Lysozyme promotes renal fibrosis through the JAK/STAT3 signal pathway in diabetic nephropathy

Abstract

Introduction: Diabetic nephropathy (DN) is a leading cause of kidney failure. Lysozyme (LYZ) is an essential component of innate immunity and exhibits antibacterial properties. However, LYZ has been reported to induce nephropathy, implying a possible association between impaired renal function and lysozyme expression.

Material and methods: Bioinformatics analysis was used to predict the hub gene associated with DN, and the differential expression of the hub gene was confirmed using a mouse model. A mouse model of streptozotocin (STZ)-induced diabetic nephropathy was established to investigate the correlation between DN and LYZ expression, and the functionality of LYZ was verified through knockdown and overexpression experiments conducted in vivo. Immunohistochemistry (IHC) was utilized to assess fibrosis-related markers and cytokines, while Masson staining was performed to assess renal fibrosis. Fibroblast proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. The role of the JAK pathway was confirmed using the JAK inhibitor AG490, and Western blot was used to investigate the underlying mechanisms.

Results: Mechanistically, 25 mM glucose promotes the expression of LYZ in fibroblastic cells, and LYZ may in turn promote the proliferation of renal interstitial fibroblasts. Western blot shows that glucose can activate STAT3 in an LYZ-dependent manner, and the JAK inhibitor AG490 can partially suppress LYZ-induced STAT3 activation. Furthermore, in vivo observations have revealed that overexpression of LYZ is associated with the senescent phenotype of renal tubular epithelial cells (RTECs).

Conclusions: Lysozyme promotes kidney fibrosis via the JAK/STAT3 signaling pathway in diabetic nephropathy, and glucose may promote fibroblast proliferation by promoting LYZ auto-secretion.

Keywords: JAK; STAT3; diabetic nephropathy; lysozyme; renal fibrosis.

Figure 1

LYZ is overexpressed in DN renal and serum samples. A – Volcano plot showing differential gene expression between the DN kidney sample and the healthy kidney sample in the dataset GSE30122. B – LYZ mRNA expression level in each sample Data represent the mean ± SD of triplicates. P-value was calculated by a two-sided Student’s t-test. ^*P < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2

LYZ expression was upregulated in the model of STZ-induced DN. A – Fasting glucose, serum creatinine, blood urea nitrogen (BUN) and urinary albumin to creatinine ratio (ACR) in each group. ^*P < 0.05 versus the control group. B – Results of PCR analysis. LYZ mRNA expression in kidney tissue derived from each of the groups. C – Representative immunostaining micrographs showing tubular LYZ expression. D – Percentage of LYZ-positive renal tubular epithelial cells (RTECs) was quantified. E – Western blot analysis of LYZ expression in each group. F – Different groups (n = 3 for each group) were used to quantify relative levels of GAPDH. We quantified the percentage of RTECs that were positive based on tubulointerstitial fibrosis (TIF) scores Data represent the mean ± SD of triplicates. The p-value was calculated by a two-sided Student’s t-test. ^*P < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

Enhanced expression of LYZ and renal fibrosis were correlated. A – Fasting glucose, B – serum creatinine, C – blood urea nitrogen (BUN), D – urinary albumin to creatinine ratio (ACR) in each group (n = 3 for each group). E – Masson’s trichrome stain showed renal fibrosis with blue color. F – A representative immunostaining micrograph shows the expression of the fibrotic markers a-smooth muscle actin (α-SMA), collagen I, and fibronectin expression in different groups (n = 3 for each group). G – Western blot analysis of the expression of α-SMA, collagen I, and fibronectin in different groups Data represent the mean ± SD of triplicates. P-value was calculated by a two-tailed Student’s t-test. ^*P < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 4

LYZ expression in fibroblast cells is enhanced by glucose. A – Cells were cultured with several concentrations of glucose (0, 5, 10, 15, 25, and 50 mM) for 12 h, 24 h, and 48 h. LYZ expression level in NRK-49F culture supernatants was analyzed by ELISA. B – PCR analysis of LYZ expression in NRK-49F cells cultured with several concentrations of glucose (0, 5, 10, 15, 25, 50 mM) for 24 h. C – Western blot analysis of LYZ expression in NRK-49F cells cultured with several concentrations of glucose (0, 5, 10, 15, 25, 50 mM) for 24 h. D – PCR analysis of mRNA expression of LYZ. E – Western blot analysis of LYZ protein expression. F – cck8 assays were performed to evaluate high glucose-induced NRK-49F cell viability when LYZ was either knocked down (upper panel) or over-expressed, with or without AG490 treatment (lower panel) Data represent the mean ± SD of triplicates. P-value was calculated by a two-tailed Student’s t-test. ^*P < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

LYZ promotes fibrosis related cytokine expression in NRK-49F cells via the JAK/STAT3 signaling axis. A, B – The levels of IL-6, TGF-α/TGF-β1, collagen I/III, and MMP2/MMP9 in LYZ knocked down (A) and LYZ overexpressed (B) NRK-49F cell culture supernatants were determined by ELISA assay. C, D – Western blot analysis of IL-6, TGF-α/TGF-β1, collagen I/III, and MMP2/MMP9 in LYZ knocked down (C) and LYZ overexpressed (D) NRK-49F cells Data represent the mean ± SD of triplicates. The p-value was calculated by a two-tailed Student’s t-test. ^*P < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 6

Overexpression of LYZ was associated with the senescence phenotype in RTECs. A – Representative immunostaining micrographs showed the tubular expression of interleukin 1β (IL-1β) and matrix metalloproteinase 2 (MMP-2), B – the percentage of positive RTECs was quantified (n = 6 for each group). C – Western blotting detected the expression of MMP-2 and transforming growth factor β1 (TGF-β1) Data represent the mean ± SD of triplicates. P-value was calculated by a two-tailed Student’s t-test. ^*P < 0.05, ^**p < 0.01, ^***p < 0.001.