AS II (with a purity of over 98%) was purchased from Shanghai Jingke Chemical Technology Co., Ltd. (Shanghai, China), and was suspended in olive oil (Osaka, Japan), as a vehicle for administration to rats. Losartan was purchased from Merck Sharp & Dohme Limited (Merck Sharp & Dohme, Australia) and suspended in 0.5% methylcellulose solution. Streptozotocin (STZ) was purchased from Sigma-Aldrich Company (Sigma-Aldrich, United States) and was dissolved in the citrate buffer (0.1 M, pH 4.5) (Chen et al., 2014).

All the animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals proposed by National Institutes of Health (NIH) and were approved by the Animal Ethics Committee of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Healthy 8-week-old male Sprague-Dawley rats weighing 200–250 g were housed under a constant 12 h light–dark cycle at a temperature between 21 and 23°C and allowed free access to food and water. Diabetes was induced in rats by a single intraperitoneal injection of STZ at 55 mg/kg, according to our previous study (Xie et al., 2020). Seventy-two hours after intraperitoneal injection of STZ, the blood glucose level was measured from the tail vein, and rats with a blood glucose level above 16.7 mmol/L were considered as diabetic rats (Xie et al., 2020). The diabetic rats were then randomly divided into four groups (n = 7/each group): 1) STZ-induced diabetic rats (STZ), received an equal volume of olive oil; 2) diabetic rats treated with losartan at 10 mg·kg⁻¹·d⁻¹; 3) diabetic rats treated with low-concentration of AS II at 3.2 mg·kg⁻¹·d⁻¹ (ASIIL); 4) diabetic treated with high-concentration of AS II at 6.4 mg·kg⁻¹·d⁻¹ (ASIIH). Normal rats were chosen as the normal control (Con, nondiabetic rats). All treatment interventions were administrated at two weeks after STZ injection and were administered via oral gavage to rats for nine weeks. Rats were kept in individual metabolic cages for 24 h urine collections at the end of 0 and 9 weeks of treatments. Rats were then anesthetized with pentobarbital sodium and the blood samples were collected through the abdominal aorta for measuring biochemical parameters by using an automatic biochemistry analyzer, and the kidneys were harvested immediately.

Urine was centrifuged at 3,500 rpm for 10 min at 4°C and the supernatants of urine were measured for the urinary albumin and urinary creatinine by an automatic biochemistry analyzer (Hitachi Model 7600-120E, Japan). Urinary albumin excretion was expressed as urinary albumin/creatinine ratio (ACR). Blood was centrifuged at 3,500 rpm for 10 min at 4°C and the supernatants of blood were measured for blood glucose (GLU) by an automatic biochemistry analyzer (Hitachi Model 7600-120E, Japan).

The kidneys were fixed in 10% buffered formalin and embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS). The protocol of HE and PAS staining was described previously (Zhai et al., 2019). Sections were dried for 30 min at 65°C and then were deparaffinized and rehydrated through dimethylbenzene (I), dimethylbenzene (II), 100% ethanol (I), 100% ethanol (II), 95% ethanol, 90% ethanol, 80% ethanol, and deionized water, 10 min for each step. Subsequently, kidney sections were stained by HE and PAS and detected by light microscopy (Leica, Germany). To assess and calculate the injury of renal histology in HE and PAS staining, 20 cortical fields (×400 magnification) were chosen randomly by two blinded investigators. The sections from each renal tissue were graded and scored for the mesangial matrix index according to the following scale: Score 0, no lesion; Score 1+, <25% of the glomerulus sclerosis; Score 2+, 25–50% of the glomerulus sclerosis; Score 3+, 50–75% of the glomerulus sclerosis; Score 4+, >75% of the glomerulus sclerosis (Fujihara et al., 2000).

Morphological and structural characteristics of podocyte were examined by transmission electron microscopy (TEM). Renal cortices were cut into 1 mm³ pieces on ice and immediately fixed in 2.5% glutaraldehyde for 2 h at 4°C and washed twice in the same buffer. After dehydration with different concentrations of ethyl alcohol, renal cortices were embedded in epoxy resin 48 h at 60°C. Ultrathin sections were stained with uranyl acetate and lead citrate and examined by TEM. The number of foot processes (FP) in kidney sections was counted by a blinded observer as previously described (Chen et al., 2014).

Paraffin-embedded kidney tissues were deparaffinized and rehydrated, xylene, ethanol, and quenched by 3% H₂O₂ for 15 min. The sections were blocked by 10% goat serum for 1 h at room temperature. Then, the sections were incubated overnight at 4°C with the following primary antibodies: WT1 (1:200, ABclonal, China), nephrin (1:1,000, Abcam, United States), cleaved-caspase-3 (1:200, Abcam, United States), p62 (1:50, Proteintech, China), LC3 (1:600, Proteintech, China), Mfn2 (1:2000, Proteintech, China), Fis1 (1:100, Abcam, United States), PINK1 (1:200, Proteintech, China), parkin (1:1,000 Proteintech, China), Nrf2 (1:200, Abcam, United States), and Keap1 (1:1,500, Servicebio, China). The secondary antibodies (Dako, United States) were added to the sections, which were incubated for 1 h at 37°C. Finally, the sections were counterstained using diaminobenzidine and hematoxylin staining and then were rinsed after dehydration. Images were then collected using a light microscope (Leica, Germany). The positive-staining cells in each kidney section were semiquantified by ImageJ software (NIH, Bethesda, MD, United States).

