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.