SD male rats were purchased from SLRC Laboratory Animal Ltd. (Shanghai, China). Rats were housed at a controlled temperature of 22 ± 2°C, relative humidity of 50–60%, 12-h light and 12-h dark cycles (light, 08:00–20:00, darkness, 20:00–08:00), and allowed free access to standard dry diet and tap water. All animals received humane care, and experimental protocols were approved by the Animal Care Committee at the Naval Medical University.

Weight 180–200 g male rats were treated with STZ (Sigma, Deisenhofen, Germany) to induce type 1 diabetes. STZ was dissolved in sterile citrate buffer (pH 4.5) and injected intraperitoneally (65 mg/kg body weight) within 10 min of preparation. The non-diabetic rats initially injected with STZ vehicle served as controls (group Con, n = 10). Diabetes mellitus was confirmed by measuring glucose levels tail venous blood using a B-glucose analyzer (HemoCue, Angelholm, Sweden) 7 days later. Rats with random blood glucose level >16.7 mmol/L were included in experiments.

The diabetic rats then received telmisartan (Merck, PHR 1855, 10 mg kg⁻¹·d⁻¹ po, group DM + Tel, n = 10) or vehicle (group DM, n = 10) by gavage for 4 weeks. Telmisartan used in this paper was obtained from Sigma-Aldrich Germany, Inc., whose purity is 98%+ (HPLC). Periodically, blood glucose and body weights were measured, and urine samples for quantitative measurement of albuminuria was collected in metabolic cages. Rats were sacrificed under anesthesia after 4 weeks, the kidneys were removed and weighed for histological analysis and protein extraction.

The urine samples were centrifuged at 10,000 rpm for 5 min to remove insoluble materials. The supernatant was aliquoted and stored at −80°C until used. ELISA kit for rat urinary albumin from Chondrex (Redmond, WA) was used according to the manufacturer’s instructions.

Creatinine Assay kit (Nanjing Jiancheng, C011-2-1) was used for the determination of creatinine in blood and urine. Ccr was calculated according to the formula: Ccr = (urinary creatinine*24 h urine volume)/(blood creatinine*24 h*60 min/h)/end weight (kg).

Kidneys were cut in a slicing microtome at 7–8 μm, and fixed with 4% paraformaldehyde in PBS for 10 min. Blocking has been performed with buffer (PBS, 2% BSA, 10% FBS) for 1 h followed by 10 min incubation with a second buffer (PBS, 0.4% Triton). Primary antibody against α-SMA (Servicebio, GB13044) or NPHS2 (Abcam, ab229037) has been incubated for 3 h at room temperature in a humidified chamber. After washing, the sections were incubated with Cy3 goat anti-Mouse IgG (H + L) (Servicebio, GB21301), HRP conjugated goat anti-Rabbit IgG (H + L) (Servicebio, GB23303) or immunofluorescent TUNEL (Servicebio, G1501) reaction in a moist chamber (dark, 37°C, 1 h). The sections were then counterstained with DAPI (Servicebio, G1012) for the detection of nuclei. Finally, the stained sections were embedded in the resistance to fluorescence quenching sealing liquid and pictured using a fluorescence microscope (NIKON ECLIPSE C1, Japan).

Human renal mesangial cells (HRMCs) were obtained from ScienCell Research Laboratories, Santiago, CA, and cultured in Mesangial Cell Medium (MsCM, ScienCell Research Laboratories). HRMCs were plated on a poly-L-lysine coated flask (2 μg/cm²), and grown at 37°C in a humidified atmosphere containing 5% CO₂. The cells in this experiment were used within 3–4 passages and were examined to ensure that they demonstrated the specific characteristics of mesangial cells. Mouse podocyte cell line MPC-5 was obtained from ATCC, Maryland, United States. The cells were grown on type I collagen in RPMI 1640 (10% FBS) with 50 U/ml IFN-γ at 33°C to 85% confluency and then transferred to 37°C without IFN-γ for 10–14 days for differentiation.

