The Anti-Apoptosis Of Podocytes And Pro-Apoptosis Of Mesangial Cells For Telmisartan in Alleviating Diabetic Kidney Injury

ABSTRACT

Podocyte damage and mesangial cell expansion are two important pathological manifestations of glomerular injury in early diabetes. Telmisartan, as an angiotensin type 1 (AT1) receptor inhibitor, could improve advanced glycation end (AGE) products or angiotensin Ⅱ (Ang Ⅱ)-induced podocyte injury including detachment or apoptosis. In this current paper, we first confirmed the protective effect of telmisartan on early diabetic kidney injury in type 1 diabetic rats. Telmisartan reduced the loss of podocin and inhibited the expression of α-SMA, reflecting its protective effect on podocyte injury and mesangial proliferation, respectively. More interestingly we observed an opposite effect of telmisartan on the cell viability and apoptosis of podocytes and mesangial cells in a high-glucose environment in vitro. The anti-apoptotic effect of telmisartan on podocytes might be related to its inhibition of swiprosin-1 (a protein that can mediate high glucose-induced podocyte apoptosis) expression. While telmisartan induced a high expression of PPARγ in mesangial cells, and GW9662 (a PPARγ antagonist) partially inhibited telmisartan-induced apoptosis and reduced the viability of mesangial cells. In addition, high glucose-induced PKCβ1/TGFβ1 expression in mesangial cells could be blocked by telmisartan. These data provide a more precise cellular mechanism for revealing the protective effect of telmisartan in diabetic kidney injury.

KEYWORDS

diabetic kidney disease, podocytes (MeSH: D050199), mesangial cells, telmisartan (PubChem CID: 65, apoptosis

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INTRODUCTION

Diabetic kidney disease (DKD), well known as a chronic kidney disease induced by diabetes mellitus (DM) type 1 or 2 (Podgórski et al., 2019), could worsen glomerular filtration rate (GFR) decreases progressively, then eventually develops into end-stage renal disease (Hou et al., 2018; Ruiz-Ortega et al., 2020; Expert Group of Chinese Society of Nephrology, 2021). There are two mechanisms that hyperglycemia mediates via the kidney podocyte injury and glomerular basement membrane (GBM) changes induced by mesangial cell expansion or proliferation (Anders et al., 2018).

Podocytes, are specialized visceral epithelial cells, lining the external layer of the GBM, which’s foot processes interdigitate forming an ultimate barrier to prevent urinary protein loss (Podgórski et al., 2019). The number and/or density of each glomerulus has been studied in patients with DM (Papadopoulou-Marketou et al., 2017). Injury to the podocytes contributes to the loss of their adhesive properties and is a major cause of DKD development (Podgórski et al., 2019). Another notable characteristic of podocytes is mature podocytes are limited proliferative cells (Podgórski et al., 2019), (Griffin et al., 2003). Losses of podocytes bring about proliferation of the mesangial cells, nevertheless, more substantial losses lead to glomerular fibrosis and increased proteinuria as subsequent denudation of the GBM (Fukuda et al., 2012). Poor glycemic control results in podocytopathy (Anders et al., 2018), morphological changes characterized by podocytes hypertrophy, podocyte epithelial-mesenchymal transdifferentiation (Chang et al., 2017), podocytes detachment (Zhang et al., 2020), podocytes apoptosis (Wang et al., 2018a) and podocytes loss, which are leading to the progressive podocytes aberrations result in the detachment of the GBM with consequent glomerulosclerosis.

Mesangial cells have a significant impact on not only the adjustment of glomerular and intraglomerular circulation but also the conservation of glomeruli, such as the defense of glomerular endothelial cells and outflow of substances from serum and fluid from microvessels (Wakisaka et al., 2021). Thickened GBM and expanded mesangial are noticeable glomerular impairments in diabetes (Papadopoulou-Marketou et al., 2017). GBM thickening is an early histopathological in DKD and is affected by the aberrant income and variation of extracellular matrix secreted by endothelial cells and podocytes (Anders et al., 2018). Hyperglycemia excites mesangial cells to proliferate and fabricate matrix (Kriz et al., 2017) via activation of transforming growth factor-β (TGFβ), which directly causes the transcriptional activation of matrix collagens (Ziyadeh et al., 2000) conducing to the expanding mesangial matrix.

