Tubule-Specific Pacs-2 Deletion Aggravates Kidney Injury in HFD/STZ-induced Mice
The PT-Pacs-2−/− mice were generated by a Cre-LoxP recombination system in our group as previously described(Li et al. 2022), which was confirmed by western blot analysis of extracted renal cortex. Mice were induced to develop DKD with HFD/STZ treatment. The protein expression of Pacs-2 was lower in diabetic Pacs-2fl/fl mice compared to that of control Pacs-2fl/fl mice, and this decrease was further aggravated in diabetic PT-Pacs-2−/− mice (Fig. 1A, B). In addition, the body weight of diabetic mice was lower than that of non-diabetic mice after the injection of STZ (Fig. 1C), and the opposite pattern was observed for the blood glucose level (Fig. 1D). Besides, diabetic Pacs-2fl/fl mice had a higher kidney weight/body weight (KW/BW) ratio than that of control Pacs-2fl/fl mice, and the level was further increased in diabetic PT-Pacs-2−/− mice (Fig. 1E), indicating the development of more severe hypertrophy in diabetic kidneys when tubular Pacs-2 was deleted. Diabetic PT-Pacs-2−/− mice also showed higher urinary albumin and NAG excretion compared to their controls (Fig. 1F). Moreover, histopathological analysis showed that kidney injury was further aggravated in diabetic PT-Pacs-2−/− mice compared with diabetic Pacs-2fl/fl mice, including notable tubular epithelial disruption, tubular basement membrane thickening, tubulointerstitial fibrosis, hypertrophy of glomeruli and increased mesangial matrix (Fig. 1G, H and I). These results indicate that deletion of Pacs-2 in proximal tubular cells promotes the renal injury and progression of DKD.
Pacs-2 Gene Deficiency Contributes to Lipid Accumulation in Tubular Cells of Diabetic Mice
Compared to diabetic Pacs-2fl/fl mice, diabetic PT-Pacs-2−/− mice had more lipid droplets and neutral lipids deposition in kidney tubules, as assessed by immunohistochemistry of Adipophilin and Oil Red O staining (Fig. 2A, B and C). Next, we investigated which molecules are associated with perturbed lipid metabolism underlying lipid accumulation in the kidney of diabetic PT-Pacs-2−/− mice. As shown in Fig. 2D, compared to control Pacs-2fl/fl mice, a notable increase of the mRNA levels of Srebp1 and its downstream acetyl-CoA carboxylase alpha (Acaca) and fatty acid synthase (Fasn), controlling de novo fatty acid synthesis in the kidney of diabetic Pacs-2fl/fl mice were observed, which accompanied with increased expression of Srebp2 with its downstream low density lipoprotein receptor (Ldlr) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), which mediated cholesterol intake and biosynthesis, respectively, and the levels were further increased in diabetic PT-Pacs-2−/− mice. In addition, the mRNA level of ATP binding cassette subfamily a member 1 (Abca1) controlling cholesterol efflux was decreased in diabetic Pacs-2fl/fl mice compared to control Pacs-2fl/fl mice, and the levels were further decreased in diabetic PT-Pacs-2−/− mice. However, there was no significant difference in the mRNA levels of peroxisome proliferator activated receptor alpha (Ppara) between diabetic Pacs-2fl/fl mice and diabetic PT-Pacs-2−/− mice. Since lipid accumulation was found to accelerate renal tubule fibrosis(Gai et al. 2019), we detected the expression of fibronectin (FN) and smooth muscle alpha-actin (α-SMA). As expect, the expression levels of FN and α-SMA in the renal cortex of diabetic PT-Pacs-2−/− mice were higher than those of diabetic Pacs-2fl/fl mice (Fig. 2E, F).