Triple immunofluorescence labeling, including terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, WT1, and 4,6-diamidino-2-phenylindole (DAPI), was used to examine the podocyte apoptosis. TUNEL assay was conducted on frozen sections of kidney samples (4 μm) according to the manufacturer’s instructions (In Situ Apoptosis Detection Kit, Merck, S7165). Afterward, the sections were incubated with the primary antibody of WT1 diluted in PBS (1:100, ABclonal, China) at 4°C overnight. Then, secondary antibodies conjugated to Alexa Fluor 488 (abcam, United States) were added to the sections for 1–2 h and counterstained with DAPI. The triple-positive cells (WT1, TUNEL, and DAPI) were identified as the apoptotic podocytes. All fluorescent images were visualized under a confocal microscope (Olympus, Japan) by two blinded investigators.

Kidney tissues were homogenized in RIPA lysis buffer containing protease and phosphatase inhibitor cocktail on ice with a homogenizer. The supernatants were collected after centrifuging at 12,000 rpm for 24 min at 4°C. Protein concentration of the supernatants was calculated by the bicinchoninic acid (BCA) kit assay (Biosharp, China). The tissue lysates, mixed with an equal amount of 5× SDS loading buffer, were denatured in boiling water for 10 min. Samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene difluoride (PVDF) membranes, which were then blocked with Tris-buffered saline Tween-20 (TBST) containing 5% nonfat milk for 1 h at RT. Next, membranes were incubated overnight at 4°C with the following primary antibodies: nephrin (1:1,000, Abcam, United States), cleaved-caspase-3 (1:1,000, Abcam, United States), p62 (1:1,000, Abcam, United States), LC3 (1:1,000, Abcam, United States), Mfn2 (1:1,000, Cell Signaling Technology, United States), Fis1 (1:1,000, Abcam, United States), PINK1 (1:200, Abcam, United States), parkin (1:1,000, Abcam, United States), Nrf2 (1:1,000, Abcam, United States), Keap1 (1:1,000, Abcam, United States), and β-actin (1:5,000, Abcam, United States). After washing for 5–10 min three times with TBST, membranes were incubated with HRP-bounded secondary antibodies (Abcam, United States) for 1 h at RT and washed 5–10 min for three times with TBST again. Finally, proteins were visualized by ECL and ImageJ was used to determine band intensity. Each blot was independently repeated three times.

Total RNA was extracted from kidney tissues using TRIzol (Invitrogen, Carlsbad, CA) and reverse-transcribed to cDNA with a Reverse Transcription Kit (Takara, Japan) following the manufacturer’s protocol. Real-time PCR was performed by Light Cycler 480 system (Roche, United States) using SYBR Green Master Mix (Vazyme, United States). The primer sequences are as follows: nephrin: 5′-CGT​GCT​GGT​GAT​GAC​TGT​ACG (forward) and 5′-CGT​TCT​TGT​TCT​CCG​ATT​GTG (reverse); cleaved-caspase-3: 5′- CTC​GGT​CTG​GTA​CAG​ATG​TCG (forward) and 5′-TGG​CTC​AGA​AGC​ACA​CAA​AC (reverse); Mfn2: 5′-ACC​ATC​AGT​AGC​CAA​TCT​GGA​C (forward) and 5′-AGA​GCA​GGG​ACA​TCT​CGT​TTC (reverse); Fis1: 5′-CTG​GAC​TCA​TTG​GAC​TGG​CTG​TG (forward) and 5′-AGG​AAG​GCG​GTG​GTG​AGG​ATG (reverse). The target gene expression was calculated using the 2^−ΔΔct method and normalized to β-actin.

Data were analyzed using GraphPad Prism software (version 7.0) and presented as means ± standard deviation (SD). Differences between two groups were compared by unpaired Student's t-tests and differences among multiple groups were performed using one-way analysis of variance (ANOVA). Data were considered statistically significant if p < 0.05.

To identify the renoprotective effects of AS II in STZ-induced diabetic rats, the levels of blood glucose (GLU) and urinary ACR were examined. Compared with the normal control rats, ACR level at baseline was markedly increased in diabetic rats (Figure 1A). However, treatment with AS II or losartan significantly reduced the level of ACR at the end of 9 weeks after STZ injection (Figure 1B). These data suggested that AS II treatment attenuated albuminuria in STZ-induced diabetic rats. However, no significant difference in the level of GLU was observed between AS II and losartan treatment groups, which indicated that AS II had no direct effect on the levels of blood glucose (Figure 1C).

