Deciphering the Pharmacological Mechanisms of Qidan Dihuang Decoction in Ameliorating Renal Fibrosis in Diabetic Nephropathy through Experimental Validation In Vitro and In Vivo

Objective QiDan DiHuang decoction (QDD) has been proven to have good efficacy in decreasing albuminuria levels, improving renal function, and inhibiting renal fibrosis in diabetic nephropathy (DN). However, the potential mechanism remains unclear. The purpose of this study was to explore the underlying mechanism of QDD for treating DN in vitro and in vivo. Methods Db/db mice were treated with QDD or saline intragastrically for 12 weeks. Non-diabetic db/m mice were used as controls. Rat renal tubular epithelial cells (NRK-52E) were cultured in high glucose conditions. ATF4 siRNA was transfected into NRK-52E cells. Different indicators were detected via UPLC, RT-PCR, western blotting, cell viability assays and apoptosis, transmission electron microscopy, histology, and immunofluorescence staining. Results Db/db mice experienced severe kidney damage and fibrosis, increased levels of PERK, eIF2α, and ATF4, and suppression of renal autophagy compared with db/m mice. The results showed a significant improvement in glucose intolerance, blood urea nitrogen, urine albumin, serum creatinine, and renal fibrosis in db/db mice with QDD treatment. Meanwhile, the application of QDD resulted in the downregulation of PERK, eIF2α, and ATF4 and the upregulation of autophagy in diabetic kidneys. In vitro, the exposure of NRK-52E cells to high glucose resulted in downregulation of the ratio of LC3-II/LC3-I and upregulation of P62, a reduction in the number of autophagosomes and upregulation of fibronectin (FN), collagen IV and TGF-β1 protein, which was reversed by QDD treatment through inhibiting ATF4 expression. Conclusions Taken together, our results suggest that QDD effectively alleviates diabetic renal injuries and fibrosis by inhibiting the PERK-eIF2α-ATF4 pathway and promoting autophagy in diabetic nephropathy.


Introduction
Diabetic nephropathy (DN) is known to be a major complication of diabetes and is also the main cause leading to kidney failure [1]. e pathogenesis of DN is complicated and multifactorial. e major pathological changes of DN manifest as mesangial expansion, thickened basement membrane, and renal fibrosis. Renal fibrosis is an important factor for kidney function decline in DN and progression to ESRD, characterized by excessive deposition of extracellular matrix components, such as collagens and fibronectin [2,3]. erefore, elucidating the mechanism of renal fibrosis and developing effective treatment methods are urgent problems to be solved.
Traditional Chinese medicine (TCM) has significant advantages and has been beneficial in treating diabetes and its complications for hundreds of years, especially in the prevention and treatment of DN [4,5]. Qidan Dihuang decoction (QDD) is an herb compound for the treatment of DN, which was developed via literature research and data analysis, and has been patented (Patent No. ZL201310015464.5). QDD is composed of Astragalus membranaceus (Huang Qi), Salvia miltiorrhiza Bunge (Dan Shen), Rehmannia glutinosa (Gaertn) (Di Huang), Dioscoreae rhizoma (Shan Yao), and Glycyrrhiza uralensis (Gan Cao). Our previous study reported that QDD has remarkable clinical efficacy in improving albuminuria levels in patients with DN [6]. In addition, QDD has a protective effect on the kidneys of diabetic rats and it can reverse the inflammatory milieu of DN serum [7]. We also found that QDD can significantly reduce serum creatinine and 24 h proteinuria in rats with DN and improve the levels of fibrosis marker proteins SMA-α and TGF-β [8]. erefore, QDD is considered a good candidate for preventing renal fibrosis in DN. However, the protective mechanism of QDD against DN is unknown.
Autophagy is a lysosomal degradation pathway. e activation of autophagy can promote the degradation of protein aggregates and damaged organelles, thereby helping maintain intracellular homeostasis [9]. Multiple studies have confirmed that inhibition of autophagy plays a pivotal role in the pathogenesis of diabetic nephropathy. Activation of autophagy is beneficial for improving kidney injury and restoring renal function. erefore, improving autophagy has become an important therapeutic target in DN treatment [10,11]. Autophagy activation can be regulated by multiple pathways, including the mechanistic target of the rapamycin (mTOR) pathway and the AMP-activated protein kinase (AMPK) pathway. However, studies have suggested that endoplasmic reticulum stress (ERS) is also a key way to regulate autophagy activity [12]. Moreover, ERS has been increasingly recognized as a critical mechanism in the pathogenesis of DN [13,14]. e unfolded protein response (UPR), mediated by endoplasmic reticulum stress, is designed to eliminate the accumulation of misfolded proteins [15]. e three main arms of the UPR are mediated by protein kinase RNA (PKR)-like ER kinase (PERK), inositolrequiring 1 (IRE1), and activating transcription factor 6 (ATF6). Studies suggested that the PERK/eIF2α/ATF4 pathway was involved in the regulation of autophagy [16]. Our previous study showed that the ERS-inducible transcription factor ATF4 promotes tubulointerstitial fibrosis by inhibiting autophagy in DN [17]. erefore, we speculate that the mechanism of QDD delaying renal fibrosis in DN is related to the regulation of autophagy via the PERK/eIF2α/ ATF4 signaling pathway.
is study aimed to explore the effect of QDD in improving renal fibrosis in DN and the effect of QDD in regulating the PERK/eIF2α/ATF4 signaling pathway and renal autophagy. We demonstrated that QDD suppressed renal fibrosis in DN by inhibiting the PERK/eIF2α/ATF4 signaling pathway and restoring autophagy activity.

