Part Ⅱ Smad3 Deficiency Improves Islet-based Therapy For Diabetes And Diabetic Kidney Injury By Promoting β Cell Proliferation Via The E2F3-dependent Mechanism

Methods

1. Animals

The Smad3-null (Smad3KO) mice are a kind gift from Prof. Chuxia Deng and maintained in heterozygotes (Smad3+/- ) in C57BL/6 background [20]. Smad3KO mice were generated by intercrossing of Smad3+/- mice. The db/db mice with mutation of Lepr (Leptin receptor) in C57BLKS background were purchased from the Animal Experimental Center of the Chinese University of Hong Kong (CUHK). All animals were maintained in a specific pathogen-free (SPF) facility with free access to food and water at a day/night cycle of 12/12 h. All animal manipulations in this study were performed following the regulations of and approved by the Ethics Committee of Animal Experiments of CUHK with the Ref. No. 18-177-MIS.

2. Streptozocin (STZ)-induced diabetic mouse model

Male C57BL/6 mice at the age of 8-10 weeks were intraperitoneally injected with 50 mg/kg/day STZ daily for 5 continuous days. 5 days post the final STZ injection, mice with a stable random blood glucose of more than 16 mM for 2 consecutive days were regarded as the successful establishment of diabetes and used as the recipient mice for islet transplantation.

3. Islet isolation, transplantation, and assessment of diabetic phenotype

Islets were isolated from male or female Smad3 knockout (KO) or wild-type (WT) mice at the age of 8-10 weeks as previously described [21]. To perform the islet transplantation, indicated several islets were transplanted under the renal capsule of male db/db mice at the age of week 4 or STZ-induced diabetic mice on day 7 post the final STZ injection following the protocol described by Szot et al [22]. Briefly, the recipient mouse was anesthetized by the anesthesia Combo (1.5% ketamine and 0.15% xylazine, 6 μL/g bodyweight). After the loss of consciousness, an incision of 1 cm was made on the skin covering the left kidney. Pull out the kidney gently and keep it moist with sterile saline during the surgery. With a 25G needle, make a small scratch on the capsule to create a nick. Indicated numbers of islets were delivered into the renal capsule by a PE50 tubing. After all, the islets were transplanted, seal the nick by cauterization with low heat. Replace the kidney and close the peritoneum with sutures. Seal the skin with staples. 100 μL Penicillin-streptomycin solution (Gibco, cat# 15140122, USA) was injected intraperitoneally to prevent infection. 100 μL Tegmic was injected intramuscularly to relieve the pain. The animal was placed in a warm environment until full recovery. The same operation procedure was also carried out in sham control mice but without islet infusion. Both random and 6 h-fasting blood glucose (RBG and FBG) levels of the tail tip blood were checked weekly with a portable glucose meter (Roche, ACCU-CHEK® Performa). The insulin resistance test (IPITT), glucose tolerance test (IPGTT), levels of HbA1c, and serum insulin were measured as previously described [9].

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4. Total urinary protein and serum creatinine

24 h urine was collected in a metabolic cage. 24 h total urinary protein was calculated by multiplying the urine volume and urinary protein concentration which was quantitated by the Quick Start Bradford Protein Assay Kit (Bio-Rad, cat# 5000201, USA). Serum creatinine was determined with the creatinine quantification kit (Stanbio Laboratory, cat# 0430-120, USA).

5. Histopathology

Renal histopathological changes were examined in paraformaldehyde-fixed, paraffin-embedded sections (4 µm) with Periodic Acid-Schiff's (PAS) reagent as previously described [23]. Mesangial matrix expansion was scored using the quantitative image program (Image-Pro Plus 7.0, Media Cybernetics) as previously described [23].

