Korean J Physiol Pharmacol 2022; 26(6): 427-438
Published online November 1, 2022 https://doi.org/10.4196/kjpp.2022.26.6.427
Copyright © Korean J Physiol Pharmacol.
Weichen Zhao, Chunyuan He, Junjie Jiang, Zongbiao Zhao, Hongzhong Yuan, Facai Wang*, and Bingxiang Shen*
Department of Pharmacy, Lu'an Hospital Affiliated to Anhui Medical University, Lu’an People’s Hospital, Lu'an, Anhui 237005, China
Correspondence to:Facai Wang
Author contributions: W.Z., F.W., and B.S. designed the research. W.Z., C.H., J.J., Z.Z., and H.Y. participated in the experiments. W.Z. and C.H. analyzed the data. W.Z. wrote the manuscript. F.W. offered funding. All authors have read and agreed to the published version of the manuscript.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pyroptosis, a form of cell death associated with inflammation, is known to be involved in diabetic nephropathy (DN), and discoid domain receptor 1 (DDR1), an inflammatory regulatory protein, is reported to be associated with diabetes. However, the mechanism underlying DDR1 regulation and pyroptosis in DN remains unknown. We aimed to investigate the effect of DDR1 on renal tubular epithelial cell pyroptosis and the mechanism underlying DN. In this study, we used high glucose (HG)-treated HK-2 cells and rats with a single intraperitoneal injection of streptozotocin as DN models. Subsequently, the expression of pyroptosis-related proteins (cleaved caspase-1, GSDMD-N, Interleukin-1β [IL-1β], and interleukin-18 [IL-18]), DDR1, phosphorylated NF-κB (p-NF-κB), and NLR family pyrin domain-containing 3 (NLRP3) inflammasomes were determined through Western blotting. IL-1β and IL-18 levels were determined using ELISA. The rate of pyroptosis was assessed by propidium iodide (PI) staining. The results revealed upregulated expression of pyroptosis-related proteins and increased concentration of IL-1β and IL-18, accompanied by DDR1, p-NF-κB, and NLRP3 upregulation in DN rat kidney tissues and HG-treated HK-2 cells. Moreover, DDR1 knockdown in the background of HG treatment resulted in inhibited expression of pyroptosis-related proteins and attenuation of IL-1β and IL-18 production and PI-positive cell frequency via the NF-κB/NLRP3 pathway in HK-2 cells. However, NLRP3 overexpression reversed the effect of DDR1 knockdown on pyroptosis. In conclusion, we demonstrated that DDR1 may be associated with pyroptosis, and DDR1 knockdown inhibited HG-induced renal tubular epithelial cell pyroptosis. The NF-κB/NLRP3 pathway is probably involved in the underlying mechanism of these findings.
Keywords: Diabetic nephropathy, Discoid domain receptor 1, NLR family pyrin domain-containing 3, Nuclear factor kappa B, Pyroptosis
Diabetic nephropathy (DN) is a serious microvascular complication that is associated with high mortality and disability . There are reports linking inflammation to kidney damage in DN [2,3]. However, the mechanism through with inflammation affects kidney damage in DN is still unclear. Efforts must be taken to identify this mechanism as it may play a crucial role in developing effective treatments for DN. Pyroptosis is a form of programmed cell death—induced in response to inflammatory responses—that has been reported to be involved in the pathogenesis of DN [4,5]. Further, pyroptosis has been reported to be accompanied by the release of pro-inflammatory factors, including interleukin-1β (IL-1β) and interleukin-18 (IL-18) [6,7]. Additionally, studies have confirmed that gasdermin D (GSDMD) is the executor of pyroptosis, and that pyroptosis is initiated by the cleavage of gasdermin D by activated caspase 1 (i.e., cleaved caspase-1), which results in the formation of N- and C-terminal domains of GSDMD (GSDMD-N and GSDMD-C, respectively). GSDMD-N then translocates to the plasma membrane and induces pore formation, thereby inducing pyroptosis. Subsequently, the cell membrane ruptures and the secretion of IL-1β and IL-18 increases [8,9]. Pyroptosis also damages high glucose (HG)-treated renal tubular epithelial cells and renal glomerular endothelial cells [10,11]. However, the mechanism underlying pyroptosis in the background of DN is not entirely understood. The nuclear transcription factor-κB (NF-κB) signaling pathway plays a vital role in the regulation of inflammatory responses. Studies have found that the activation of NF-κB is involved in renal inflammation and fibrosis during the progression of DN [12,13]. Furthermore, recent studies have shown that the NLR family pyrin domain-containing 3 (NLRP3) inflammasome is directly involved in renal inflammation that leads to the progression of diabetic glomerular damage [14,15]. Studies have also shown that NF-κB and NLRP3 inflammasomes are involved in pyroptosis of cells in several diseases [16-18], including renal tubular pyroptosis in DN [10,19]. Hence, regulating the NF-κB/NLRP3 inflammasome signaling pathway could affect the pathogenesis of DN.