Cell Counting Kit-8 (CCK-8) was used to measure cell proliferation and cell viability. Cells were seeded in each well of a 96-well culture plates (5 × 10³/well). After the treatment, 10 μl CCK-8 (Beyotime, Shanghai, China) was added and incubated for 1 h at 37°C. Absorbance was measured using a microplate reader (Thermo Fisher Sci-entific, Waltham, MA, United States) at a wavelength of 450 nm.

Cells were plated and grown until they reached 60% confluence, and then treated with high glucose (50 mmol/L) or telmisartan. After 96 h, the collected cells were washed with cold PBS and resuspended in binding buffer. Annexin V-FITC and PI (eBioscience, Santiago, CA, United States) were added to the cellular suspension as in the manufacturer’s instructions, and a sample fluorescence of 10,000 cells was analyzed by flow cytometry conducted with FACScan (Becton, Dickinson and Company, Franklin, NJ, United States).

The renal cortex, HRMCs and MPC-5 were homogenized in Tissue or Cell Protein Extraction Reagent (Beyotime) supplemented with protease and phosphatase inhibitors (Merck, Whitehouse Station, NJ, United States). Samples were separated on a 10% SDS PAGE and transferred to nitrocellulose membrane (Pall Corporation, NY, United States). The membrane was blocked with 5% bovine serum albumin and blotted with antibody. Anti-PKCβ1 (Cell Signaling Technology, 46,809), anti-swiprosin-1 (Abcam, ab24368), TGF-β1 (Abcam, ab215715), Tubulin (Beyotime, AT819) and GAPDH (Beyotime, AF5009) were used at a concentration of 1:1,000. Proteins were visualized using an IRDye-conjugated anti-mouse or anti-rabbit secondary antibodys (Rockland, Limerick, PA, United States) at 1:5,000. Using ODYSSEY INFRARED IMAGING SYSTEM (LI-COR) to analyses the results.

Total RNA was isolated from cells with TRIzol reagent (Invitrogen, Carlsbad, CA, United States) followed by chloroform isopropanol extraction and ethanol precipitation, and 1 μg of total RNA was reverse-transcribed using the PrimeScript RT reagent kit (Takara Bio, Otsu, Japan). RT-PCR was performed by the Thermal Cycler Dice Real Time System TP800 (Takara Bio, Otsu, Japan) by use of SYBR Green fluorescence signals. The following primers were used:

Data processing was analyzed by Origin 6.1 (OriginLab, Northampton, MA) and expressed as mean ± SD of at least three independent experiments. Statistical significance was determined using ANOVA. A value of p < 0.05 was considered statistically significant.

STZ-injected rats produced characteristic symptoms of diabetes at the 4th weeks, including declined body weight gain (Figure 1A) hyperglycemia (Figure 1B), increased kidney-to-body weight ratio, 24-h urine protein and urea nitrogen (Figures 1C,D,F), and decreased creatinine clearance rate (Figure 1E). Telmisartan treatment significantly increased body weight (Figure 1A) and creatinine clearance rate (Figure 1E), decreased blood glucose (Figure 1B), urine protein (Figure 1D) and urea nitrogen (Figure 1F). Although the kidney-to-body weight ratio of the telmisartan treatment group was reduced, there was no significant difference between these two groups (Figure 1C). Histological analysis showed that extracellular matrix deposition (Figure 1G) and glomerular volume (Figure 1H) were amplified in diabetic rats, while telmisartan attenuated extracellular matrix deposition significantly (Figure 1G).

To further estimate the effect of telmisartan on early glomerular damage of diabetic rats, we observed the changes in podocytes loss and mesangial matrix expansion. NPHS2, also known as podocin, is a characteristic protein molecule located on the slit diaphragm of podocytes. The decreased expression of NPHS2 in diabetic glomeruli represents the damage and loss of podocytes (Tanoue et al., 2021), and telmisartan could alleviate the down-regulation of NPHS2 (Figure 2A). α-SMA, an indicator of the activation of mesangial cells under hyperglycemia to secrete extracellular matrix, could be inhibited after telmisartan treatment (Figure 2B). In addition, telmisartan reduced the number of apoptotic cells in the glomeruli of early diabetic rats (Figure 2C).

