Human Umbilical Cord Mesenchymal Stem Cells Inhibit Pyroptosis of Renal Tubular Epithelial Cells through miR-342-3p/Caspase1 Signaling Pathway in Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the microvascular complications of diabetes. Recent studies suggest that the pyroptosis of renal tubular epithelial cell plays a critical role in DN. Currently, effective therapeutic strategies to counteract and reverse the progression of DN are lacking. Mesenchymal stem cells (MSCs) represent an attractive therapeutic tool for tissue damage and inflammation owing to their unique immunomodulatory properties. However, the underlying mechanisms remain largely unknown. In the present study, we found that human umbilical cord MSCs (UC-MSCs) can effectively ameliorate kidney damage and reduce inflammation in DN rats. Importantly, UC-MSC treatment inhibits inflammasome-mediated pyroptosis in DN. Mechanistically, we performed RNA sequencing and identified that miR-342-3p was significantly downregulated in the kidneys of DN rats. Furthermore, we found that miR-342-3p was negatively correlated with renal injury and pyroptosis in DN rats. The expression of miR-342-3p was significantly increased after UC-MSC treatment. Moreover, miR-342-3p decreased the expression of Caspase1 by targeting its 3′-UTR, which was confirmed by double-luciferase assay. Using miRNA mimic transfection, we demonstrated that UC-MSC-derived miR-342-3p inhibited pyroptosis of renal tubular epithelial cells through targeting the NLRP3/Caspase1 pathway. These findings would provide a novel intervention strategy for the use of miRNA-modified cell therapy for kidney diseases.


Introduction
Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes [1,2] and has been a major cause of end-stage renal disease (ESRD) in patients [3]. Currently, effective therapeutic strategies to counteract and reverse the progression of DN are lacking. DN is caused by continuous increase of blood sugar, accompanied by the production of proteinuria (>0.5 g/24 h). Moreover, DN has been categorized into two stages: microalbuminuria and macroalbuminuria [3,4]. The histopathological characteris-tics of DN are glomerular hyperfiltration rate, thickening of the renal tubular basement membrane, mesangial expansion, and extracellular matrix deposition, which leads to interstitial fibrosis and eventually develops into severe diffuse and nodular glomerulosclerosis of remnant glomeruli and kidney failure [5,6].
In the classic pyroptosis pathway, the inflammasome sensors detect diverse microbial signals and activate Cas-pase1 through the apoptosis-associated speck-like protein containing a CARD (ASC) adaptor. Then, cleaved-Caspase1 breaks the autoinhibitory interactions between gasdermin D's (GSDMD) N-terminal and C-terminal and triggers the release of cytokines interleukin-18 (IL-18) and interleukin-1β (IL-1β). The released GSDMD-N domain binds to the plasma membrane and generates membrane pores, which results in cell swelling and eventual lysis [7]. The NLRP3 inflammasome has been implicated in the pathogenesis of chronic kidney disease, acute kidney injury, and DN [8,9]. Recent studies indicate that inflammasome-mediated pyroptosis is involved in the development of DN. High blood sugar-induced pyroptosis is closely related to renal fibrosis, glomerulosclerosis, and renal tubular damage. Tubular epithelial cell pyroptosis is a risk factor to tubular injury in DN [10]. Xie et al. found that the lncRNA GAS5/miR-452-5p pathway reduces oxidative stress and pyroptosis in highglucose-stimulated renal tubular cells [11]. In patients with diabetic kidney disease (DKD), the expression levels of IL-1β, IL-18, and NLRP3 were significantly increased in renal biopsy samples, correlated with the aggravating of albuminuria, suggesting that inflammasome may play a critical role in the progress of DN [12].
Mesenchymal stem cells (MSCs) are stromal cells with self-renewing and multiple differentiation ability, which can be isolated from various tissues, such as umbilical cord, bone marrow, and adipose tissue [13,14]. Human umbilical cord MSCs (UC-MSCs) have advantages including low immunogenicity, noninvasive harvest procedure, and easy expansion in vitro [15]. Currently, the safety and efficacy of MSCs have been applied into varieties of diseases, including graft-versus-host disease, osteoarthritis, systemic lupus erythematosus, rheumatoid arthritis, diabetes, inflammatory bowel disease, and autoimmune diseases [16,17].
MSCs can attenuate diabetic lung fibrosis via adjusting Sirt3-mediated stress responses in rats [18]. Our previous study indicated that UC-MSCs ameliorate DN by inhibiting renal inflammation, fibrosis, and apoptosis [19,20]. However, it is still unclear whether MSCs can inhibit pyroptosis in DN.
In our study, we used a rat model of DN to explore the efficacy and mechanism of UC-MSC-based protection against renal injury in DN. We identified miR-342-3p, a downregulated miRNA in DN rat kidney, by highthroughput sequencing and uncover the beneficial effects of UC-MSCs on the treatment of DN. Mechanistically, UC-MSCs can inhibit pyroptosis of renal tubular epithelial cells through the miR-342-3p/Caspase1 signaling pathway. These findings provide novel insights into the mechanisms of DN and new strategies for the treatment of DN.

