Study on effect of pancreatic kininogenase on diabetic nephropathy-induced fibrosis via Notch1/Hes1/Pten/Akt signaling pathway

Abstract

Objective: To elucidate the mechanism by which pancreatic kininogenase (PKase) impacts renal fibrosis in diabetic nephropathy through modulation of the Notch1/Hes1 and Pten/Akt pathways.

Methods: This study employed in vivo models and cellular assays to investigate PKase's effects on cellular viability, apoptosis, and oxidative stress. Assay kits were used to assess these parameters, while protein expression levels were measured via Western Blot and RT-qPCR. Histological changes in kidney tissues were analyzed using HE and Masson's staining. Fibrosis markers-including E-cadherin, vimentin, α-SMA, Collagen I, TGF-β, and fibronectin-were evaluated through immunofluorescence and immunohistochemistry.

Results: After eight weeks of PKase treatment, significant improvements in blood glucose levels and associated symptoms were observed in diabetic nephropathy rats. Both in vivo and in vitro results demonstrated that PKase treatment inhibited the expression of diabetic nephropathy markers, including vimentin, α-SMA, FN, Collagen I, and TGF-β, while increasing the expression of E-cadherin. Additionally, the expression of Notch1, Hes1, and phosphorylated Akt (p-Akt) was upregulated, and Pten expression was suppressed, all of which were reversed by PKase treatment. Furthermore, both analyses indicated that PKase alleviated Jagged1-induced apoptosis and oxidative stress, and mitigated tubulointerstitial fibrosis.

Conclusion: PKase appears to ameliorate diabetic nephropathy-induced renal fibrosis by activating the Pten/Akt pathway and inhibiting the Notch1/Hes1 pathway, suggesting its potential as a therapeutic agent in diabetic nephropathy.

Clinical trial number: Not applicable.

Keywords: Diabetic nephropathy; Notch1/Hes1 pathway; PKase; Pten/Akt pathway; Renal fibrosis.

Fig. 1

The influence of PKase on the viability and apoptosis of HK-2 cells. A. Assessment of HK-2 cell viability. B-C. Flow cytometric analysis of apoptosis rate in HK-2 cells. D-F. WB analysis was used to assess the levels of apoptotic proteins in HK-2 cells. N = 3, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001

Fig. 2

The effect of PKase on the Pten/Akt signaling pathway regulated by Notch1/Hes1 in HK-2 cells. A-E WB analysis of pathway protein expression levels. F-H RT-qPCR assessment of relative expression levels of pathway proteins. N = 3, * ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001

Fig. 3

PKase’s effect on the expression of fibrotic-associated proteins in HK-2 cells. A-L Expression of E-cadherin, Vimentin, α-SMA, Collagen I, TGF-β and FN in HK-2 cells, Scale = 50 μm. N = 3, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001

Fig. 4

Establishment of diabetic nephropathy model and evaluation of renal function. Illustrates the evaluation of PKase’s influence on renal function within the diabetic model. (A) Evaluation of blood glucose levels in the model group and control group rats. (B) Measurement of blood glucose levels in rats of each group within eight weeks. C-G Determination of concentrations of SCr, BUN, TG, TC in rat serum and 24-hour microalbuminuria in urine after eight weeks. H HE staining, Scale = 400 μm. N = 6, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001

Fig. 5

The influence of PKase on modulating Notch1/Hes1 pathway and its influence on the Pten/Akt signaling pathway in the diabetic nephropathy model. Illustrates the examination of pathway protein expression in the diabetic model. A-E. WB analysis of the expression levels of Notch1, Hes1, Pten and p-Akt. F-H RT-qPCR assessment of the expression of Notch1, Hes1 and Pten. N = 6, * ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001

Fig. 6

The impact of PKase on apoptotic proteins and oxidative stress in DN renal tissues through the modulation of the Notch1/Hes1-regulated Pten/Akt signaling pathway. PKase’s influence on the expression of apoptotic proteins in kidney tissues. A-C WB analysis of apoptosis protein expression. D-F The levels of oxidative stress factors were assessed using assay kits. N = 6, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001

Fig. 7

PKase mediates the effect of Pten/Akt signaling pathway on renal fibrosis in DN through Notch1/Hes1. A-G Immunohistochemical examination of the expression of kidney tissue-related proteins associated with renal fibrosis, Scale = 100 μm. H-I Assessment of renal fibrosis extent using Masson’s staining, Scale = 400 μm. N = 6, ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, **P < 0.0001