Independent and interactive roles of hirudin and HMGB1 interference in protecting renal function by regulating autophagy, apoptosis, and kidney injury in chronic kidney disease

Abstract

Chronic kidney disease (CKD) is a progressive disorder characterized by renal fibrosis, inflammation, and dysregulated autophagy and apoptosis. High-mobility group box 1 (HMGB1) plays a crucial role in regulating autophagy in CKD. Hirudin, a potent thrombin inhibitor, has demonstrated antifibrotic and anti-inflammatory properties, but its effects on autophagy and apoptosis in CKD remain unclear. In this study, a rat model of renal interstitial fibrosis (RIF) and an HK-2 cell culture model were established to assess the effects of varying doses of hirudin and HMGB1 interference. Molecular and histological analyses, including RTqPCR, Western blot, TUNEL staining, hematoxylin-eosin (H&E) staining, immunofluorescence, and immunohistochemistry (IHC), were performed to assess renal injury, fibrosis, apoptosis, and autophagy-related markers. Hirudin treatment significantly reduced the expression of LC3, ATG12, ATG5, α-SMA, COL1A1, caspase-3, and caspase-9 while increasing P62 levels (p<0.05). It also lowered the renal coefficient (p<0.001) and apoptosis levels. The optimal effective concentration of hirudin in vitro was determined to be 4.8 ATU/mL (p<0.001). HMGB1 interference suppressed autophagy and apoptosis, as indicated by decreased LC3-II/LC3-I, ATG12, ATG5, caspase-3, and caspase-9 levels, increased P62 expression (p<0.001), and reduced apoptosis. However, simultaneous HMGB1 interference in hirudin-treated cells weakened the therapeutic effects of hirudin, leading to increased autophagy and apoptosis markers, decreased P62 levels, and a higher renal coefficient. These findings indicate that hirudin exerts protective effects in CKD by modulating autophagy and apoptosis, potentially through HMGB1 regulation. These findings highlight the therapeutic potential of targeting these mechanisms in renal dysfunction and underscore the necessity for further research to support clinical applications.

Figure 1.

Effect of hirudin on autophagy and apoptosis in UUO rats. A) Expression of LC3, ATG12, ATG5 and P62 mRNA in UUO rat kidney tissue was observed by RTqPCR. B) Western blot analysis of ATG12, ATG5 and P62 protein expression in UUO rat kidney tissue. C) UUO rat renal co-efficiency. D) TUNEL detected apoptosis in renal tissues.

Figure 2.

Effect of hirudin on renal fibrosis and apoptosis in UUO rats was investigated by immunofluorescence, immunohistochemistry, and H&E. A) Immunofluorescence was used to detect the level of α-SMA and immunohistochemistry was used to detect the levels of COL1A1, caspase-3, and caspase-9; scale bar: 50 µm. B) H&E staining for detection of RIF.

Figure 3.

In vitro screening of the optimal effective concentration of hirudin and exploring the influence of hirudin and HMGB1 on CKD. A) The viability of HK-2 cells treated with different concentrations of hirudin detected by CCK-8. B) The expressions of LC3, ATG12, ATG5 and P62 mRNA in HK-2 cells was dected by RTqPCR. C) Western blot analysis of LC3-II/ LC3-Ⅰ, ATG12, ATG5 and P62 protein levels in HK-2 cells. D) HK-2 cell apoptosis detection by TUNEL; scale bar: 50 µm. E) Immunofluorescence is showing the levels of caspase-3 and caspase-9.

Figure 4.

The effect of hirudin and HMGB1 in CKD rats was investigated in vivo. A) The expression of LC3, ATG12, ATG5 and P62 mRNAs in UUO rat kidney tissues verified by RTqPCR. B) Western blot analysis of LC3-II/ LC3-Ⅰ, ATG12, ATG5 and P62 protein levels in UUO rat kidney tissue. C) Renal coefficient of UUO rat renal tissue. D) Apoptosis of kidney tissue in UUO rats was detected by TUNEL.

Figure 5.

The effect of hirudin and HMGB1 in CKD rats was investigated by immunofluorescence, immunohistochemistry, and H&E. A) Immunofluorescence detection of the level of α-SMA and IHC detection of the level of COL1A1, caspase-3, and caspase-9. B) H&E staining for detection of RIF.