Asprosin Promotes Human Renal Tubular Epithelial Cells Apoptosis by Inhibiting Autophagy

doi:10.21203/rs.3.rs-5211042/v1

Download PDF

Article

Asprosin Promotes Human Renal Tubular Epithelial Cells Apoptosis by Inhibiting Autophagy

https://doi.org/10.21203/rs.3.rs-5211042/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Asprosin, a recently discovered adipokine, is a glucotropic hormone involved in the pathogenesis of diabetes and closely associated with diabetic kidney disease (DKD). Renal tubular epithelial cell injury is one of the important pathological characteristic of DKD. However, the precise molecular mechanism remains unclear. In this study, we validated the role of Asprosin in proximal tubular epithelial cells injury in DKD. The expression level of Asprosin was found to be higher in kidney tissues and plasma of DKD patients than in the healthy group. Additionally, the kidney tissues of DKD mouse and HK-2 cells treated with high glucose showed elevated Asprosin expression. Moreover, Asprosin intervention in HK-2 cells led to insufficient autophagy and increased apoptosis. These findings suggest that Asprosin exacerbates autophagy disturbance and induces apoptosis in HK-2 cells under high glucose conditions, and our further studies verified that Asprosin promotes HK-2 cell apoptosis by inhibiting autophagy. Thus, our findings demonstrate for the first time that elevated glucose levels can upregulate Asprosin in both kidney tissue and plasma. Moreover, Asprosin can enhance apoptosis in HK-2 cells by inhibiting autophagy, aggravate autophagy dysregulation and apoptosis caused by high glucose, and promote injury in renal tubular epithelial cells.

Health sciences/Endocrinology

Health sciences/Nephrology

Health sciences/Pathogenesis

Asprosin

Autophagy

Apoptosis

HK-2

DKD is a complication of renal microvascular injury caused by diabetes, which has emerged as the primary cause of chronic kidney disease in China and is associated with a heightened risk of mortality among diabetic patients [1–3]. The etiology of DKD is ntricate, with tubular epithelial cells (TECs) injury serving as a pivotal factor in its onset and progression [6, 8]. Studies have revealed that early-stage DKD patients may exhibit abnormalities in TEC function and morphology, including epithelial mesenchymal transformation, apoptosis and detachment. These aberrations ultimately contribute to renal fibrosis facilitate the advancement of DKD towards end-stage renal disease [9–12]. However, further investigation is still required to elucidate the mechanism underlying tubular injury in DKD patients.

Asprosin is a newly discovered adipokine mainly synthesized and released by white adipose tissue during periods of fasting [13]. Studies have revealed that, apart from white adipose tissue, Asprosin is also distributed in various tissues including kidney, liver, pancreas, brain and other tissues [14], exerting diverse effects on glucose metabolism. Within the central nervous system, Asprosin can directly act on the hypothalamic neuronal network to stimulate appetite and increase food intake behavior [19]. In the liver, Asprosin activates the GPCR-cAMP-PKA pathway leading to continuously release excess glucose by hepatic cells and sbusequent elevation in blood glucose levels [20]. In the pancreas, Asprosin can cause insulin secretion disorder through TLR4/JNK-mediated inflammatory response, leading to elevated blood glucose levels[21]. Numerous studies have demonstrated that Asprosin is implicated in various of diabetic complications, including diabetic retinopathy and peripheral arterial disease (PAD) [18, 22]. Recent research has also revealed that the increased level of Asprosin level is a significant risk factor for the development of DKD and positively correlated with insulin resistance index [23–26]. Based on these findings, we hypothesize that Asprosin may play a role in the pathogenesis and progression of DKD, however, its specific mechanism remains unclear.

The process of autophagy is a highly conserved and tightly regulated intracellular degradation pathway, crucial for maintaining cellular homeostasis by eliminating senescent and damaged organelles as well as misfolded proteins [27]. Under normal physiological conditions, proximal tubular epithelial cells possess abundant lysosomes and actively participate in endocytosis, resulting in a heightened level of basal autophagy [28]. In vivo studies have demonstrated that mice with specific deficiency of Atg5 in the proximal renal tubular exhibit an accumulate a large number of misfolded proteins and deformed organelles, resulting to renal tubular injury, inflammation, fibrosis and other conditions [29,30]. In vitro studies have also found that autophagy defects lead to the aggregation of protein aggregates and inclusion bodies as well as apoptosis in proximal tubular cells [31, 32]. Renal tubular function is a pivotal determinant in the prognosis of renal diseases, with proximal tubular cells frequently serving as primary targets of renal injury. The impairment of proximal tubular autophagy plays a cricial role in the pathogenesis and progression of renal diseases such as DKD [33]. Studies have found that upon acute pathological stimuli like ischemia/reperfusion injury and toxic drug induction, proximal tubular epithelial cells can rapidly upregulate autophagy activity to safeguard against degeneration and acute ischemic injury in proximal renal tubules [35]. However, autophagy disorder can cause the accumulation of a large number of abnormal mitochondria within proximal tubular cells, leading to the generation of reactive oxygen species (ROS). Consequently, this triggers inflammatory reactions and contributes to the development of renal fibrosis and other renal diseases [34]. Recent studies have revealed that elevated glucose levels can induce autophagy dysfunction in proximal tubular epithelial cells, resulting in impaired function and apoptosis [36]. Inhibition of autophagy exacerbates apoptosis and interstitial fibrosis of renal tubular epithelial cells in obstructed kidney [37], while promoting autophagy can prevent renal tubular interstitial fibrosis [38]. However, further investigation is required to elucidate the specific mechanism involved.