To reveal the different effects of telmisartan on podocytes and mesangial cells, we used human renal mesangial cells (HRMCs) and mouse podocyte cell (MPC-5) lines to investigate the effect of telmisartan on cell viability in a high-glucose (HG, 50 mmol/L) environment, respectively. As the results showed, cell viability of MPC-5 was significantly decreased when stimulated with HG after 96 h, while telmisartan could restore cell viability at the concentration of 10 μM. The cell viability of MPC-5 began to decrease significantly after 48 h of HG stimulation, while telmisartan (10 μM) could fully restore this decrease at three time points of 48, 72 and 96 h (Figure 3A). Surprisingly, cell viability of HRMCs was significantly decreased when stimulated with telmisartan dose-dependently even in a normal glucose medium. Telmisartan also could not improve the decreased viability of HRMCs induced by HG (Figure 3B). Furthermore, we investigated whether the effect of telmisartan on podocytes and mesangial cells was related to its known target angiotensin II receptor. Here, angiotensin II did not reduce cells viability, and telmisartan also only damaged the viability of mesangial cells (Figure 3C). Meanwhile, HG and telmisartan did not affect AT₁ receptor mRNA expression in these 2 cell lines (Figure 3D). Telmisartan is highly selective for AT₁ receptor, while AT₂ receptor may be activated compensatory by angiotensin II. As the results shown, HG-induced down-regulation of AT₂ receptor in podocytes could be inhibited by telmisartan. However, the mRNA expression of AT₂ receptor in mesangial cells was not affected by HG or telmisartan (Figure 3E).

As telmisartan could reduce glomerular cell apoptosis in diabetic rats and restore the decreased viability of podocytes induced by HG, we further confirmed that telmisartan attenuated MPC-5 apoptosis induced by HG in vitro via measuring the ratio of apoptotic Anexin-V and IP-stained cells by cytometry (Figures 4A,B). We have previously reported that swiprosin-1 participates in the apoptosis of podocytes in early diabetic kidney injury (Wang et al., 2018b). Here, we also found that telmisartan decreased the expression of swiprosin-1 in diabetic renal cortex (Figure 4C) and HG-stimulated MPC-5 (Figure 4D).

The above results have shown that telmisartan reduces glomerular α-SMA and directly inhibits the cell viability of HRMCs. We further investigated whether telmisartan itself could induce mesangial cells apoptosis. Under HG stimulation, telmisartan could further increase the apoptosis of HRMCs significantly (Figure 5A). Besides, telmisartan itself could also directly induce apoptosis of HRMCs in normal glucose medium (Figure 5B). PPARγ, as another target that could be activated by telmisartan, is widely involved in the apoptosis of various cells (Ayza et al., 2020). Telmisartan-induced HRMCs apoptosis and cell viability damage were significantly inhibited by the PPARγ blocker GW9662 (Figures 5B,C). Interestingly, telmisartan specifically induced high expression of PPARγ in HRMCs but not in MPC-5 (Figure 5D).

TGF-β is a major mediator of matrix expansion in diabetic glomerulus and its up-regulation stimulated by HG in mesangial cells requires PKCβ1 (Wu et al., 2009). As the results showed below/above, telmisartan inhibited the up-regulation of PKCβ1 in renal cortex of diabetic rats (Figure 6A). Specifically, HG induced the protein expression of PKCβ1 in HRMCs but not in MPC-5, and telmisartan could reduce PKCβ1 expression induced by HG in HRMCs (Figure 6B). The mRNA expression of PKCβ1 induced by HG was also suppressed by telmisartan in HRMCs (Figure 6C). Reversely, HG down-regulated the mRNA expression of PKCβ1 in MPC-5, which could be partially restored by telmisartan (Figure 6C). Similarly, HG specially induced TGFβ1 expression in HRMCs but not MPC-5, and telmisartan significantly inhibited HG-induced TGFβ1 in HRMCs (Figure 6D).