Early intervention with hypoglycemic and antihypertensive treatment is beneficial to delay the occurrence and development of DKD (Martins and Norris, 2001). Especially recommended in normal blood pressure adults with DM and albuminuria is an angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) (Liew et al., 2020). Blockade of the renin–angiotensin system ameliorates the expression of ANGPTL2 and integrin which maintain the glomerular barrier (Tawfik et al., 2021). The reason telmisartan was chosen is that it is described as more efficient than other ARB drugs in mitigating proteinuria (Naruse et al., 2019; Guo et al., 2020). Moreover, telmisartan can decrease cisplatin-induced nephrotoxicity such as podocyte apoptosis and autophagy-associated protein expression levels (Malik et al., 2015). Fascinatingly, telmisartan has such characteristics taking into account its twin role of AT1 receptor blocking action and peroxisome proliferator-activated receptor gamma (PPARγ) partial agonistic property (Balakumar et al., 2012). The goal of this paper was to examine the protective influence of telmisartan on podocyte injury and mesangial expansion at the early stage of type 1 DM, respectively.

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MATERIALS AND METHODS

1. Animals

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.

2. Diabetic Model and Treatment

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 vehicles served as controls (group Con, n = 10). Diabetes mellitus was confirmed by measuring glucose levels in tail venous blood using a B-glucose analyzer (HemoCue, Angelholm, Sweden) 7 days later. Rats with random blood glucose levels>16.7 mmol/L were included in the 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 were collected in metabolic cages. Rats were sacrificed under anesthesia after 4 weeks, the kidneys were removed and weighed for histological analysis and protein extraction.

3. Urinary Albumin

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.

4. Creatinine Clearance Rate

The 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).

5. Immunohistochemical and TUNEL Staining

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).

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6. Cell Culture

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.

7. Cell Viability and Proliferation Assay

Cell Counting Kit-8 (CCK-8) was used to measure cell proliferation and cell viability. Cells were seeded in each well of 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 Scientific, Waltham, MA, United States) at a wavelength of 450 nm.

8. Annexin V and Propidium Iodide Staining

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 a binding buffer. Annexin V-FITC and PI (eBioscience, Santiago, CA, United States) were added to the cellular suspension as per 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).

9. Western Blotting

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 a nitrocellulose membrane (Pall Corporation, NY, United States). The membrane was blocked with 5% bovine serum albumin and blotted with antibodies. 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 IRDye-conjugated anti-mouse or anti-rabbit secondary antibodies (Rockland, Limerick, PA, United States) at 1: 5,000. Using ODYSSEY INFRARED IMAGING SYSTEM (LI-COR) to analyze the results.

10. Statistical Analyses

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.

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DISCUSSION

Telmisartan is a selective AT1 receptor blocker that has been used clinically for reducing elevated blood pressure and urinary protein excretion in hypertensive patients (Baden et al., 2008; Mann et al.,2009). Several clinical trials have suggested that telmisartan is effective to reduce proteinuria in patients with macroalbuminuria and delaying the onset and progression of diabetic nephropathy (Makino et al., 2005; Nakamura et al., 2010; Fujita et al., 2011; Schmieder et al., 2011). In the present study, oral treatment with telmisartan in STZ-induced diabetes rats prevented the onset of early abnormalities in renal and overall including the decrease in body weight, blood glucose, and urine protein. These results confirmed that telmisartan has renoprotection in early-stage diabetic nephropathy mice. More importantly, this study found that the protective effect of telmisartan on diabetic glomeruli was reflected in the anti-apoptotic and pro-apoptotic effects on podocytes and mesangial cells, respectively.

Advanced glycation end products (AGE) could cause podocyte DNA injury and detachment partly through stimulation of the Ang II-AT1R axis, thus supplying an innovative beneficial feature of telmisartan in DKD (Fukami et al., 2013). In normotensive, low-grade proteinuric glomerular diseases, treatment with telmisartan in the early stage of disease, attenuates glomerular and tubulointerstitial damage (Villa et al., 2011). And several pathways are probably linked to the pleiotropic consequences including growth factor signaling, mammalian target of rapamycin signaling, protein ubiquitination, the Wnt-beta catenin pathway, and hypoxia signaling (Villa et al., 2011).

Recently, we reported that swiprosin-1 (Wang et al., 2018b), another name for EF-hand domain containing 2 (EFhd2), played a critical part in the progression of DKD initiated after the induction, while it located in podocytes of the mouse glomerulus. Swiprosin-1 absence ameliorated mitochondria-dependent podocyte apoptosis stimulated by hyperglycemia or high glucose through the p38 MAPK signaling pathway. Here, we also found that telmisartan inhibited hyperglycemia or high-glucose-induced expression of swiprosin-1 both in vivo and in vitro, which indicated the anti-apoptosis effect of telmisartan on podocytes may be related to the regulation of swiprosin-1 expression.