In addition, studies have found that Asprosin has the ability to bind to TLR4 receptor of pancreatic β cells, thereby inhibit autophagy through AMPK-mTOR pathway. Furthermore, it promote inflammatory response and apoptosis in β cells [15, 16]. Moreover, Asprosin activates endoplasmic reticulum stress response and induces inflammatory response in skeletal muscle cells via PKCδ / SERCA2 pathway [17]. It also inhibits autophagy and promote apoptosis in retinal microvascular endothelial cells [18]. However, further investigation is required to determine whether Asprosin can contribute to the development of DKD by regulating autophagy in proximal tubular epithelial cells.

The objective of this study is to investigate the role of Asprosin in regulating autophagy and apoptosis of proximal tubular epithelial cells in DKD, thereby further elucidating the key pathophysiological mechanisms underlying Asprosin’s involvement in the occurrence and development of DKD. Additionally, this research aims to identify novel potential targets for the clinical prevention and treatment of DKD.

2.1 Plasma and renal tissue asprosin expression level in patients from DKD increased.

In order to investigate the alterations of Asprosin in plasma and renal tissue in DKD patients, this study collected demographic data and measured plasma Asprosin levels using ELISA in Control group and DKD group. The results revealed a significant elevation of plasma Asprosin levels in DKD patients (Table 1).

Table 1

Comparison of general data and plasma Asprosin levels between the two groups
Indicators	Control	DKD	t(X2)	P
Gender (Male/Female)	13/18	13/18
Age (Years)	73.77 ± 5.03	73.58 ± 7.07	0.124	0.902
Height (m)	1.61 ± 0.07	1.61 ± 0.07	0.280	0.781
Weight (Kg)	58.46 ± 7.86	59.25 ± 9.06	0.366	0.716
BMI(Kg/m²)	22.37 ± 1.53	22.81 ± 2.12	0.932	0.355
SBP(mmHg)	134.9 ± 3.86	134.13 ± 4.15	0.760	0.450
DBP(mmHg)	85.23 ± 3.34	83.61 ± 4.94	1.492	0.140
Asprosin(ng/mL)	0.36 ± 0.02	0.39 ± 0.02	8.789	< 0.0001

In addition, We collected renal tissues from DKD patients who underwent renal biopsy. Results of Periodic Acid-Schiff (PAS) staining results revealed renal tubular atrophy and increased glomerular mesangial matrix (Fig. 1a). Immunohistochemical (IHC) staining was employed to assess the expression of Asprosin in normal renal tissue and DKD patients’ renal tissue. The findings demonstrated a significant increase in Asprosin expression in the renal tissue of DKD patients (P < 0.001) (Fig. 1b,c).

The findings of our study demonstrate a significant elevation in plasma and renal tissue Asprosin expression levels among the patients with DKD when compared to the healthy control population.

2.2 The expression of Asprosin in the kidney tissue of DKD model mice is elevaated.

2.2.1 The establishment of DKD Mouse Model

In this study, a total of 20 SPF male C57BL/6J mice were randomly assigned to the Control group (n = 10) and DKD group (n = 10), which were induced by a combination of high-fat and high-sugar diet along with intraperitoneal injection of streptozotocin (STZ). The general conditions of both groups were assessed, revealing that compared to the control group, the DKD group exhibited a significant decrease in kidney weight/body weight ratio as well as significantly elevated blood pressure, blood glucose and 24-hour urinary protein excretion. These differences were found to be statistically significant (P < 0.05) according to Table 2. PAS staining results demonstrated glomerular sclerosis and increased glomerular mesangial matrix in the renal tissue of DKD mice (Fig. 2.1).

Table 2

General data of two groups of mice
Indicators	Control	DKD	t (X²)	P
Kidney/Body weight(%)	8.02 ± 0.32	5.65 ± 0.50	8.896	P < 0.0001
SBP(mmHG)	113.40 ± 6.58	138.00 ± 19.49	2.674	0.028
DBP(mmHG)	76.40 ± 11.41	90.60 ± 5.55	2.502	0.037
RBG(mmol/L)	7.94 ± 0.93	28.90 ± 2.61	16.900	P < 0.0001
24-UTP(ng)	86.39 ± 23.81	811.3 ± 131.50	12.130	P < 0.0001

The aforementioned results demonstrate the successful establishment of the DKD mouse model.

2.2.2. The expression level of Asprosin in renal tissue of DKD model mice is elevated.

The Western Blot and q-PCR results revealed a significant upregulation of Asprosin protein (P < 0.0001) and mRNA (P < 0.01) expression levels in the renal tissue of DKD group mice compared to those in Control group mice (Fig. 2.2 ).

In conclusion, our findings demonstrate an increased expression level of Asprosin in the renal tissue of DKD model mice.

2.3 High glucose induced the expression of Asprosin in HK-2 cells.

To investigate the effect of high glucose on HK-2 cells, we treated HK-2 cells with high glucose or hypertonic solution for 24h. Western Blot results showed that high glucose increased the expression level of Asprosin in HK-2 cells (P < 0.05) (Fig. 3a,b). This was further supported by q-PCR results (P < 0.001) (Fig. 3c).

In summary, our result shows high glucose can promote the expression of Asprosin in HK-2 cells.

