerative diseases and cancer (29,30). Interestingly, in the present study, the mTOR signaling pathway acted as a critical regulator involved in Cx43/autophagy-mediated podocyte injury in DN. It was observed that mTOR signaling was activated after stimulation with HG in vivo and in vitro, indicating that an mTOR-dependent mechanism may be involved in Cx43-mediated podocyte injury in DN, which is consistent with previous results (31,32). Furthermore, after inhibition of Cx43 function by Cx43 siRNA, the p-mTOR/mTOR protein ratio decreased markedly, followed by a decrease in autoph-agic flux, indicating that Cx43 may regulate autophagy flux by negatively affecting mTOR signaling, which may be partially responsible for podocyte injury in DN.
In addition, Cx43 was found to be regulated by autophagic flux. It has been reported that the initiation of autophagy includes the formation of the phagophore, the initial sequestering compartment, which expands into an autophagosome. Completion of the autophagosome is followed by fusion with lysosomes and degradation of its contents, allowing complete flux, or flow, through the entire pathway. As RP promotes initiation of the autophagic flux, LC3I to LC3II turnover is increased in the glomeruli. Of note, LC3II is also a selective substrate of autophagy. Hence, LC3II degradation is blocked in the presence of CQ, which inhibits the acidification of organelles and, subsequently, autophagosome-lysosome fusion (33). This may explain why CQ treatment caused a greater accumulation of LC3II in the HG + CQ group when compared with the HG + RP group in the present study (even though CQ is an inhibitor of autophagy). Accordingly, compared with the HG group, the level of Cx43 increased significantly in the HG + CQ group. On the contrary, the level of Cx43 decreased significantly in the HG + RP group, indicating that a negative feedback mechanism is involved in Cx43-modulated podocyte injury to maintain the homeostasis of podocytes.
In summary, the findings of the present study uncovered a previously unknown mechanism implicated in podocyte injury under DN conditions. It was confirmed that upregulation of Cx43 expression negatively regulates autophagy by activating the mTOR signaling pathway in DN, while inhibiting Cx43 expression improved impaired autophagic flow and reduced podocyte damage. Hence, the Cx43-autophagy loop may be used as a novel therapeutic target for the development of new drugs for DN.