Discoid domain receptor 1 (DDR1), a type of receptor tyrosine kinase, is expressed extensively in various tissues, such as the brain, lungs, kidneys, and placenta . DDR1 is involved in a series of physiological and pathological processes, including migration, proliferation, survival, differentiation, and extracellular matrix remodeling . Recent studies have shown that DDR1 is significantly upregulated in the aortic tissues of rats with diabetes and in HG-treated human umbilical vein endothelial cells (HUVECs) . Studies have also confirmed that
Streptozotocin (STZ) and D-glucose were purchased from Sigma-Aldrich (Taufkirchen, Germany). Hematoxylin-eosin (H&E) staining kits (Roche, Indianapolis, IN, USA), Propidium iodide (PI), HRP-labeled anti-rabbit secondary antibodies, HRP-labeled anti-mouse secondary antibodies and anti-β-actin antibodies were purchased from Beyotime Biotechnology (Shanghai, China). Enzyme linked immunosorbent assay (ELISA) kits for IL-1β and IL-18 were procured from Zhongshan Biotechnology (Beijing, China). Antibodies against GSDMD, caspase-1, and NLRP3 were purchased from Proteintech (Wuhan, China). Further, antibodies against IL-1β, IL-18, and DDR1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against NF-κB and phosphorylated NF-κB (pNF-κB) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). siDDR1, Real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) kits, and the Lipofectamine 3000 were purchased from RiboBio (Guangzhou, China).
The animal experimental protocol has been approved by the Experimental Animal Ethics Committee (Changsha, China), in line with internationally recognized ethical standards. Male specific pathogen-free rats of the Sprague–Dawley strain (body weight, 200 ± 20 g) were obtained from the Hunan SJA Laboratory Animal Co., Ltd (Changsha, China), and were housed room temperature (25 ± 1°C) with free access to food and water. The rats were randomly divided into two groups, i.e., control and DN. The rats in these two groups were fed for 12 weeks under the same conditions. The DN rats were administered a single intraperitoneal injection of STZ (60 mg/kg) . To confirm the successful establishment of the DN model, glucose levels were measured in the blood collected from the tail vein of the DN group rats. The blood glucose was higher than 16.7 mM for 3 consecutive days, indicating the establishment of a successful DN model. The control rats were intraperitoneally injected with an equal volume of citrate buffer, pH 4.4.
Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm). The sections were stained with H&E, and the morphological changes in the glomeruli and tubular interstitium were observed using a fluorescence microscope (Olympus, Nanjing, China).
Albumin excretion, plasma creatinine level, and plasma cystatin-C level were measured using kits (W Systems, Minneapolis, MN, USA), as per the manufacturer's instructions. The glomerular volume, fractional mesangial area, and tuft area were analyzed using Image-Pro Plus (Media 149 Cybernetics, Silver Springs, MD, USA).