2.4 Asprosin induced and aggravated high glucose-induced apoptosis in HK-2 cells.

In order to explore the effect of Asprosin on HK-2 cell apoptosis and obtain the optimal concentration of Asprosin intervention, this study used different concentrations of Asprosin (0nm/L, 25nm/L, 50nm/L, 100nm/L, 150nm/L, 200nm/L) to stimulate HK-2 cells for different time (0h, 24h, 48h, 72h). Western Blot results showed that Asprosin could increase the expression of Cleaved Caspase-3 in HK-2 cells in a certain range in a concentration and time dependent manner (P < 0.05) (Fig. 4a, b). Under the light microscope, we could observe that the HK-2 cells in the 50nm/L Asprosin treatment group shrank in volume and deformed, and although the cell membrane was intact, foaming phenomenon occurred, and some cells wrinkled and fell off (Fig. 4c), which was further supported by flow cytometry (P < 0.05) (Fig. 4d). Further study found that compared with the HG group, the expression level of Cleaved Caspase-3 in HK-2 cells in the HG + Asprosin group was significantly increased (P < 0.001) (Fig. 4e), which was further supported by flow cytometry (P < 0.0001) (Fig. 4f).

In summary, the above results show that Asprosin can induce apoptosis of HK-2 cells, and can further aggravate the apoptosis of HK-2 cells induced by high glucose.

2.5 Asprosin may induce apoptosis in HK-2 cells by regulating autophagy.

In order to further explore the mechanism of Asprosin-induced apoptosis in HK-2 cells, RNA-seq was performed on HK-2 cells after stimulated with 50nm/L Asprosin for 24h. Pearson correlation algorithm was used to detect the sample correlation, the results showed that the sample correlation was good (R > 0.990) (Fig. 5a). One-way ANOVA was used to screen differentially expressed genes (threshold = 1.5 times, P < 0.05), and the results showed that a total of 191 differentially expressed genes were identified between the two groups, including 127 up-regulated genes and 64 down-regulated genes (Fig. 5b). KEGG enrichment analysis results showed that these differentially expressed genes were mainly involved in intracellular physiological processes such as autophagy and apoptosis, and in various glycolipid metabolism processes, which were closely related to insulin resistance, diabetes and diabetic complications, and were mainly enriched in autophagy and apoptosis-related pathways such as AMPK, mTOR, PI3K/Akt, HIF-1, FoxO, NF-kB (Fig. 5c).

In summary, the RNA-seq results suggest that autophagy-related pathways, especially AMPK/mTOR, PI3K/Akt may have a causal relationship with Asprosin-induced apoptosis in HK-2 cells, which still needs further study.

2.6 Asprosin induced and aggravated high glucose-induced autophagy disorder in HK-2 cells.

In order to explore the effect of Asprosin on HK-2 cell autophagy and obtain the optimal concentration of Asprosin intervention, different concentrations of Asprosin (0nm/L, 25nm/L, 50 nm/L, 100 nm/L, 150 nm/L, 200 nm/L) were used to stimulate HK-2 cells for different time (0h, 24h, 48h, 72h).The results of Western Blot showed that Asprosin could inhibit the autophagy of HK-2 cells in a certain range in a concentration and time dependent manner (P < 0.05) (Fig. 6a,b). In order to further explore whether Asprosin could promote the autophagy disorder of HK-2 cells induced by high glucose, HK-2 cells were cultured in high glucose (30mm/L) DMEM medium for 24h to simulate the high glucose environment in vitro of HK-2 cells with DN, and HK-2 cells cultured in normal glucose concentration (or high glucose) were treated with 50nm/L Asprosin or GST. The results of Western Blot showed that Asprosin could aggravate the autophagy disorder of HK-2 cells induced by high glucose (P < 0.05) (Fig. 6c).In addition, the results of GFP-LC3 immunofluorescence (IF) showed that Asprosin could aggravate the autophagosome formation disorder of HK-2 cells induced by high glucose (P < 0.001) (Fig. 6d).

In summary, this study demonstrated that Asprosin could induce the autophagy disorder of HK-2 cells and aggravate the autophagy disorder of HK-2 cells induced by high glucose.

2.7 Autophagy agonist rapamycin reversed autophagy disorder and apoptosis induced by Asprosin.

Rapamycin (Rap), as a conventional autophagy activator, can promote the autophagy level of HK-2 cells by blocking the mTOR pathway. To prove that Asprosin promotes apoptosis of HK-2 cells by inhibiting autophagy, we pretreated HK-2 cells with Rap (1ng/mL). Western Blot results showed that Rap successfully activated the autophagy of HK-2 cells and significantly reversed the autophagy disorder and apoptosis of HK-2 cells induced by Asprosin (P < 0.05) (Fig. 7a). This was further supported by GFP-LC3 IF and flow cytometry results (P < 0.01) (Fig. 7b,c,d).

In summary, this study demonstrated that Asprosin can induce apoptosis of Hk-2 cells by inhibiting autophagy.

2.8 Autophagy inhibitor 3-MA aggravated autophagy disorder and apoptosis induced by Asprosin.

3-Methyladenine (3-MA) is a conventional autophagy inhibitor, which can cause autophagy disorder in HK-2 cells by inhibiting the formation of autophagosomes. In order to further explore the role of autophagy in Asprosin-induced apoptosis of HK-2 cells, we pretreated HK-2 cells with autophagy inhibitor 3-MA (2mm/L). Western Blot results showed that 3-MA successfully inhibited autophagy in HK-2 cells, and could aggravate Asprosin-induced autophagy disorder and apoptosis of HK-2 cells (P < 0.05) (Fig. 8a). This result was further supported by GFP-LC3 IF and flow cytometry results (P < 0.05) (Fig. 8b,c, d).