QDD Preparation and UPLC-QTOF-MS Analysis of QDD.
QDD is comprised of five herbs: 30 g of Astragalus membranaceus, 15 g of Rehmannia glutinosa (Gaertn), 15 g of Dioscoreae rhizoma, 15 g of Salvia miltiorrhiza bunge, and 5 g of Glycyrrhiza uralensis. e herbs were soaked in 500 ml distilled water for 30 minutes and boiled twice for one hour per time. After filtering with sterile gauze, the extraction liquid was centrifuged, and the supernatant was dried in a vacuum freeze dryer and stored at −80°C.
About 100 mg sample was added into 500 μL extract, homogenized for 4 minutes, placed in an ice-water bath, sonicated for 1 hour, and then allowed to stay at −20°C for 1 hour. e sample was centrifuged at 12000 rpm for 10 minutes at 4°C. en, 200 μl of supernatant was taken for the test. e target compound was analyzed by UPLC-QTOF-MS, and 0.1% formic acid (solvent A) and acetonitrile 0.1% formic acid (solvent B) were used for gradient elution. Linear gradient elution was conducted as follows: 0-3.5 minutes at 5-15% B, 3.5 to 6.5 minutes at 15-30% B, 6.5 to 12.5 minutes at 30-70% B, 12.5 to 18 minutes at 70-100% B, and 18 to 22 minutes at 100% B. In addition, based on the response value, methylnissolin 3-O-glucoside, trifolirhizin, berberrubine, and prazewaquinone A were selected for quantitative analysis.

Animal Model and
Treatments. C57BL/KsJ leptin receptor-deficient (db/db) and db/m Six-week-old male mice were purchased from the Model Animal Research Center of Nanjing University. Both db/db and db/m mice were raised at 21 ± 3°C in a 12-hour light/dark cycle environment. Before the experiment, all experimental mice were adaptively fed for 1 week, and they had free access to food and water. Six db/m mice were used as controls. According to the random number table method, 12 db/db mice were divided into the db/db group and db/db + QDD group, with six mice in each group. Mice in the db/db + QDD group were given QDD by gavage at 10 g/kg/day, whereas the mice in the db/db group and db/m group were administered saline. e body weight and blood glucose of the mice were measured once a week, and basic data such as the health status, food intake, and water consumption of the mice were observed and recorded. Twelve weeks later, the mice were moved to the euthanasia 2 Evidence-Based Complementary and Alternative Medicine workstation and anesthetized with sodium pentobarbital (60 mg/kg iP). After the collection of blood samples from the orbital venous, the renal tissues of the mice from each group were immediately collected according to the different testing protocols. e blood glucose test in this experiment was performed with a blood glucose meter (Roche Diabetes Care GmbH, UK). e animal experiments were approved by the Animal Experiment Ethics Committee of Jinan University (approval number: 2019022501).