Materials and Methods
2.1. Animal Experiment. Male SD rats at age of 6 weeks (body weight of 200-220 g) were purchased from Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). All animal procedures were approved by the Provincial Center for Food and Drug Safety Evaluation and Animal Experiment (Permit number: 202020108).
The SD rats were randomly divided into 4 groups: control group, control+UC-MSC group, diabetic nephropathy group (DN group), and DN+UC-MSC group. After one-week adaptive feeding, a DN model was induced by intraperitoneal injection of streptozotocin (STZ, S0130, Sigma-Aldrich, USA) for once (60 mg/kg dissolved in 0.1 M citrate buffer, pH 4.5). The control groups were injected an equal amount of citrate buffer. The blood glucose levels were monitored for three consecutive days, and rats with blood glucose levels ≥ 16:7 mmol/L were considered diabetic. The 24 h urine protein and urine creatinine were measured at 6 weeks after STZ injection, and 24 h urine protein ≥ 30 mg/ kg was verified as DN. UC-MSCs (2 × 10 6 ) in 500 μL were administered via the tail vein twice at 9 weeks and 10 weeks, while the control group received the equal volume of PBS. The rats were euthanized at 11 weeks, and the kidney and blood were harvested for further analysis.
2.2. Renal Function. Serum creatinine and urea nitrogen levels were measured using the kit purchased from Nanjing Jiancheng Bioengineering Institute (C011-1-1, C013-1-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions. The 24 h urine of each rat was collected with a metabolic cage. Urinary albumin levels were measured with the BCA kit (P0012S, Beyotime, China) according to the manufacturer's instructions. Urine creatinine levels were measured with the kit according to the procedure (C011-2-1, Nanjing Jiancheng Bioengineering Institute, China).

Cell Culture and Treatment.
The UC-MSCs were freshly isolated from human umbilical cord tissue after deliveries in Renmin Hospital of Wuhan University (Permit number: WDRY2019-G001). UC-MSCs were isolated, amplified, and identified to meet the characteristics of MSCs using methods described previously [19]. In brief, the umbilical cord was cut into small pieces and cultured in serum-free medium (Lonza, MD, Walkersville) supplemented with serum alternatives and L-glutamine at 37°C in a 5% CO 2 incubator. The UC-MSCs were identified by expression of a specific panel of cell surface markers (CD105 + , CD73 + , CD90 + , CD34 -, CD11b -, CD45 -, CD19 -, and HLA-DR -) using Human MSC Analysis Kit (562245, BD Biosciences, USA). For multipotent differentiation potential, UC-MSCs were cultured in different inducing media (6114531, 6114541, and 6114551, DAKEWE, China), respectively. The fifth generation (P5) of UC-MSCs was used in animal experiments. To obtain the UC-MSC-conditioned medium (UC-MSC-CM), the supernatant was collected after 72 h of cell culture, filtered through a 0.22 μm syringe filter, and stored at -80°C. miR-342-3p mimic and miR-342-3p inhibitor were synthesized by RiboBio. UC-MSCs were transfected using Tran-sIntroTM EL Transfection Reagent (FT201-02, TransGen, China) according to the manufacturer's instructions.
2.4. Histology, Immunofluorescence, and Immunohistochemistry. For histological analyses, the kidney tissue was cut longitudinally, fixed in 4% formaldehyde, and embedded into 5 μm thick sections for hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS). For immunofluorescence staining, kidney paraffin slices were dewaxed into water. Then, the antigen was retrieved with EDTA antigen retrieval buffer (pH 8.0). After cooling, the slides were washed with PBS (pH 7.4) for 3 times. After the sections were slightly dried, a special pen was used to draw a circle around the slices and the autofluorescence quencher was added to slices for 5 minutes. BSA was dropwise added to slices and incubated. Then, slices were incubated at 4°C overnight with 1 : 100 Ly6G (1 : 200, 87048S, Cell Signaling Technology, USA) or CD11b (1 : 500, ab133357, USA), respectively. After washing with PBS for 3 times, the secondary antibody (Boster, China) was added to the slices and incubated at room temperature for 50 minutes in the dark. After washing with PBS for 3 times, DAPI staining was performed. Then, images were detected by a fluorescence microscope (Eclipse Ci-E, Nikon, Japan).
The expression of each gene was calculated by the 2 -ΔΔCt method. β-Actin and U6 were used as the internal reference.
2.8. Enzyme-Linked Immunosorbent Assay (ELISA). The concentrations of IL-18 and IL-1β in DN rat kidney were measured using the ELISA kit (CSB-E04610r and CSB-E08055r, CUSABIO, China) according to the manufacturer's instructions. Cytokine levels were presented by cytokine concentration/albumin concentration.
2.9. miRNA Sequencing and Analysis. The kidney tissue of the DN and control groups was submitted to Majorbio Platform for RNA extraction, quantity control, and highthroughput sequencing. The data were analyzed on the online platform of Majorbio Cloud Platform.
2.10. Statistical Analysis. All data were presented as mean ± standard deviation (SD). Differences between groups were analyzed using an unpaired Student t test (for comparison between two samples) or analysis of variance (ANOVA) (for multiple comparisons). Statistical calculations were performed using GraphPad Prism. Statistical significance was accepted at p < 0:05.