In summary, this study demonstrated that Asprosin can induce apoptosis of Hk-2 cells by inhibiting autophagy.

In this study, we found that: (1) Compared with healthy people, the levels of circulating Asprosin and its expression in proximal tubule epithelial cells of DKD patients were increased. (2) The expression of Asprosin in the renal tissue of DKD model mice exhibited a significant increase when compared to that in normal group. (3) The presence of a high glucose environment can induce an upregulation in the presence of Asprosin in HK-2 cells. (4) Asprosin can induce apoptosis of HK-2 cells by inhibiting autophagy.

Asprosin is a novel adipokine encoded by two exons of the FBN1 gene [13], which is distributed in various tissues such as kidney, liver, pancreas, brain and testis [14], and is closely related to the process of glycolipid metabolism. Asprosin can penetrate the blood-brain barrier and directly act on the feeding center of hypothalamus, leading to weight gain and increased blood glucose levels. Furthermore, it can activate the GPCR-cAMP-PKA pathway, leading to continuous over-release of glucose by hepatocytes, causing an increase in blood glucose levels [20]. Additionally, it induce insulin resistance in skeletal muscle cells through PKCδ-related pathway [39], enhances GLUT8 expression, and promote glucose uptake in testis[40]. Moreover, Asprosin inhibit autophagy of pancreatic β cells through AMPK-mTOR pathway to promote β cell apoptosis, and thus participate in the occurrence and development of diabetes [16].

Besides diabetes, Asprosin can also contribute to the development of various diabetes complications. Studies have revealed that Asprosin can inhibit autophagy and promote apoptosis of human retinal microvascular endothelial cells,thereby participating in the development of diabetic retinopathy [18]. Additionally, it can activate the TGF-β signaling pathway and play a role in the development of peripheral artery disease of lower limbs among diabetic patients [22]. Furthermore, it is closely associated with the occurrence of DKD. Research has demonstrated that serum Asprosin levels are independently correlated with early renal injury markers UACR, and are positively correlated with disease duration, BMI, SBP, BUN, Cr, UA, UAE, HOMA-IR, insulin levels, HbA1c, IL-6, and TNF-α, and negatively correlated with eGFR [16, 25, 42, 45].

In this study, we have confirmed the elevated expression of Asprosin in the circulation of DKD patients and in the renal tissue of DKD mice. Furthermore, we have successfully validated this finding for the first time in the renal biopsy tissue from DKD patients. Through our in vitro experiments, we have demonstrated that high glucose stimulation can induce a significant increase in Asprosin expression in HK-2 cells. Based on this findings, it is plausible to speculate that the heightened levels of circulating Asprosin in individuals with diabetic nephropathy (DN), as well as the upregulation of Asprosin in renal tubular epithelial cells induced by high glucose exposure, may play a crucial role in both the occurrence and progression of DKD. However, further investigations are warranted to elucidate the precise underlying mechanisms. [16, 24].

Asprosin plays an important role in various physiological processes such as apoptosis, cell autophagy, inflammatory response, endoplasmic reticulum stress, etc [17, 18, 21, 65]. Apoptosis is a kind of programmed death, which is closely related to the occurrence and development of many important kidney diseases, including DN, renal fibrosis, and acute kidney injury [46–50]. Studies have found that apoptosis of proximal tubular epithelial cells is one of the important features of DN [48,51–55], but the involvement of Asprosin in the apoptosis of proximal tubular epithelial cells in DKD patients remains unclear. The results of this study demonstrate that Asprosin significantly enhances the expression of Cleaved Caspase-3 and augments the rate of apoptosis in HK-2 cells. Additionally, Asprosin exacerbates high glucose-induced apoptosis in HK-2 cells, which may serve as a crucial mechanism underlying its involvement in the occurrence and development of DKD. This finding is consistent with previous studies that have reported a dose-dependent induction of in MIN6 cells and human primary pancreatic cells by Asprosin in vitro [16, 21]. However, other studies have found that Asprosin exerts a protective effect on mesenchymal stromal cells (MSCs) against oxidative stress-induced apoptosis through the ERK1/2-SOD2 pathway[56], as well as attenuates cardiomyocyte injury induced by high glucose concentration [42]. We speculate that this discrepancy may be attributed to the distinct receptors and signaling pathways of Asprosin in various tissues. To further elucidate the mechanism underlying Asprosin- induced apoptosis of HK-2 cells, we employed RNA-seq to investigate the gene transcription changes following Asprosin stimulation. The results revealed that upon Asprosin stimulation, differentially expressed genes were primarily enriched in autophagy pathways such as AMPK and mTOR, suggesting a potential involvement of Asprosin in regulating of autophagy process of HK-2 cells.

Autophagy is a highly conserved and highly regulated dynamic intracellular degradation pathway, which is crucial in maintaining the steady state and function of cells [27,28]. Autophagy is a dynamic balance process, and excessive inhibition or over-activation can cause autophagy imbalance and lead to cell injury. Studies have confirmed that autophagy imbalance can be involved in various kidney diseases, including diabetic nephropathy, lupus nephritis, and renal fibrosis [57–59]. The proximal tubular is an important part of the renal unit, and has a high level of basal autophagy. Studies have found that a large number of misfolded proteins and deformed organelles can appear in mouse kidney cells with proximal tubular specific knockout of Atg5, which can cause kidney injury [29,30]. The effect of Asprosin on autophagy in HK-2 cells was investigated in this study. Western Blot results revealed that upon stimulation with varying concentrations of Asprosin for different durations, there was a significant reduction in the expression of LC3 II and an increase of P62 level, indicating the inhibitory role of Asprosin on autophagy in HK-2 cells. We further observed that Asprosin exacerbates the high glucose-induced autophagy dysfunction in HK-2 cells, as evidenced by the lentiviral GFP-LC3-infected stable strain of HK-2 cells under high glucose conditions. This finding is consistent with previous literature demonstrating that Asprosin suppresses autophagy of mouse MIN6 cells via the AMPK-mTOR pathway[16].