Western
Blotting. Proteins were extracted from kidney tissue samples and NRK-52E cells using the RIPA method.

Oral Glucose Tolerance Test (OGTT).
Mice were fasted for 6 h and then given glucose solution by gavage at 2.0 g/ kg.
en, at 0, 30, 60, 90, and 120 minutes, blood was drawn from the tail vein, and the blood glucose at each time was measured with a blood glucose meter. Finally, the blood glucose curve was drawn and its area under the curve (AUC PG ) was calculated. Area of blood glucose curve (AUC PG ) � 15 × FPG + 30 × PG 30 + 30 × PG 60 + 30 × PG 90 + 15 × PG 120.
2.6. Biochemical Analysis. Serum creatinine (Scr) and blood urea nitrogen (BUN) were measured by an automatic chemical analyzer (AU480; Beckman Coulter Inc). A blood glucose meter (Roche Diabetes Care GmbH, UK) was used to detect blood glucose. In addition, mouse metabolic cages were used to collect mouse urine for 24 hours, and urinary albumin was detected by ELISA.

Cell Viability
Assay. NRK-52E cells (5 × 10 3 cells/well) were seeded into 96-well plates and treated with high glucose for 48 hours or QDD for 12 hours. e viability was evaluated with a Cell Counting Kit 8 (Dojindo Laboratories, Kyushu, Japan).

Cell Apoptosis.
e cells were trypsinized without EDTA and collected. e cells were washed twice with PBS, and the supernatant was discarded after centrifugation. e experiment was performed according to the instructions of the Annexin-V-FITC detection kit (K201-100, BioVision, USA). In short, 100 μL of binding buffer was added to prepare the cell suspension. en, 2.5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added to the cell suspension. All samples were incubated in a dark environment at room temperature for 15 minutes, and 200 μl binding buffer was gently mixed with the cells. Flow cytometry was used to detect the level of cell apoptosis (version 10.0, FlowJo, FACS CaliburTM, BD, USA).

Histopathology and Immunofluorescence.
e kidney was quickly removed and washed with physiological saline, fixed in 4% paraformaldehyde for 30 minutes, and dehydrated. Finally, it was infiltrated by xylene and embedded in paraffin.
e samples were cut into 5 μm thick paraffin sections. Following conventional staining steps, the sections were stained with HE, Periodic acid-Schiff (PAS), and Masson's trichrome. e mesangial expansion index was measured to evaluate the quantifications of PAS staining. Positive signals were quantified using Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD) in six randomly selected visual fields at 200× magnification. e mesangial expansion index was expressed as a percentage positive area of the entire glomerulus. Collagen fibrils area Evidence-Based Complementary and Alternative Medicine ratio was measured to evaluate the quantifications of Masson staining. e collagen deposition in the kidney was assessed by Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD). For each experimental group, six fields were randomly selected at 200× magnification. Collagen fibrils area ratio was expressed as a percentage of blue fibrotic area relative to the entire area.
Immunofluorescence staining was used to detect the expression of renal light chain 3 (LC3B). After antigen retrieval and blocking, the kidney tissue sections were incubated with anti-LC3B (1 : 200, CST) at 4°C for 12 hours. After returning to room temperature, the samples were washed with PBS and incubated with ALexa Fluor 594-conjugated goat anti-rabbit IgG, FITC-conjugated goat anti-mouse IgG, and cy3-conjugated goat anti-rabbit IgG at 37°C for 1 hour. DAPI under dark conditions was used to label the cell nucleus, and the sample was washed with PBS. A laser scanning confocal fluorescence microscope was used to observe the sections and collect images (UltraVIEW VoX; PerkinElmer, Inc). e fluorescence activity was measured to evaluate the quantifications of immunofluorescence staining. e fluorescence activity of LC3B was expressed as the percentage integrated optical density of the entire area. For each experimental group, six fields were randomly selected and the mean fluorescence activity was calculated.