Overexpression of PACS-2 Ameliorates Lipid Accumulation, CE and FFA content increase in HK-2 Cells Induced by HGPA
To confirm the role of PACS-2 in regulation of lipid accumulation, the overexpression of PACS-2 in HK-2 cells was established by transfection with PACS-2 plasmid. A significant increase of PACS-2 expression was observed (Fig. 3A). Overexpression of PACS-2 ameliorated lipid accumulation in HGPA-treated HK-2 cells as determined by Oil red O staining (Fig. 3B, C). As shown in Fig. 3D and G, this beneficial effect of regulating lipid metabolism was further verified by the examination of TC, FC and FFA. PACS-2 overexpression resulted in a significant reduction of CE and FFA in HK-2 cells exposed to HGPA ambiance compared to control. However, no change was seen in the FC and TC content (Fig. 3E).
Identification of SOAT1 as a downstream molecule of PACS-2 and the expression of SOAT1 was increased in the kidney of diabetic mice
Next we explored the mechanism by which PACS-2 regulated lipid metabolism in the diabetic kidney. By transcriptome analysis of renal cortex, 219 differentially expressed genes (DEGs) were identified between renal cortex of control Pacs-2fl/fl mice and control PT-Pacs-2−/−mice, of which 127 genes were up-regulated while 92 genes were down-regulated in control PT-Pacs-2−/−mice (Fig. 4A). The heat map of DEGs involved in lipid metabolism showed that Soat1, an ER resident protein converting FC to CE, was notably upregulated in control PT-Pacs-2−/− mice compared with control Pacs-2fl/fl mice (Fig. 4B), and this finding was further confirmed by real time qRT-PCR, immunofluorescence and western blot analysis, respectively (Fig. 4C, D and F). Immunofluorescence images delineated that the expression of SOAT1 was mainly localized in tubular cells but not in glomeruli. And the increased fluorescence intensity of SOAT1 was observed in diabetic Pacs-2fl/fl mice compared to control Pacs-2fl/fl mice. Furthermore, Pacs-2 deletion in tubular cells further increased the expression of SOAT1 (Fig. 4D, E). Similar results were found by western blot analysis (Fig. 4F, G). Besides, by western blot analysis, the intensity of the bands reflected as the expression of N-terminal cleavage product of SREBP1 and SREBP2 (n-SREBP1 and n-SREBP2), which represented the activation of SREBP1 and SREBP2, was notably increased in diabetic PT-Pacs-2−/−mice compared to diabetic Pacs-2fl/fl mice. This data indicated that deletion of Pacs-2 may activate SREBP1 and SREBP2, the key transcription factors of lipid metabolism, by increasing the expression of SOAT1 in diabetic conditions.
Treatment of HK-2 Cells with PACS-2 and SOAT1 siRNA Regulates the Expression of n-SREBPs and Lipid Accumulation
Since SOAT1 may play a critical role in the process of PACS-2 modulating the lipid accumulation in tubular cells under DKD condition, next we further detected the effect of silencing PACS-2 and SOAT1 by siRNA treatment in vitro. Results showed that gene silencing of PACS-2 increased expression of SOAT1 and activation of SREBPs in HK-2 cells exposed to HGPA ambiance compared to cells treated with control siRNA. While the effect was blocked partially in HK-2 cells co-transfected with SOAT1 siRNA (Fig. 5A, B). By real time qRT-PCR analysis, a notably increased expression of SREBP1, ACACA, FASN, SREBP2 and decreased expression of ABCA1 were found in HK-2 cells treated with HGPA plus PACS-2 siRNA, but the effect was reversed partially by treating with SOAT1 siRNA. In addition, an increased tendency of the mRNA level of LDLR and HMGCR was seen, despite no statistical difference (Fig. 5C). Furthermore, silencing of PACS-2 promoted intracellular lipid deposition and increased the levels of CE and FFA in HK-2 cells exposed to HGPA ambiance, however this effect was partially blocked by treatment with SOAT1 siRNA (Fig. 5D, E, F and I). No change was seen in the content of FC and TC (Fig. 5G, H). These data indicated that reduced PACS-2 could activate SREBPs and promote lipid accumulation by SOAT1 pathway.