Autophagy is closely associated with apoptosis. Studies have revealed that autophagy exert a protective effect on cell by inhibition of apoptosis, or induce apoptosis in damaged cells [46]. In this study, our findings demonstrate that the autophagy agonist Rap can ameliorate the Asprosin-induced disruption of autophagy and subsequently alleviate the Asprosin-induced apoptosis in HK-2 cells. Conversely, autophagy inhibitor 3-MA could exacerbate the Asprosin-induced disturbance of autophagy and further aggravate Asprosin-induced apoptosis in HK-2 cells. Our study provides initial evidence that Asprosin promotes apoptosis of HK-2 cells by suppressing cellular autophagy, which may represent an important mechanism underlying the development of DKD.. finding aligns with previous literature reports indicating that Asprosin inhibits autophagy and enhances β-cell apoptosis[16].

In conclusion, our study has provided the first evidence that Asprosin expression is upregulated in both renal tissue and circulation of patients with DKD, as well as in response to high glucose stimulation. Moreover, we have discovered that Asprosin exerts a promotive and aggravating effect on apoptosis of renal tubular epithelial cells by inhibiting autophagy, which may serve as a crucial mechanism underlying the development of diabetic nephropathy. However, further investigations are warranted to elucidate the specific receptors and pathways through which Asprosin regulates autophagy in HK-2 cells.

4.1. Cell Culture and Treatments

The HK-2 cells (Human renal proximal tubular epithelial cells) and the HK-2 cell stable strain infected with GFP-LC3 lentivirus were maintained by our research group. They were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, PAN, Africa), and incubated in a cell culture incubator at 37°C with 5% CO2 for cultivation.

The HK-2 cells were transfected with mRFP-GFP-LC3 lentivirus (Genechem, Shanghai, with puromycin resistance) following the manufacturer's protocol. Following 48–96 hours of infection, the cells were cultured in fresh medium containing puromycin at a final concentration of 1µg/mL to select for the LC3 stable expression cell line.

4.2. Grouping and processing of plasma samples from patients with diabetic nephropathy

A total of 25 patients from the physical examination center of the First Affiliated Hospital of Chongqing Medical University from January 2021 to January 2022 were selected as the control group. The inclusion criteria were: (1) Meeting the diagnostic criteria for normal glucose tolerance; (2) Age 18–85 years old. The exclusion criteria were: (1) Patients with cardiovascular or respiratory diseases; (2) Patients with chronic hepatitis or severe liver damage; (3) Patients with autoimmune diseases; (4) Patients with systemic infectious diseases within 3 months.

A total of 40 inpatients in the Department of Nephrology of the First Affiliated Hospital of Chongqing Medical University from January 2021 to January 2022 were selected in the DKD group. The inclusion criteria were: (1) Meeting the diagnostic criteria of T2DM: random blood glucose ≥ 11.1mmol/L, or fasting blood glucose ≥ 7.0 mmol/L, or 2h venous blood glucose ≥ 11.1 mmol/L after glucose load; (2) Meeting the diagnostic criteria of DKD: urinary albumin/creatinine ratio was repeatedly examined within 3 to 6 months, and 2 out of 3 times were ≥ 30 mg/g; (3) Age was 18 to 85 years old. The exclusion criteria were: (1) Patients with cardiovascular or respiratory diseases; (2) Patients with chronic hepatitis or severe liver damage; (3) Patients with autoimmune diseases; (4) Patients with systemic infectious diseases within 3 months.

This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (K2023-430). The population was matched in age, body mass index (BMI), and education level. All subjects were fasted overnight and blood samples were collected, centrifuged, and stored at -80°C for subsequent studies.

4.3. Collection and preparation of kidney tissue specimens

Renal biopsy tissues from patients with DKD and adjacent normal renal tissues from patients with renal cell carcinoma were collected, and the tissues were fixed in formaldehyde solution at room temperature for 45 minutes, dehydrated by gradient ethanol method, and embedded in paraffin. The wax blocks were repaired into small trapezoidal blocks, pre-cooled for 40 minutes, and cut into 2µm thickness sections.This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (K2023-430).

4.4. Grouping and treatment of mice

Twenty male SPF C57BL/6J mice weighing 20–22 g were selected and kept in an animal room with a light-dark cycle of 12 h, 24°C, and 50% relative humidity, in a cage of 5 mice per cage. After 1W adaptive feeding, the mice were randomly divided into control group (n = 10) and DKD group (n = 10). The control group was given a normal diet, and the DKD group was given a high-fat and high-sugar diet for 8 weeks. Then the mice were fasted for 12h, and the mice in the DKD group were given an intraperitoneal injection of 40mg/kg STZ, and the mice in the control group were given an intraperitoneal injection of the same dose of sodium citrate buffer. After 7 days of continuous injection, random blood glucose (RBG) was measured by tail vein blood collection. The mice with RBG of 13.5–25mmol/L were selected for the formal experiment. The above methods were continued for 4W, during which blood pressure, body weight, RBG and 24h-urine total protein (24h-UTP) were recorded weekly. Finally, RBG ≥ 16.7 mmol/L and 24h-UTP ≥ 200ng were used as the modeling standard. This study complies with the ARRIVE guidelines and was approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (K2023-430). After the intervention, all mice were anesthetized and their kidney tissue specimens were obtained, stored in a refrigerator at -80°C for long-term preservation and for subsequent experiments.