Transmission Electron Microscopy.
After the experiment, the cells were collected, and the samples were immediately fixed in 2.5% glutaraldehyde. e samples were rinsed 4 times with 0.1 mol/L phosphate buffer and then fixed with 1% osmium tetroxide at 4°C. After dehydration, the samples were embedded in Durcupan ACM. Finally, the samples were stained with uranyl acetate and lead citrate and sectioned. A transmission electron microscope was used to observe the autophagosomes (JEOL-100CXII, JEOL, Japan). Ten fields of view from each group were selected randomly for observation.

Statistical Analysis.
e data are expressed as the mean ± SE, and SPSS software 22.0 (SPSS, Inc) was used for statistical analysis. e student's t-test was applied to determine significant differences between two independent groups. For multiple groups, one-way ANOVA with Tukey's post hoc test was used. P < 0.05 was considered statistically significant.

QDD Improved Kidney Injury in Db/Db Mice.
To evaluate the effects of QDD on the renal function of db/db mice, body weight, urinary albumin urea nitrogen, serum creatinine, and glucose tolerance were measured in each group. e results suggested that db/db mice showed obvious glucose intolerance compared with db/m mice. e glucose intolerance of the QDD group was significantly improved (Figures 3(a)-3(f )). Compared to the db/m group, the body weight of the db/db group and QDD group were obviously increased (Figure 4(a)). In addition, the urinary albumin, urea nitrogen, and creatinine levels of the db/db group were obviously increased compared with the db/db group. e QDD group showed significantly reduced urinary albumin, urea nitrogen, and creatinine compared with the db/db group (Figures 4(b)-4(d)). We used HE, PAS, and Masson staining methods to detect pathological changes in the kidney tissue. Mesangial matrix and glomerular volume expansion, mesangial cell proliferation, thickening of the basement membrane, and evident renal fiber accumulation were observed in the kidney tissue of the db/db mice. Compared to with the db/db group, QDD treatment alleviated these pathological injuries (Figures 4(e)-4(h)). According to the results above, QDD improved glucose intolerance, renal function, urinary albumin, and pathological injuries.

QDD Ameliorated Renal Fibrosis and Inhibited the PERK-eIF2α-ATF4 Pathway in Db/Db Mice.
To clarify the effects of QDD on renal fibrosis, the expression of FN, Col-IV, and TGF-β1 was evaluated by western blot. e western blot results revealed that the expression levels of FN, TGF-β1, and Col-IV increased significantly in the db/db group compared with the db/m group. QDD remarkably reduced FN, TGF-β1 and Col-IV expression in the kidney compared with the db/db group ( Figure 5(a)). To further explore the potential mechanism of QDD in the treatment of DN, we tested the expression of PERK, eIF2α, and ATF4, which are related to endoplasmic reticulum stress. e results showed that the mRNA expression of PERK, ATF4 and protein expression of PERK, p-eIF2α and ATF4 in the kidney tissue of db/db mice were markedly increased, while the mRNA level and total protein expression of eIF2α was not changed in the db/db mice. After QDD treatment, the mRNA expression of PERK, ATF4 and protein expression of PERK, p-eIF2α and ATF4 showed a significant decline (Figures 5(a)  Our results suggested that protein eIF2α had high activity rather than total amount changes and QDD has effects on phosphorylation of eIF2α. ese results indicated that QDD can improve renal fibrosis and inhibit the PERK-eIF2α-ATF4 pathway in db/db mice.