Human renal tubular (HK-2) cells—cultured for at most 10 generations—were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The HK-2 cells were cultured in a Dulbecco's modified eaglemedium (DMEM; Gibco Life Technologies, Grand Island, NY, USA) containing 5.5 mM glucose, supplemented with 10% fetal bovine serum (Gibco Life Technologies) and 1% penicillin/streptomycin (Beyotime Institute of Biotechnology) at 37°C in an atmosphere 5% CO2. In the experiments, the control cells were cultured in the presence of 5.5 mM D-glucose for 48 h, whereas HG cells were exposed to 33 mM D-glucose for 48 h. Subsequently, for DDR1 knockdown experiments, HK-2 cells were divided into three groups, i.e., HG (exposed to 33 mM D-glucose for 48 h), DDR1-negative control (exposed to 33 mM D-glucose for 48 h after transfection with siControl for 24 h), and DDR1 siRNA (exposed to 33 mM D-glucose for 48 h after transfection with siDDR1 for 24 h). Lipofectamine 3000 was used to transfect the pcDNA3.1-NLRP3 overexpression vector (OVE-NLRP3) and the pcDNA3.1-NC vector (OVE-NC) into DDR1 siRNA cells. The vectors were designed and synthesized by Shanghai Gene Pharmaceutical Co., Ltd (Shanghai, China).
HK-2 cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology) at 25°C in the dark for 10 min, followed by PI staining (5 mg/ml) at 25°C in the dark for 10 min. Fluorescence microscopy was used to verify the high membrane permeability in HK-2 cells.
The concentrations of IL-1β and IL-18 in the plasma of the rats and the HK-2 cells culture supernatants were determined using ELISA kits, as per the manufacturers’ instruction.
The rat kidney tissues and HK-2 cells were independently lysed in RIPA buffer supplemented with 1% phenylmethanesulfonyl fluoride (Beyotime Biotechnology). The proteins extracted from these samples were quantified using commercial bicinchoninic acid protein assay kits (Beyotime Biotechnology). Forty micrograms of individual samples were loaded into each lane of sodium dodecyl sulfate-polyacrylamide gels and resolved by electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich). Following which, the membranes were blocked for 2 h using 5% non-fat milk at room temperature (25 ± 1°C) and incubated overnight with primary antibodies (anti-caspase-1 [1:500 dilution], anti-GSDMD [1:1,000], anti-IL-1β [1:1,000], anti-IL-18 [1:1,000], anti-DDR1 [1:1,000], anti-NF-κB [1:1,000], anti-p-NF-κB [1:500], anti-NLRP3 [1:1,000], and anti-β-actin [1:1,000]) at 4°C. Subsequently, the PVDF membranes were washed with TBS-Tween and incubated with HRP-labeled anti-rabbit (1:5,000) or anti-mouse (1:5,000) secondary antibodies for 2 h at room temperature on a shaker. Chemiluminescence was visualized using ECL kits (Beyotime Biotechnology). Protein bands on the membrane were imaged on ChemiDoc XRS system and the corresponding intensities were evaluated using ImageJ (Bio-Rad, Philadelphia, PA, USA).
Total RNA was extracted from HK-2 cells using the Trizol reagent (TaKaRa, Beijing, China), in accordance with the instructions provided by the manufacturer, and the concentration and purity of this RNA were determined using the absorbance at 260 nm. This RNA was then reverse transcribed into cDNA using 7500 Sequence Detector system (Applied Biosystems, Foster City, CA, USA), in accordance with the manufacturer’s instructions. For RT-qPCR, 2 μl of cDNA was used as template, and a SYBR Green‒based methodology was used employed. The following conditions were used for RT-qPCR: 95°C for 10 min; 40 cycles of 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. Quantitative analysis was performed using the 2–ΔΔCt method. The primers used for RT-qPCR were synthesized by Sangon Biotech Co., Ltd (Shanghai, China), andtheir sequences were listed in Table 1.