UC-MSC Administration Attenuates STZ-Induced DN.
To investigate the effect of UC-MSCs on DN rats, we established a STZ-induced DN model. At 10 weeks after the model was established, the DN groups were injected with 2 × 10 6 UC-MSCs via the tail vein, while the control group was injected with an equal volume of PBS. A detailed experimental scheme is shown in Figure 2(a). Animals were sacrificed, the kidney tissue and blood were collected at 12 weeks. We detected the biochemical indicators related to renal function such as 24 h urinary protein, serum creatinine, and serum urea nitrogen and calculated the creatinine clearance. Results indicated that the renal function of DN rats was significantly attenuated after UC-MSC treatment (Figures 2(b)-2(e)).
In addition, H&E staining showed that treatment with UC-MSCs remarkably reduced the morphological and especially ameliorated the vacuolar degeneration of renal tubular epithelial cells (Figure 2(f)). Meanwhile, PAS staining showed extracellular matrix deposition in DN rats, which was attenuated by UC-MSC treatment (Figure 2(g)). Taken together, these results suggest that UC-MSC treatment ameliorated STZ-induced renal injury.

UC-MSC Administration Inhibits Inflammation in DN
Rats. Next, we investigated the infiltration of inflammatory cells in DN, including neutrophils and macrophages. The results indicated that the numbers of Ly6G + cells in UC-MSC treatment rats were significantly lower than those in the DN groups (Figure 3(a)). Furthermore, accumulation  Stem Cells International of CD11b + cells was observed in DN rats compared to the control groups. In the UC-MSC treatment groups, the number of CD11b + cells obviously decreased (Figure 3(b)). Then, we test the concentration of IL-1β and IL-18 by ELISA. Compared with the DN groups, the levels of IL-1β and IL-18 decreased in the UC-MSC treatment groups (Figures 3(c) and 3(d)).
These findings indicate that UC-MSC administration inhibits inflammation in DN.    (Figures 4(a)-4(c)). Furthermore, we also observed significantly increased production of mRNA and protein of pyroptosis-related molecules in DN rats compared to the control groups, which was relieved by UC-MSC treatment (Figures 4(d)-4(f)). Taken together, UC-MSCs inhibit renal pyroptosis via NLRP3/Caspase1-mediated pyroptosis signaling.