Mesangial cell proliferation and excessive deposition of extracellular matrix proteins have been ascertained as contributing to the development of DKD (Lee et al., 2004). Previous studies showed that high glucose could induce the expression of mesangial extracellular matrix proteins under hyperglycemia (Taniguchi et al., 2013). α-SMA is generally used to differentiate mesangial cells from other glomerular cells in STZ-induced diabetes mice, and increased α-SMA expression could be the marker of mesangial cells' phenotypic shifts from the non-activated phase to the proliferative, secretory activated phase (Niu et al., 2014). Here, we found that telmisartan decreased α-SMA expression in the diabetic glomerulus. In addition, it has been reported that mesangial cell proliferation has a significant impact on the pathogenesis of DKD (Zeng et al., 2013). Our results in this study were reflecting the time- and dose-dependent depressed effect of telmisartan on mesangial cell proliferation related to proapoptotic characteristics.

Clinical evidence recommends that telmisartan is more efficient than losartan in ameliorating proteinuria in hypertensive persons with DKD, which may be related to its ability to partially agonize PPARγ (Bichu et al., 2009). Furthermore, these beneficial changes such as the prevention of renal atrophy and fibrosis of telmisartan were connected with a diminishing in the expression of TGFβ1 and other proinflammatory and profibrotic cytokine genes via the PPARγ/HGF activation (Kusunoki et al., 2012), independent of Ang II type 1 receptor blockade. Here, we also found telmisartan specifically activated PPARγ gene expression in mesangial cells, and the pro-apoptotic effect caused by telmisartan to mesangial cells could be alleviated by PPARγ inhibitors.

PKCβ1 is one of the extensively expressed families of serine–threonine kinases that transduce a wide range of cellular progressions by substrate-specific phosphorylation (Newton, 1995). It has been reported that not only increased PKCβ activity but also its mRNA levels are observed in human diabetic nephropathy biopsies (Langham et al., 2008). Hyperglycemia-induced PKCβ expression and activation has pleiotropic effects in mesangial cells, including the promoting excessive accumulation of ECM proteins (Brownlee, 2001). Studies have shown that inhibition of PKCβ attenuates glomerular hypercellularity and extracellular matrix expansion in db/db mice and glomerular dysfunction in STZ-rats (Ishii et al., 1996; Koya et al., 2000). Likewise, PKCβ inhibitor attenuated platelet-derived growth factor (PDGF)-driven mesangial cell proliferation and collagen production (Tokuyama et al., 2011). In our study, telmisartan reduced the upregulation of PKCβ1 mRNA and protein expression in hyperglycemia-stimulated mesangial cells. In addition, TGFβ1 expression in mesangial cells induced by high glucose could also be inhibited by telmisartan.

Both AT1 and AT2 receptors, well known as seven transmembrane-spanning G protein-coupled receptors, have been cloned and pharmacologically illustrated (Touyz and Berry, 2002). The AT1 receptors can be selectively antagonized by telmisartan, while the AT1 receptor blockers can induce the expression of AT2 receptors (Touyz and Berry, 2002). Studies have shown that AT1 receptors exert their influences by restraining cell growth, and by provoking apoptosis (Horiuchi et al., 1997; Touyz et al., 1999). Moreover, AT2 receptors induce cell apoptosis in a specific conformation through p38 MAPK-mediated apoptotic signaling (Miura and Karnik, 2000). In our present paper, expression of AT1 and AT2 mRNA was unchanged in cultured mesangial cells stimulated with telmisartan or hyperglycemia. Therefore, telmisartan-induced mesangial cell apoptosis and decreased expression of PKCβ1 might not be mediated by AT1 and AT2 receptors.

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In conclusion, telmisartan attenuated early glomerular injury in type 1 diabetic rats by inhibiting podocyte apoptosis and promoting mesangial apoptosis. The antiapoptotic effect of telmisartan in podocytes may be related to its inhibition of swiprosin-1 expression, meanwhile, the proapoptotic effect on mesangial cells was partially associated with its agonistic effect on PPARγ. Additionally, telmisartan selectively blocked the expression of PKCβ1/TGFβ1 in mesangial cells but not in podocytes. Advanced studies are necessitated to elucidate the opposite but beneficial effects of telmisartan on podocytes and mesangial cells and the underlying molecular mechanisms.

Xin Wei 1, Yabin Ma2, Ya Li 3 , Wenzhao Zhang4 , Yuting Zhong5 , Yue Yu6 , Li-Chao Zhang5, Zhibin Wang4 and Ye Tu2

1 Department of Clinical Pharmacy, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China,

2 Department of Pharmacy, Shanghai East Hospital, Tongji University, Shanghai, China,

3 Department of Clinical Pharmacy, Clinical Trial Center, The First Affiliated Hospital of Shandong First Medical University and Shandong Provincial Qianfoshan Hospital, Jinan, China,

4 Department of Critical Care Medicine, School of Anesthesiology, Naval Medical University, Shanghai, China,

5 Department of Pharmacy, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai, China,

6 Institute of Vascular Disease, Shanghai TCM-Integrated Hospital, Shanghai, China