QDD Promoted Autophagy in Db/Db Mice.
We detected the expression of the autophagy-related proteins mTOR, p-mTOR, and LC3 in db/db mice. e expression of p-mTOR in the kidneys of db/db mice significantly increased, and the ratio of LC3-II/LC3-I markedly decreased compared with that in db/dm mice. After QDD treatment, db/db mice showed downregulation of p-mTOR and   upregulation of the ratio of LC3-II/LC3-I (Figure 6(a)). In addition, the results of immunofluorescence also showed that the expression of LC3B in the kidneys of db/db mice was decreased compared with that in db/dm mice. QDD significantly increased the expression of LC3B (Figure 6(b)).
Our results demonstrated that QDD can inhibit the PERK-eIF2α-ATF4 pathway and promote autophagy in db/db mice.   Evidence-Based Complementary and Alternative Medicine

QDD Alleviated HG-Induced NRK-52E Cells Injury.
To determine whether QDD affected cell viability, we used a CCK-8 assay to assess the effect of QDD on the viability of NRK-52E cells. NRK-52E cells were treated with high glucose (HG) combined with QDD at various concentrations, ranging from 200 μg/mL to 2400 μg/mL. Treatment with QDD at concentrations from 200 μg/mL to 2000 μg/mL markedly improved the survival rate of the NRK-52E cells. However, a significant reduction in cell viability and survival rate was observed in the cells treated with 2400 μg/ml QDD (Figure 7(a)). Based on these findings, QDD at 200 μg/mL, 1200 μg/mL, and 2000 μg/mL concentrations were used in the subsequent experiments. Additionally, we also observed the effect of QDD on the apoptosis rate of the cells and found that QDD can significantly reduce the level of cell apoptosis (Figure 7(b)).

QDD Inhibited the Expression of ATF4 and Col-IV and Promoted Autophagy in HG-Treated NRK-52E Cells.
To clarify the effect of QDD on ATF4, fibrosis levels, and autophagy in HG-treated NRK-52E cells, we used western blotting to detect the expression of ATF4, Col-IV, and the autophagy-related proteins P62, LC3-I, and LC3-II. e results indicated that the protein expression of ATF4, P62, and fibrosis marker protein Col-IV was remarkably upregulated in HG-treated NRK-52E cells compared with cells cultured in NG conditions. At the same time, the ratio of LC3-II/LC3-I decreased markedly in HG-treated NRK-52E cells. e cells exposed to HG combined with 200 μg/mL, 1200 μg/mL, or 2000 μg/mL QDD showed downregulation of ATF4 and Col-IV and upregulation of the ratio of LC3-II/ LC3-I. However, HG-induced cells treated with 1200 μg/mL or 2000 μg/mL QDD showed a significantly reduced level of P62 (Figures 8(a) and 8(b)). Moreover, we also detected the changes in the autophagic vesicles in NRK-52E cells by transmission electron microscopy. In each group, 10 regions were randomly selected for observation, and the number of autophagosomes was calculated and counted. e results showed that compared with NRK-52E cells cultured in NG conditions, the number of autophagosomes in NRK-52E cells cultured in HG conditions was significantly reduced. e number of autophagosomes in the cells cultured under HG conditions with 2000 μg/mL QDD was obviously increased compared with that in the cells cultured under HG conditions (Figure 8(c)). Our results verified that QDD inhibited the expression of ATF4 and Col-IV and promoted autophagy in HG-treated NRK-52E cells.

QDD Improved Fibrosis and Autophagy Levels by
Inhibiting ATF4 in HG-Treated NRK-52E Cells. To further understand the exact mechanism of QDD in alleviating renal tubulointerstitial fibrosis, we transfected NRK-52E cells with siATF4. e protein levels of the autophagy-related proteins P62 and p-mTOR were significantly decreased, and the ratio of LC3-II/LC3-I was obviously increased in the high glucose group transfected with siATF4 compared with the siNC group. Meanwhile, the expression levels of the fibrosis-related proteins FN, TGF-β1, and Col-IV were also reduced significantly in the high glucose group transfected with siATF4 compared with the siNC group. Interestingly, compared with the HG group transfected with siATF4, the expression of the p-mTOR protein was significantly downregulated, and the expression of FN, TGF-β1, and Col-IV also decreased significantly in the QDD (2000 μg/mL) combined with siATF4 group (Figure 9(a)). In addition, we also detected the level of autophagic vesicles by transmission electron microscopy. Compared with the high glucose group transfected with siNC, the number of autophagic vesicles was obviously increased in the high glucose group transfected with siATF4. Moreover, incubation in HG conditions with 2000 μg/mL QDD combined with transfection of siATF4 significantly enhanced the number of autophagic vesicles compared with that in HG conditions transfected with siATF4 alone (Figure 9(b)). ese results suggested that QDD improved the level of fibrosis by inhibiting ATF4 and regulating autophagy in NRK52E cells.

