Alpha-kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

Abstract

Background: Alpha-kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1β in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1β and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined.

Methods: Immunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1β, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1β and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD.

Results: The heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68⁺ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1β in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1β, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA.

Conclusions: Our data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Keywords: Alpha‐kinase 1; Diabetic nephropathy; Pyroptosis; Tubular injury.

Fig. 1

A Clusters of DEGs in the renal tissues of diabetic nephropathy and control mice. Each column represents a mouse sample and each row represents a gene. Red color indicates upregulation, and blue shows downregulation. Each group has 3 samples. B Biological process enrichment analysis of DEGs. C Protein–protein interaction (PPI) network of potential target genes of ALPK1. DEGs differentially expressed genes, DN diabetic nephropathy, NC negative control

Fig. 2

A IHC staining of ALPK1 (a, b), Caspase-1 (c, d), GSDMD (e, f) and CD68 (g, h) in renal tissues (scale bar, 50 μm; magnification ×200). A1 Renal cortical relative expression of ALPK1 in renal biopsies of patients with GML and DN. A2 Quantification of the number of CD68⁺ macrophages in renal biopsies of patients with GML and DN. Blood glucose levels (B) and 24-h urine protein levels (C) in DN and GML patients. Correlations between ALPK1 expression and the number of CD68⁺ macrophages (D), eGFR (E) and tubular atrophy (IFTA) scores (F). Correlations between the number of CD68⁺ macrophages per field and eGFR (G). Correlations between the number of CD68⁺ macrophages per field and tubular atrophy (IFTA) scores (H). GML glomerular minimal lesion, DN diabetic nephropathy, AOD Average Optical Density, AU arbitrary units, eGFR estimated glomerular filtration rate, IFTA interstitial fibrosis and tubular atrophy. Data were presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Fig. 3

A Kidney sections stained with HE in control (a) and DN mice (b). Loss of brush border (black arrow shows) and vacuolar degeneration (white arrow shows) were seen. IHC staining of ALPK1 (c, d), F4/80 (e, f), fibronectin (g, h) and α-SMA (i, j) in renal tissues (scale bar, 50 μm; magnification ×200). B Blood glucose levels. C Western blotting and densitometric analysis of ALPK1 (C1), NF-κB P65 (C2), phospho-NF-κB P65 (C3), GSDMD-FL (C4), GSDMD-NT (C5), cl-IL-1β (C6) and α-SMA (C7) expressions in kidney tissues of control and DN mice. D Serum IL-1β levels. E Urinary IL-1β levels. DN diabetic nephropathy. Experiments were performed in triplicate. Data were presented as mean ± SD (*P < 0.05, **P < 0.01)

Fig. 4

A, A1 The protein levels of ALPK1 in HK-2 cells treated with 5.5, 10, 20 or 30 mM d-glucose for 24 h were determined by western blot. B, B1 The protein levels of ALPK1 in HK-2 cells treated with 30 mM d-glucose for different periods of time (0, 12, 24 or 48 h) were determined by western blot. Experiments were performed in triplicate. Data were presented as mean ± SD (ns no significance, **P < 0.01, ****P < 0.0001)

Fig. 5

HK-2 cells were treated with ALPK1 siRNA or non-target siRNA for 6 h prior to 24 h-HG (30 mM) treatment. A Densitometry analysis shows knocking down ALPK1 using siRNA transfection downregulated ALPK1 (A1), caspase-1 (A2), caspase-1 P20 (A3), GSDMD (A4), GSDMD-NT (A5) and α-SMA (A6) which increased under high glucose environment. B, B1 Flow cytometry analysis shows high glucose (5.63%) increased proportion of pyroptotic HK-2 cells compared with the control (3.41%) and the proportion was reversed after knockdown of ALPK1 (3.98%). Quadrant 2 presented ratio of pyroptosis cells. Experiments were performed in triplicate. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (compared with LG group). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 (compared with HG group)

Fig. 6

HK-2 cells were treated with ALPK1 siRNA or non-target siRNA for 6 h prior to 24 h-HG (30 mM) treatment. A Representative images of immunofluorescence staining for NF-κB P65 (green) with nuclei marked by DAPI (blue). ALPK1 knockdown inhibited the nuclear translocation of NF-κB P65 (arrow shows) (scale bar, 50 μm; magnification ×400). B, B1 The protein level of phospho-NF-κB P65 in four groups were determined by western blot. C, C1 Western blot determined cellular distribution of P65 in HK-2 cells. Experiments were performed in triplicate. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (compared with LG group). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 (compared with HG group)

Fig. 7

HK-2 cells were treated with ALPK1 siRNA or non-target siRNA for 6 h prior to 24 h-HG (30 mM) treatment. A Representative images of immunofluorescence staining for GSDMD (green) with nuclei marked by DAPI (blue) show knockdown of ALPK1 inhibited translocation of GSDMD to the cell membrane (arrow shows) induced by high glucose (scale bar, 50 μm; magnification ×400). B The protein levels of IL-1β and IL-18 in HG-treated HK-2 cells were determined by western blot. C IL-18 level in supernatant of HK-2 cells was measured by ELISA. Experiments were performed in triplicate. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (compared with LG group). ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 (compared with HG group)