We performed histological examination of renal tissues from all groups. As shown in HE and PAS staining (Figures 2A,B), diabetic rats were characterized with severe pathological changes, such as the accumulation of extracellular matrix (ECM) deposition, mesangial matrix expansion, and cell proliferation. For AS II- and losartan-treated diabetic rats, the situation of these pathological changes was significantly improved, as shown by quantitative analysis (Figures 2C,D). Furthermore, we observed the changes in podocytes morphology through TEM to further verify the role of AS II in DN progression. As presented in Figures 2E,F, the diabetic rats exhibited apparent podocyte loss, FP fusion, and effacement at 9 weeks after STZ injection. However, AS II treatment considerably reversed these changes in STZ-induced diabetic rats.

To further assess the protective effects of AS II on podocytes in diabetic rats, we analyzed the expression of nephrin, which is a podocyte-specific protein and essential for the functional and morphological integrity of glomerular filtration barrier (Wang et al., 2018). As shown in Figures 3A,B, the number of nephrin-positive puncta was consistently decreased in diabetic rats compared with the normal rats, indicating the number of podocytes per glomerulus was dramatically downregulated and glomerular filtration barrier function was damaged. However, this change was notably revered by AS II and losartan treatment. Consistent with these observations, western blot and RT-qPCR results also confirmed that nephrin expression was decreased in diabetic rats, while AS II and losartan treatment remarkably reversed these changes (Figures 3C–E). These data suggested that AS II protected against diabetic podocyte abnormalities in function and structure.

Triple immunofluorescence labeling, including TUNE, WT1, and DAPI, was used to examine the podocyte apoptosis in diabetic rats. Cleaved-caspase-3 (c-caspase-3) is an active form of caspase-3. It is well known for its role in apoptosis pathway, which is related to the deterioration of DN (Ghosh et al., 2009). In this study, we observed the expression of c-caspase-3 protein was significantly elevated in STZ-induced type 1 DN model compared to the normal rats, whereas AS II and losartan treatment dramatically reversed this change, as reflected by immunohistochemistry staining (Figures 4A,B). Moreover, western blot and RT-qPCR also showed the expression of c-caspase-3 was increased in diabetic rats, while AS II and losartan administration inhibited these effects (Figures 4C–E). Furthermore, double immunofluorescent staining of WT1 and TUNEL showed increased podocyte apoptosis in the kidneys of diabetic rats, but this change was reversed by AS II and losartan treatment (Figure 4F). These results demonstrated the significant antiapoptotic effect of AS II on podocytes in diabetic rats.

To preferably understand the protective effects of AS II on mitochondrial biology, the changes of mitochondrial morphology and dynamics were observed by TEM, immunohistochemistry staining, western blot, and RT-qPCR assays. In Figure 5A, enlarged mitochondria in podocytes in parallel with the destruction of mitochondria cristae were found in diabetic rats, whereas these changes achieved amelioration partly after 9 weeks AS II and losartan treatment.

Given that Fis1 is essential for mitochondrial fission, while mitochondrial fusion is mediated by Mfn2, we then investigated the expression of Fis1 and Mfn2 to evaluate the effect of AS II on mitochondrial dynamics. The results showed that diabetic rats exhibited a significant reduction of Mfn2 protein, while Fis1 expression was upregulated compared with nondiabetic rats. However, AS II and losartan treatment increased the expression of Mfn2 and conversely decreased the expression of Fis1 in the kidneys of diabetic rats (Figures 5B–K). In summary, these data indicated the protective effect of AS II on mitochondrial morphology and dynamics changes in diabetic rats.

We further examined the level of autophagy-related proteins in kidney tissues, such as LC3 and P62 proteins, to verify the effect of AS II on autophagy in DN. As is shown in Figures 6A–D, the expression of LC3 in diabetic rats was less than that in normal control rats. On the contrary, P62 expression was significantly increased after STZ injection. Nevertheless, AS II and losartan treatment obviously reversed the expression of LC3 and attenuated the level of P62 protein. Consistently, western blot results also showed that the ratio of LC3-II/LC3-I was decreased in diabetic rats, along with the increased P62 expression, while these alterations were partially reversed after 9-week treatment of AS II and losartan (Figures 6E–G). These data suggested that AS II exerted a beneficial effect on DN via reversing insufficient autophagy in diabetic rats.

Moreover, the expressions of PINK1 and Parkin related to mitophagy were apparently decreased in diabetic rats, whereas AS II and losartan treatment led to the enhancement of PINK1 and Parkin expression (Figures 7A–G). Because the expression of PINK1 is positively regulated by Nrf2 under oxidative stress conditions (Liu et al., 2018), we then investigated the level of Nrf2 and its upstream regulator Keap1 in the kidneys of diabetic rats. In our study, both immunohistochemistry staining and western blot results showed that the expression of Nrf2 was downregulated in diabetic rats, accompanied by increasing Keap1 level. However, AS II and losartan treatment increased the expression of Nrf2, further decreasing Keap1 level (Figures 8A–G). Taken together, the above results elucidated that the enhancement of antioxidant stress ability and mitophagy activation through comodulation of Nrf2 and PINK1 might be one of the possible mechanisms for the renoprotective effects of AS II.