4.5. Immunofluorescent staining

After washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes.Then wash with PBS and incubated with DAPI for five minutes in the absence of light. After washing again, the slides were sealed with anti-fluorescence quenching agent, then collected the images under inverted fluorescence microscope.

4.6. Protein extraction and Western Blot Analysis.

Total cell lysates were prepared with RIPA lysate buffer containing protease and phosphatase inhibitors (P0013B, Beyotime), 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel was prepared using a commercial kit (PG113, EpiZyme). Denaturing samples were separated through the SDS-PAGE gel and transferred to PVDF membrane, which was then blocked with Blot Blocking Buffer (YWB0500, Yoche) for fifteen mimutes. PVDF membrane was probed with the corresponding primary and secondary antibodies. ECL chemiluminescence system was used to detect the immunoreactive bands and the band intensity was quantified using Image J software.

4.7. Antibodies

We used the following antibodies for western blot analysis, immunohistochemistry or Immunofluorescent staining: β-actin (Proteintech,66000-1-Ig), Asprosin (MA544582,Thermo), Cleaved Caspase-3 (#9661,Cell Signaling), SQSTM1/P62 (ab109012,abcam), LC3B(ab192890,abcam), Alexa FluorTM 594 goat anti-rabbit IgG (#A-11012, invitrogen), Peroxidase-Conjugated Goat anti-Rabbit IgG(ZB-2301,ZSGB-BIO), HRP-conjugated Affinipure Goat Anti-Mouse IgG(SA00001-1,Proteintech).

4.8. Statistical Analysis

Data were presented as mean ± standard error (SEM). All experiments were repeated three times, and data were processed and analyzed using GraphPad Prism software. Comparisons between two groups were performed using the t-test, and comparisons between multiple groups were performed using one-way ANOVA. A statistically significant difference was considered when P < 0.05.

4.9. Ethical approval and consent to participate

The study was conducted according to the Declaration of Helsinki guidelines and was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (K2023-430). Informed consent was obtained from all subjects agreed to participate in this study and answered the questionnaire.

Conflict of interest:

The authors have declared that no conflict of interest exists.

Author Contribution

Z. wrote the main manuscript text . L. contributed to formal analysis and data curation.D. contributed to supervision, funding acquisition and conceptualization.All authors reviewed the manuscript.

Acknowledgements:

We are grateful to all the patients for their selffess dedication and health care workers who provided clinical diagnosis and treatment to nephrology patients. This study was funded by the National Natural Science Foundation of China (no.82470758)

Data Availability

All original data will be provided subsequently if necessary. If someone wants to request the data from this study, please contact author Zheng Shuran at [email protected] or [email protected].