Table 1 . Primer sequence of the gene.
F, forward; R, reverse; NF-κB, nuclear transcription factor-κB; NLRP3, NLR family pyrin domain-containing 3; GSDMD, gasdermin D; IL, interleukin.
SPSS 20.0 (IBM Co., Armonk, NY, USA) was used to analyze the experimental data. The data were represented as mean ± standard error of the mean (SEM). The differences between two groups were analyzed using the Student's t-test, and one-way analysis of variance was used to analyze the differences among multiple groups.
In DN rats, we analyzed the effect of hyperglycemia on pyroptosis in the kidney tissues. Analysis of renal pathology in DN rats revealed glomerular and tubulo-interstitial alterations (Fig. 1A). Additionally, the DN rats exhibited high albumin/creatinine ratio, plasma cystatin-C levels, glomerular volume, fractional mesangial area, and tuft area compared to control rats (all p < 0.01) (Fig. 1B–F). Western blotting revealed that the expression of cleaved caspase-1, GSDMD-N, IL-1β, and IL-18 was increased. Further, ELISA revealed that the concentrations of IL-1β and IL-18 were significantly higher in the DN rats than in the control rats (all p < 0.01) (Fig. 2A–D). These findings indicated the occurrence of pyroptosis in the renal tissues of the DN rats. To further explore the role of pyroptosis in DN, we treated HK-2 cells with HG to mimic DN conditions. Consistent with the
As DDR1—an inflammatory protein—is known to play an important role in numerous pathophysiological processes, we investigated the role of DDR1 in pyroptosis. Higher expression of DDR1 was observed in the DN rat kidney tissues and HG-treated HK-2 cells (all p < 0.01) (Fig. 3), suggesting that DDR1 may be an important regulator of kidney damage in DN.
Based on the observation that the expression of DDR1 is upregulated in HG-treated HK-2 cells, we speculated that DDR1 might play a role in HG-induced pyroptosis of HK-2 cells as well. Validation experiments clearly showed that the expression of DDR1 was decreased in HG-treated HK-2 cells after transfection with siDDR1 (p < 0.01) (Fig. 4A, B). Subsequently, PI staining was performed to analyze the effect of DDR1 on membrane rupture during pyroptosis. The increase in the percentage of PI-positive cells in HG-treated cells was suppressed in the background of DDR1 knockdown (p < 0.01) (Fig. 4C, D). Furthermore, DDR1 knockdown significantly suppressed the HG-induced elevation in the levels of cleaved caspase-1, GSDMD-N, IL-1β, and IL-18 (p < 0.05 or p < 0.01) (Fig. 4E, F). The concentrations of IL-1β and IL-18 decreased after silencing DDR1 in the background of HG treatment (compared to HG group) (all p < 0.01) (Fig. 4G, H).
Previous studies have shown that the NF-κB/NLRP3 pathway plays a role in HG-induced pyroptosis. Therefore, we investigated the role of DDR1 in HG-induced pyroptosis of HK-2 cells and assessed the expression of the NF-κB/NLRP3 pathway intermediaries using Western blotting. The expression of p-NF-κB and NLRP3 was upregulated in the DN rat kidney tissues (Fig. 5A, B) and HG-treated HK-2 cells (all p < 0.01) (Fig. 5C, D). Moreover, DDR1 knockdown effectively inhibited the NF-κB/NLRP3 pathway, as indicated by the downregulated expression of p-NF-κB and NLRP3 (p < 0.05 or p < 0.01) (Fig. 5C, D). Hence, all data consistently revealed that DDR1 knockdown resulted in inhibited pyroptosis, and indicated that DDR1 is possibly involved in regulating the NF-κB/NLRP3 pathway.