Discussion
e db/db mouse model of type 2 diabetes has typical pathophysiological characteristics that parallel the development of DN in humans, showing obvious renal tubular interstitial fibrosis [18,19]. In this study, QDD treatment improved glucose intolerance and alleviated kidney damage and renal tubular interstitial fibrosis in the kidneys of db/db mice. Meanwhile, we found that the PERK/eIF2α/ATF4 pathway was activated and the autophagy activity was suppressed in DN mice and HG-treated NRK-52E cells. QDD treatment can enhance autophagy suppression and inhibit the activated PERK/eIF2α/ATF4 pathway. Our study provides the first evidence for the outstanding role of QDD in regulating PERK/eIF2α/ATF4 pathway-mediated renal autophagy to resist diabetic kidney damage and prevents renal tubulointerstitial fibrosis. e representative cartoon with the potential mechanisms of QDD for DN is shown in Figure 10.
e symptoms related to diabetes are named "Xiao Ke" in traditional Chinese medicine (TCM). According to the theory of TCM, Qi-Yin deficiency with blood-stasis syndrome is an important syndrome of DN. QDD, comprised of Astragalus membranaceus, Salvia miltiorrhiza Bunge, Rehmannia glutinosa (Gaertn), Dioscoreae rhizoma and Glycyrrhiza uralensis, has the effect of invigorating Qi, nourishing Yin and promoting blood circulation. Astragalus has antidiabetic effects [20,21]. Researchers reported that Astragalus polysaccharide upregulates the expression of SIRT1 by inhibiting miR-204, ER stress, and apoptosis in high glucose-induced diabetic retinopathy and metabolic memory models, thereby delaying the development of diabetic retinopathy [22]. At the same time, Astragalus polysaccharides can reduce the apoptosis rate of cardiomyocytes by inhibiting the expression of ATF6 and PERK, which are related to the ER stress pathway in diabetic cardiomyopathy rats and high glucose-treated H9C2 cells [23]. Rehmannia has the function of nourishing yin and invigorating the kidney in TCM theory and has been widely used for the treatment of diabetes, osteoporosis, and cardiovascular diseases for a long time [24]. Chen et al. found that catalpol, an active ingredient extracted from Rehmannia, can reduce podocyte damage in diabetic nephropathy by stabilizing the podocyte skeleton and improving autophagy of damaged podocytes [25]. Rhizoma dioscoreae was first described in the classical medical material book "Shennong Classic Chinese Medicine." is herb has different pharmacological effects, such as lowering blood sugar and relieving asthma and diarrhea [26][27][28]. Salvia miltiorrhiza can invigorate qi and promote blood circulation. Researchers reported that Salvia miltiorrhiza can prevent diabetic nephropathy. e mechanism is not only related to the regulation of the metabolome but may also be related to the inhibition of wnt/β-catenin and TGF-β1 signaling [29]. In this study, we have identified the potential compounds of QDD, including methylnissolin 3-O-glucoside, trifolirhizin, berrubine, and prazewaquinone A through UPLC-Q/TOF-MS. Methylnissolin 3-O-glucoside is derived from astragalus membranaceus. However, as far as we know, no studies about the effect of methylnissolin 3-O-glucoside have been published yet. Trifolirhizin is a natural flavonoid glycoside,   which has the function of anti-inflammatory and regulating autophagy. A previous study confirmed that trifolirhizin can induce autophagy via AMPK/MTOR pathway to prevent and treat colorectal cancer [30]. Berberrubine, a primary metabolite of berberine [31], was demonstrated to have the effect of suppressing the expression of gluconeogenic genes, reducing hepatic gluconeogenesis, and lowering blood glucose [32]. Przewaquinone A is one of the hydroxylated metabolites of tanshinone IIA [33], which has been proven to ameliorate the thickening of the glomerular basement membrane and the collagen deposition in the renal tissues of streptozotocin-induced diabetic rats via attenuating PERK/ p-elf2α/ATF-4 signaling activities [34]. We confirmed the efficacy of QDD in preventing renal fibrosis in type 2 diabetic db/db mice. However, the mechanism of QDD preventing renal fibrosis and postponing the progression of DN needs further research.