miR-342-3p Is Identified as the Most Closely Related to
Pyroptosis and DN. MicroRNAs (miRNAs) have a potential role in regulating the pathogenesis of several diseases, including DN [23]. To elucidate the key miRNAs involved in DN, we analyzed miRNA transcripts using RNA sequencing (RNA-seq). The heatmap analysis revealed differentially expressed miRNAs with statistical significance (FDR value < 0:05, fold change > 1) between the DN and control groups ( Figure 5(a)). We validated 7 candidate miRNAs by qRT-PCR and found that miR-342-3p was identified as the most significantly downregulated miRNA in the kidney of DN rats ( Figure 5(b)). Importantly, significant negative correlations of miR-342-3p with 24 h urinary protein were observed in rat urine (Figure 5(c)). Additionally, miR-342-3p expression was negatively correlated with mRNA levels of Caspase1 and NLRP3 in DN rat kidney (Figures 5(d) and 5(e)). Next, we detected the expression of miR-342-3p between HEK293T and UC-MSCs. qRT-PCR results revealed that miR-342-3p was highly expressed in UC-MSCs ( Figure 5(f)). Moreover, UC-MSC treatment obviously increased miR-342-3p expression in the kidney of DN rats, as compared with untreated controls (Figure 5(b)). These results reveal the potential function and candidate biomarker attributes of miR-342-3p in DN and suggest that UC-MSC-derived miR-342-3p may play an important role in protection against DN.

UC-MSC-Derived miR-342-3p Inhibits NLRP3
Inflammasome Activation In Vitro through Targeting the 3′ -UTR of Caspase1. To explore the biological functions of miR-342-3p, we predicted the potential targets of miR-342-3p related to inflammasome using miRanda. Interestingly, we found that miR-342-3p has potential target sites with Caspase1 ( Figure 6(a)). To evaluate the binding of miR-342-3p with Caspase1, the wide-type 3 ′ -UTR sequence and the mutated one were cloned into pmirGLO vectors, respectively. The reporter constructs were cotransfected  Stem Cells International into HEK293T with miR-342-3p mimic or mimic NC. The dual-luciferase assay showed that miR-342-3p decreases pmirGLO-Caspase1-WT activity by binding to the target sequence. Furthermore, the inhibition of luciferase was abolished after mutation of the binding sites ( Figure 6(b)).
Current studies have suggested that pyroptosis-induced cell death promotes several diabetic complications, including DN [25]. Li et al. found that pyroptosis-associated proteins, including GSDMD, NLRP3, Caspase1, and IL-1β, were upregulated in renal tubules [26]. In HG-treated mouse podocytes, GSDMD promotes pyroptosis-regulated cell death [27]. One of the important findings in our study is that UC-MSC treatment attenuates the pyroptosis in DN. Our in vivo study showed that the expression of markers of NLRP3 inflammasome activation, such as Caspase1, ASC, and IL-1β in DN rat kidney, was significantly increased. Moreover, UC-MSC treatment notably reduced the expression of NLRP3, Caspase1, ASC, and IL-1β. Then, we explored the mechanism of effects of UC-MSC treatment in vitro. In HG-induced NRK52E cells, UC-MSC-CM significantly inhibited the expression of NLRP3, Caspase1, and ASC. Meanwhile, UC-MSC-CM reduced the release of IL-1β and IL-18 in the supernatant. These results provide a potential therapeutic approach of UC-MSCs to prevent the activation of inflammasome and pyroptosis in DN.
More recently, microvesicles and exosomes derived from MSCs contain cytokines, growth factors, and miRNAs [28]. miRNAs are a class of noncoding endogenous RNAs that negatively regulate gene expression through binding to the 3 ′ -UTR of target gene [29]. To explore the miRNAs involved in DN, we performed RNA sequencing on control and DN kidney tissue and selected the differentially expressed miRNA closely related to DN, among which miR-342-3p was the top one downexpressed miRNA in DN. Recently, some studies have revealed that miR-342-3p has been reported to participate in the progression of DKD [30,31]. In particular, Jiang et al. found that miR-342-3p suppresses renal interstitial fibrosis in DN by targeting SOX6 [31]. Our in vivo study showed that miR-342-3p was significantly downregulated in DN rats, whereas it was highly expressed in UC-MSCs. Importantly, we found that the miR-342-3p/ Caspase1 pathway can regulate the pyroptosis of renal tubular epithelial cells in DN rats, which is consistent with the previous studies [32].
Moreover, our in vitro study showed that the expression of NLRP3 and Caspase1 was significantly downregulated by miR-342-3p-overexpressed UC-MSC-CM in high-glucoseinduced NRK52E cells. Although it is unclear whether UC-MSC-derived miR-342-3p can also suppress renal interstitial fibrosis, our in vivo and in vitro data suggest that UC-MSCderived miR-342-3p may be a promising strategy for the treatment of DN, through inhibiting pyroptosis of renal tubular epithelial cells by targeting the NLRP3/Caspase1 pathway.

Conclusions
In conclusion, our study identified miR-342-3p as the most closely related to pyroptosis and DN. UC-MSC-derived miR-342-3p was shown to inhibit the activation of NLRP3