To confirm the role of NLRP3 in DDR1-induced pyroptosis, we overexpressed NLRP3 in HK-2 cells. The success of overexpression was verified by RT-qPCR. Our results showed that the expression of NF-κB, caspase-1, GSDMD, IL-1β, and IL-18 that had been suppressed in response to DDR1 knockdown was upregulated upon overexpressing NLRP3 (all p < 0.01) (Fig. 6). Taken together, our results show that DDR1 knockdown inhibited HG-induced pyroptosis of renal tubular epithelial cells by regulating the NF-κB/NLRP3 pathway, and that overexpression of NLRP3 partially reversed these effects of DDR1 knockdown.
DN is one of the main causes of chronic kidney failure. In recent years, the incidence of DN has been reported be associated with significantly increased risk of end-stage renal disease (60%), a condition whose pathogenicity is associated with multiple factors . However, the mechanism underlying the pathogenesis of this condition has not been completely elucidated. Pyroptosis is a recently discovered form of cell death that is different from apoptosis and necrosis . In the context of pyroptosis, the classical signaling pathway mediated by caspase-1 is the most widely studied . HG has been reported to significantly upregulate the expression of caspase-1 in renal tubular epithelial cells and renal glomerular endothelial cells [10,11]. Moreover, the silencing of caspase-1 has been reported to effectively inhibit HG-induced pyroptosis, thereby attenuating DN-induced kidney damage . Studies have also reported that in STZ-treated rats, the expression of pyroptosis-related proteins (cleaved caspase-1, GSDMD-N, IL-1β, and IL-18) was significantly upregulated in the kidney tissues [30,31]. Both
As the regulatory mechanism of pyroptosis in DN is not yet fully understood, we explored the role of the inflammatory protein DDR1 in pyroptosis in the context of DN. DDR1, a receptor tyrosine kinase, has emerged as a focus of interest in cardiovascular diseases [32,33]. In the context of endothelial senescence, the expression of DDR1 was upregulated—accompanied by a decrease in cell adhesion and migration—in diabetic rats and HG-treated endothelial cells . Additionally, DDR1 silencing significantly improved endothelial function . DDR1 has also been shown to accelerate renal fibrosis and renal cell apoptosis in DN rats . These findings suggest that DDR1 is involved in diabetic vascular disease. We found that DDR1 expression was upregulated
NF-κB has been identified as a key inflammatory pathway in the pathogenesis of DN-associated inflammation in both animal model and human clinical studies [40,41]. In DN rats, inhibition of NF-κB activation has been reported to downregulate the expression of inflammatory cytokines, thus preventing DN progression . Activation of the NF-κB pathway is a prerequisite for the activation of the NLRP3 inflammasome, a phenomenon induced in response to the upregulated expression of NLRP3, member of the NOD-like receptor protein family that is responsible for forming protein complexes in the cytoplasm that detect various cell signals related to immunity and cell death, and regulating cell function [43-45]. NLRP3 offers a platform to activate caspase-1, cleaving it into enzymatically active caspase-1, thereby promoting the maturation of the IL-1β and IL-18 precursors and the secretion of active IL-1β and IL-18 proteins . In this study, the expression of the NF-κB/NLRP3 pathway intermediaries was upregulated, both
In conclusion, the data from our study prove that DDR1 is upregulated in DN rats and in HG-treated HK-2 cells, and have established the effects of DDR1. Further, DDR1 knockdown inhibited the progression of DN by attenuating the HG-induced pyroptosis of HK-2 cells, which was partially mediated through the NF-κB/NLRP3 pathway. However, the mechanism through which DDR1 affects the activation of the NF-κB pathway could not be clarified in this study. This provides scope for future studies to elucidate the mechanism through which DDR1 affects the activation of the NF-κB pathway to develop new avenues for effective DN treatment.
This work was supported by Anhui Province Natural Science Foundation of Colleges and Universities (No. KJ2021A0342) and Anhui Medical University Scientific Research Fund Project (No. 2020xkj231).
The authors declare no conflicts of interest.
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