Stress-induced autophagy mainly serves as an adaptive and defense mechanism for cell survival to maintain cell homeostasis [35,36]. Researchers have confirmed that inhibiting the autophagy of cells in the renal tubules and glomeruli can promote the development of diabetic nephropathy, glomerular interstitial fibrosis and focal segmental glomerulosclerosis [37][38][39]. Guo et al. found that dihydromyricetin can promote autophagy in NRK-52E cells and reduce renal interstitial fibrosis by regulating miR-155-5p/PTEN signaling and the PI3K/AKT/mTOR signaling pathway in diabetic nephropathy [40]. Zhang et al. found that microRNA-22 accelerates renal tubular interstitial fibrosis by regulating PTEN and inhibiting autophagy in NRK-52E cells in diabetic nephropathy [41]. Our results showed a reduction in the number of autophagosomes in HG-treated NRK-52E cells. Furthermore, qPCR, western blotting, and immunofluorescence indicated that the expression of LC3 was decreased and the expression of p-mTOR was increased in the kidneys of db/db mice. Db/db mice showed critical tubular interstitial fibrosis. Similarly, in HG-treated NRK-52E cells, in addition to the decrease in LC3 expression and increase in p-mTOR expression were observed, the expression of P62 and Col-IV proteins also increased. After QDD treatment, the abnormalities of these indicators were reversed. ese results suggested that QDD is of great significance for the treatment of tubulointerstitial fibrosis by regulating autophagy in DN.
Emerging evidences focus on the relationship between endoplasmic reticulum stress (ERS) and autophagy. PERK-eIF2α pathway has been reported to participate in the regulation of autophagy [42,43]. Knockdown of PERK can reduce ERS-mediated autophagy activity and regulation of myocardial fibrosis in H9c2 cardiomyoblasts [44]. A recent study suggested the PERK-eIF2α pathway has a critical role in autophagy activation and fibrotic changes during ER stress in renal tubular cells [45]. In our previous study, we confirmed that inhibiting the expression of ATF4 can promote autophagy in NRK-52E cells, thereby reducing tubular interstitial fibrosis in DN [17]. It is increasingly recognized that the activation of endoplasmic reticulum stress (ERS) plays an important role in the development and progression of tubular interstitial fibrosis in DN [46][47][48]. In the present study, our results showed that the expression of PERK, eIF2α, and ATF4 in the kidney tissue of db/db mice was obviously upregulated. To clarify the exact mechanism, we inhibited the expression of ATF4 in NRK-52E cells and transfection of the siATF4 plasmid enhanced autophagy, downregulated the expression of TGF-β1, FN, and Col-IV, and improved cell viability. However, of note, ATF4 siRNA only decreased one-third of ATF4 protein levels in NRK-52E cells cultured in HG conditions. Knockdown of ATF4 partly restored autophagy levels in HG-exposed NRK-52E cells.
us, we hope to explore the effect of QDD combined with siATF4 on autophagy and fibrosis levels in HG-treated NRK-52E cells. Although the combination of QDD and siATF4 did not suppress ATF4 expression markedly compared with siATF4 alone, the combination of QDD and siATF4 has a trend to inhibit the expression of ATF4 more compared with siATF4 alone. We found that intervention with QDD combined with transfection of siATF4 significantly improved autophagy activity and alleviated tubular