Thomas, M. C. et al. Diabetic kidney disease[J]. Nat. Rev. Dis. Primers. 1, 15018 (2015). Published 2015 Jul 30.
Yang, C. et al. CKD in China: Evolving Spectrum and Public Health Implications[J]. Am. J. Kidney Dis. 76 (2), 258–264 (2020).
Alicic, R. Z., Rooney, M. T. & Tuttle, K. R. Diabetic Kidney Disease: Challenges, Progress, and Possibilities[J]. Clin. J. Am. Soc. Nephrol. 12 (12), 2032–2045 (2017).
Antini, C., Caixeta, R., Luciani, S. & Hennis, A. J. M. Diabetes mortality: trends and multi-country analysis of the Americas from 2000 to 2019[J]. Int. J. Epidemiol. 53 (1), dyad182 (2024).
Li, X. et al. Podocyte injury of diabetic nephropathy: Novel mechanism discovery and therapeutic prospects[J]. Biomed. Pharmacother. 168, 115670 (2023).
Mohandes, S. et al. Molecular pathways that drive diabetic kidney disease[J]. J. Clin. Invest. 133 (4), e165654 (2023). Published 2023 Feb 15.
Petrazzuolo, A. et al. Broadening horizons in mechanisms, management, and treatment of diabetic kidney disease[J]. Pharmacol. Res. 190, 106710 (2023).
Chen, S. J., Lv, L. L., Liu, B. C. & Tang, R. N. Crosstalk between tubular epithelial cells and glomerular endothelial cells in diabetic kidney disease[J]. Cell. Prolif. 53 (3), e12763 (2020).
Liu, W. et al. Micheliolide ameliorates diabetic kidney disease by inhibiting Mtdh-mediated renal inflammation in type 2 diabetic db/db mice[J]. Pharmacol. Res. 150, 104506 (2019).
Bhattacharjee, N., Barma, S., Konwar, N., Dewanjee, S. & Manna, P. Mechanistic insight of diabetic nephropathy and its pharmacotherapeutic targets: An update[J]. Eur. J. Pharmacol. 791, 8–24 (2016).
Qi, X. M., Wang, J., Xu, X. X., Li, Y. Y. & Wu, Y. G. FK506 reduces albuminuria through improving podocyte nephrin and podocin expression in diabetic rats[J]. Inflamm. Res. 65 (2), 103–114 (2016).
Magri, C. J. & Fava, S. The role of tubular injury in diabetic nephropathy[J]. Eur. J. Intern. Med. 20 (6), 551–555 (2009).
Duerrschmid, C. et al. Asprosin is a centrally acting orexigenic hormone[J]. Nat. Med. 23 (12), 1444–1453 (2017).
Kocaman, N. & Kuloğlu, T. Expression of asprosin in rat hepatic, renal, heart, gastric, testicular and brain tissues and its changes in a streptozotocin-induced diabetes mellitus model[J]. Tissue Cell. 66, 101397 (2020).
Yuan, M., Li, W., Zhu, Y., Yu, B. & Wu, J. Asprosin: A Novel Player in Metabolic Diseases[J]. Front. Endocrinol. (Lausanne). 11, 64 (2020). Published 2020 Feb 19.
Wang, R. & Hu, W. Asprosin promotes β-cell apoptosis by inhibiting the autophagy of β-cell via AMPK-mTOR pathway[J]. J. Cell. Physiol. 236 (1), 215–221 (2021).
Jung, T. W. et al. Asprosin attenuates insulin signaling pathway through PKCδ-activated ER stress and inflammation in skeletal muscle[J]. J. Cell. Physiol. 234 (11), 20888–20899 (2019).
Shabir, K. et al. Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway. Int. J. Mol. Sci. 24 (1), 227. 10.3390/ijms24010227 (2022). PMID: 36613673; PMCID: PMC9820073.
Sohn, J. W. Network of hypothalamic neurons that control appetite[J]. BMB Rep. 48 (4), 229–233 (2015).
Romere, C. et al. Asprosin, a Fasting-Induced Glucogenic Protein Hormone[J]. Cell. 165 (3), 566–579 (2016).
Lee, T., Yun, S., Jeong, J. H. & Jung, T. W. Asprosin impairs insulin secretion in response to glucose and viability through TLR4/JNK-mediated inflammation[J]. Mol. Cell. Endocrinol. 486, 96–104 (2019).
You, M. et al. Asprosin induces vascular endothelial-to-mesenchymal transition in diabetic lower extremity peripheral artery disease[J]. Cardiovasc. Diabetol. 21 (1), 25 (2022). Published 2022 Feb 15.
Mirr, M. et al. Serum Asprosin Correlates with Indirect Insulin Resistance Indices[J]. Biomedicines. 11 (6), 1568 (2023). Published 2023 May 28.
Wang, R., Lin, P., Sun, H. & Hu, W. Increased serum asprosin is correlated with diabetic nephropathy[J]. Diabetol Metab Syndr. ;13(1):51. Published 2021 May 1. (2021).
Zhang, H., Hu, W. & Zhang, G. Circulating asprosin levels are increased in patients with type 2 diabetes and associated with early-stage diabetic kidney disease[J]. Int. Urol. Nephrol. 52 (8), 1517–1522 (2020).
Klionsky, D. J. et al. Autophagy in major human diseases.[J]. EMBO J. 40 (19), e108863 (2021).
Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J. & Simons, K. Endocytosis in filter-grown Madin-Darby canine kidney cells[J]. J. Cell. Biol. 109 (6 Pt 2), 3243–3258 (1989).
Ma, Z. et al. p53/microRNA-214/ULK1 axis impairs renal tubular autophagy in diabetic kidney disease[J]. J. Clin. Invest. 130 (9), 5011–5026 (2020).
Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues[J]. Cell. 147 (4), 728–741 (2011).
Yamamoto, T. et al. Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule[J]. Autophagy. 12 (5), 801–813 (2016).
Kimura, T. et al. Autophagy protects the proximal tubule from degeneration and acute ischemic injury[J]. J. Am. Soc. Nephrol. 22 (5), 902–913 (2011).
Mackensen-Haen, S., Bader, R., Grund, K. E. & Bohle, A. Correlations between renal cortical interstitial fibrosis, atrophy of the proximal tubules and impairment of the glomerular filtration rate[J]. Clin. Nephrol. 15 (4), 167–171 (1981).
Takahashi, A. et al. Autophagy guards against cisplatin-induced acute kidney injury[J]. Am. J. Pathol. 180 (2), 517–525 (2012).
Kimura, T. et al. Autophagy protects the proximal tubule from degeneration and acute ischemic injury[J]. J. Am. Soc. Nephrol. 22 (5), 902–913 (2011).
Kim, W. Y. et al. The role of autophagy in unilateral ureteral obstruction rat model[J]. Nephrol. (Carlton). 17 (2), 148–159 (2012).
Nam, S. A. et al. Autophagy attenuates tubulointerstital fibrosis through regulating transforming growth factor-β and NLRP3 inflammasome signaling pathway[J]. Cell. Death Dis. 10 (2), 78 (2019). Published 2019 Jan 28.
Jung, T. W. et al. Asprosin attenuates insulin signaling pathway through PKCδ-activated ER stress and inflammation in skeletal muscle[J]. J. Cell. Physiol. 234 (11), 20888–20899 (2019).
Maurya, S., Krishna, A., Lal, B. & Singh, A. Asprosin promotes steroidogenesis and spermatogenesis with improved glucose metabolism in adult mice testis[J]. Andrologia. 54 (11), e14579 (2022).
Lu, Y. et al. Asprosin aggravates vascular endothelial dysfunction via disturbing mitochondrial dynamics in obesity models[J]. Obes. (Silver Spring). 31 (3), 732–743 (2023).
Deng, X. et al. Higher Serum Asprosin Level is Associated with Urinary Albumin Excretion and Renal Function in Type 2 Diabetes[J]. Diabetes Metab. Syndr. Obes. 13, 4341–4351 (2020). Published 2020 Nov 13.
Xu, L. et al. Association Between Serum Asprosin and Diabetic Nephropathy in Patients with Type 2 Diabetes Mellitus in the Community: A Cross-Sectional Study[J]. Diabetes Metab. Syndr. Obes. 15, 1877–1884 (2022). Published 2022 Jun 18.
Goodarzi, G. et al. Circulating levels of asprosin and its association with insulin resistance and renal function in patients with type 2 diabetes mellitus and diabetic nephropathy[J]. Mol. Biol. Rep. 48 (7), 5443–5450 (2021).
Fleisher, T. A. Apoptosis[J]. Ann. Allergy Asthma Immunol. 78 (3), 245–250 (1997).
Woo, D. Apoptosis and loss of renal tissue in polycystic kidney diseases[J]. N Engl. J. Med. 333 (1), 18–25 (1995).
Kumar, D., Robertson, S. & Burns, K. D. Evidence of apoptosis in human diabetic kidney[J]. Mol. Cell. Biochem. 259 (1–2), 67–70 (2004).
Zhao, X. C., Livingston, M. J., Liang, X. L. & Dong, Z. Cell Apoptosis and Autophagy in Renal Fibrosis[J]. Adv. Exp. Med. Biol. 1165, 557–584 (2019).
Bengatta, S. et al. MMP9 and SCF protect from apoptosis in acute kidney injury[J]. J. Am. Soc. Nephrol. 20 (4), 787–797 (2009).
Kelly, D. J. et al. Attenuation of tubular apoptosis by blockade of the renin-angiotensin system in diabetic Ren-2 rats[J]. Kidney Int. 61 (1), 31–39 (2002).
Ortiz, A., Ziyadeh, F. N. & Neilson, E. G. Expression of apoptosis-regulatory genes in renal proximal tubular epithelial cells exposed to high ambient glucose and in diabetic kidneys[J]. J. Investig Med. 45 (2), 50–56 (1997).
Lo, C. S. et al. Heterogeneous Nuclear Ribonucleoprotein F Stimulates Sirtuin-1 Gene Expression and Attenuates Nephropathy Progression in Diabetic Mice[J]. Diabetes. 66 (7), 1964–1978 (2017).
Cai, T. et al. Calcium Dobesilate Prevents Diabetic Kidney Disease by Decreasing Bim and Inhibiting Apoptosis of Renal Proximal Tubular Epithelial Cells[J]. DNA Cell. Biol. 36 (4), 249–255 (2017).
Gilbert, R. E. Proximal Tubulopathy: Prime Mover and Key Therapeutic Target in Diabetic Kidney Disease[J]. Diabetes. 66 (4), 791–800 (2017).
Zhang, Z. et al. Asprosin improves the survival of mesenchymal stromal cells in myocardial infarction by inhibiting apoptosis via the activated ERK1/2-SOD2 pathway[J]. Life Sci. 231, 116554 (2019).
Yang, D. et al. Autophagy in diabetic kidney disease: regulation, pathological role and therapeutic potential[J]. Cell. Mol. Life Sci. 75 (4), 669–688 (2018).
Wang, L. & Law, H. K. The Role of Autophagy in Lupus Nephritis[J]. Int. J. Mol. Sci. 16 (10), 25154–25167 (2015). Published 2015 Oct 22.
Lin, F. Autophagy in renal tubular injury and repair[J]. Acta Physiol. (Oxf). 220 (2), 229–237 (2017).