6. Immunohistochemistry

Islets-bearing kidney or pancreas was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm. After rehydration, the sections were treated with microwave-mediated antigen retrieval in 0.01 M citrate (pH 6.0) for 10 min and blocking in 5% BSA for 1 h. Primary antibody incubation was performed overnight at 4 °C. For fluorescent staining, a fluorescent secondary antibody was applied at room temperature (RT) for 1 h. For DAB-mediated thermogenesis, an additional step of 3% H2O2 blocking for 15 min was included before the antigen retrieval. After primary antibody incubation, HRP-conjugated polymer (Envision+ System, Dako, USA) was added. For staining of E2F3, antigen retrieval was performed in EDTA-Tris solution (pH 9.0). Antibodies used in immunohistochemistry were detailed in Table S2. The staining-positive area was quantitated with Image J software (v1.47, NIH, USA).

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7. Measurement of grafted β cell mass in the islets-bearing kidney

An index of average β cell area per section was used to semi-quantitatively quantify the β cell mass in the islet graft. Briefly, the islets-bearing kidney (paraffin-embedded) was sectioned successively at 4 μm along the sagittal axis. Continuous sections at 160 μm intervals were fluorescently immunostained for insulin. At least 6 sections with insulin-positive staining were used for the calculation of β cell area per section in each mouse. The insulin-positive β cell area in each section was quantified with Image J software (v1.47, NIH, USA). The index of average β cell area per section in each mouse was calculated by dividing the total insulin-positive area into all sections by the number of insulin-positive sections.

8. Primary culture of islet cells

The isolated islets were dissociated into single cells and seeded into the 96-well plate in 200 μL of RPMI 1640 (Gibco, USA) + 10% FBS (Gibco, USA) + 1% Penicillin-streptomycin (Gibco, USA) supplemented with 20 mM glucose. Islet cells were cultured for the indicated time in an incubator set at 37 °C, 5% CO2, and 100% humidity. For adenovirus-mediated knockdown of E2F3, dissociated islet cells were cultured in a medium containing adenovirus overexpressing short hairpin RNA (shRNA) targeting mouse E2F3 (shE2F3) or control non-specific shRNA (shown) at a titer of 10 pfu per cell for 2 days followed by medium replacement. The stem sequence of the shE2F3 duplex was 5’-CATCCATGCTCTATTCTGT-3’. Dispersed cells from 50 islets were seeded per well for immunostaining and RT-PCR, and 100 islets per well for Western blot, which were conducted as previously described [9].

9. In vivo and in vitro BrdU labeling

To perform in vivo BrdU labeling, the mice were intraperitoneally injected with 50 mg/kg/day BrdU (in PBS) for 7 days from the 2nd day after islet transplantation and sacrificed 2 h after the last injection. For in vitro BrdU labeling, the dispersed islet cells were cultured for 48 h followed by replacement with fresh medium containing 10 μM BrdU and cultured for an additional 24 h. Fluorescent immunostaining against BrdU in the tissue and cultured cells was performed as previously described [9].

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10. RNA-Seq

Islets were isolated from Smad3WT-db/db, Smad3KO-db/db, Smad3WT-db/m, and Smad3KO-db/m mice. For each genotype, islets from more than 5 mice were pooled for RNA isolation. RNA was prepared with the miRNeasy Mini Kit (Qiagen, Germany). RNA-seq and the downstream data analysis were described previously [9]. The expression level of a given gene was presented as the fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed gene (DEG) was defined as ≥ 2 folds variance of FPKM between two groups. Upregulated and downregulated DEGs were combined and used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis with the online DAVID bioinformatics resources (v6.8) with default settings. The GOTERM_BP_DIRECT sub-category was employed for GO analysis.

11. Immunoprecipitation

To perform immunoprecipitation in mouse islets, about 500 freshly-isolated islets were lysed in 500 mL of RIPA buffer. A rabbit anti-Smad3 antibody (2 μg) or rabbit IgG isotype was added and incubated at 4 °C overnight with gentle rotation. Then, 20 μL of protein A agarose beads (Beyotime, cat# P2051, China) was added and incubated for 2 h at 4 °C. After washing robustly with fresh RIPA buffer, the bounded protein was released by denaturing in 1xSDS loading buffer and subjected to Western blot analysis. To avoid the influence of IgG heavy chain, a light chain-specific mouse anti-rabbit IgG (HRP conjugated, Abmart, cat# M21006, China) was used to recognize the precipitated Smad3 protein.