No competing interests reported.

Download PDF

Reviewers agreed at journal
11 Nov, 2024
Reviews received at journal
08 Nov, 2024
Reviewers agreed at journal
06 Nov, 2024
Reviewers invited by journal
25 Oct, 2024
Editor assigned by journal
25 Oct, 2024
Editor invited by journal
25 Oct, 2024
Submission checks completed at journal
24 Oct, 2024
First submitted to journal
05 Oct, 2024

You are reading this latest preprint version

Asprosin Promotes Human Renal Tubular Epithelial Cells Apoptosis by Inhibiting Autophagy

Status:

Version 1

Abstract

Figures

1. Introduction

2. Result

2.1 Plasma and renal tissue asprosin expression level in patients from DKD increased.

2.2 The expression of Asprosin in the kidney tissue of DKD model mice is elevaated.

2.2.1 The establishment of DKD Mouse Model

2.2.2. The expression level of Asprosin in renal tissue of DKD model mice is elevated.

2.3 High glucose induced the expression of Asprosin in HK-2 cells.

2.4 Asprosin induced and aggravated high glucose-induced apoptosis in HK-2 cells.

2.5 Asprosin may induce apoptosis in HK-2 cells by regulating autophagy.

2.6 Asprosin induced and aggravated high glucose-induced autophagy disorder in HK-2 cells.

2.7 Autophagy agonist rapamycin reversed autophagy disorder and apoptosis induced by Asprosin.

2.8 Autophagy inhibitor 3-MA aggravated autophagy disorder and apoptosis induced by Asprosin.

3. Discussion

4. Materials and Methods

4.1. Cell Culture and Treatments

4.2. Grouping and processing of plasma samples from patients with diabetic nephropathy

4.3. Collection and preparation of kidney tissue specimens

4.4. Grouping and treatment of mice

4.5. Immunofluorescent staining

4.6. Protein extraction and Western Blot Analysis.

4.7. Antibodies

4.8. Statistical Analysis

Declarations

Conflict of interest:

Author Contribution

Acknowledgements:

Data Availability

References

Additional Declarations

Status:

Version 1