12. Chromatin immunoprecipitation-ChIP

To perform ChIP in mouse islets, about 2000 islets were collected from 8-10 weeks old C57BL/6 mice and used as one ChIP preparation. The freshly isolated islets were stimulated with 10 ng/mL TGF-β1 (Gibco, USA) for 15 min in RPMI 1640 + 1% FBS +1% Penicillin-streptomycin. ChIP was performed with the SimpleChIP® Plus Enzymatic Chromatin IP Kit (CST, USA) following the recommended instruction. Antibodies and primers used in the ChIP assay are detailed in Table S1 and S2.

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13. Dual-luciferase reporter assay

Mouse E2F3 promoter (-1000 to +451 bp) was cloned into the pGL3-basic vector (pGL3-E2F3.Pro). Concurrently, point mutation of the Smad3 binding site was conducted to generate the pGL3- E2F3.Pro. Mut. To perform the dual-luciferase reporter assay, HEK293T cells were seeded into the 24-well plate at a density of 5 x 104 cells/well. The next day, cells were transfected with 500 ng pGL3-Basic or pGL3-E2F3.pro or pGL3-E2F3.pro.mut combined with pcDNA3.1-Smad3, which overexpresses mouse Smad3, or not (as indicated in Figure 10F) by phosphate calcium method. The pEGFP-C1 vector was supplemented to balance the transfection dose in each treatment. 10 ng pGL4.73.huc/SV40 vector expressing renilla luciferase was included in each treatment to normalize the transfection efficiency. 6 h post-transfection, the medium was changed followed by incubation for an additional 48 h. The cells were lysed, and luciferase activity was determined with the Dual-Luciferase ® Reporter Assay System (Promega, USA) and quantified in a luminometer (VICTORTM X4 2030 Multilabel Reader, PerkinElmer, USA). The transcriptional activity of the cloned E2F3 promoter was presented as the value ratio of firefly luciferase to renilla luciferase. Each treatment was performed in triplicates.

14. Statistics

Data were presented as mean ± SD and analyzed with SPSS software (16.0). The normality of the data was checked by the One-Sample Kolmogorov-Smirnov test. The homogeneity of variances among groups was tested by the Levene statistic. For comparison between the two groups, the Student’s t-test was used. For comparison among multiple groups, one-way ANOVA with an LSD test was used if an equal variance was assumed. For the equal variance that wasn’t assumed, an alternative Welch test statistic was used followed by multiple comparisons with Tamhane’s T2 test. P < 0.05 was considered statistically significant.

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Conclusions

Smad3 deficiency largely improves islet replacement therapy for both T1DM and T2DM and protects against diabetic kidney injury. Enhanced E2F3-dependent β cell proliferation may be a key mechanism of Smad3KO islet therapy in both T1DM and T2DM. Thus, Smad3KO β cell replacement therapy may be a better therapeutic strategy for diabetes clinically.

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Hong-Lian Wang1,2, Biao Wei2, Hui-Jun He2, Xiao-Ru Huang2,3, Jing-Yi Sheng2,4, Xiao-Cui Chen2,5, Li Wang1, Rui-Zhi Tan1, Jian-Chun Li1, Jian Liu1, Si-Jin Yang6, Ronald CW Ma2, and Hui-Yao Lan2,7

1 Research Center for Integrated Medicine and Department of Nephrology, Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, 646000, China.

2. Department of Medicine and Therapeutics, and Li Ka Shing Institute of Health Sciences, the Chinese University of Hong Kong, Hong Kong, 999077, China.

3. Guangdong-Hong Kong Joint Laboratory on Immunological and Genetic Kidney Diseases, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, 510080, China.

4. State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Sciences & Medical Engineering, Southeast University, Nanjing, China.

5. Key Laboratory of Prevention and Management of Chronic Kidney Disease of Zhanjiang City, Institute of Nephrology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong, 524001, China.

6. National Traditional Chinese Medicine Clinical Research Base, Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan, 646000, China.

7. CUHK-Guangdong Provincial People’s Hospital Joint Research Laboratory on Immunological and Genetic Kidney Diseases, the Chinese University of Hong Kong, Hong Kong, 999077, China.