The Reduction of Uromodulin, Complement Factor H, and Their Interaction Is Associated with Acute Kidney Injury to Chronic Kidney Disease Transition in a Four-Time Cisplatin-Injected Rat Model

Abstract

Uromodulin is recognized as a protective factor during AKI-to-CKD progression, but the mechanism remains unclear. We previously reported that uromodulin interacts with complement factor H (CFH) in vitro, and currently aimed to study the expression and interaction evolution of uromodulin and CFH during AKI-to-CKD transition. We successfully established a rat model of AKI-to-CKD transition induced by a four-time cisplatin treatment. The blood levels of BUN, SCR, KIM-1 and NGAL increased significantly during the acute injury phase and exhibited an uptrend in chronic progression. PAS staining showed the nephrotoxic effects of four-time cisplatin injection on renal tubules, and Sirius red highlighted the increasing collagen fiber. Protein and mRNA levels of uromodulin decreased while urine levels increased in acute renal injury on chronic background. An extremely diminished level of uromodulin correlated with severe renal fibrosis. RNA sequencing revealed an upregulation of the alternative pathway in the acute stage. Renal CFH gene expression showed an upward tendency, while blood CFH localized less, decreasing the abundance of CFH in kidney and following sustained C3 deposition. A co-IP assay detected the linkage between uromodulin and CFH. In the model of AKI-to-CKD transition, the levels of uromodulin and CFH decreased, which correlated with kidney dysfunction and fibrosis. The interaction between uromodulin and CFH might participate in AKI-to-CKD transition.

Keywords: AKI-to-CKD transition; cisplatin; complement activation; complement factor H; uromodulin.

Figure 1

The four-time intraperitoneal injection of low-dose cisplatin (CP) induced AKI-to-CKD transition in rats. (a) Study design overview. Rats (6 weeks old) were injected with 3.2 mg/kg cisplatin every 14 days as the CP group and 6 of them were randomly sacrificed at days 0, 4, 14, 46 and 56; and 3 at days 18, 28, 32 and 42. The normal control (NC) group was injected with the same volume of sterile 0.9% NaCl and was maintained for 56 days. Changes in (b) SCR, (c) BUN, (d) urinary KIM-1/UCRE and (e) urinary NGAL/UCRE levels in rats during the 56 days (n = 6). (f) Representative photomicrographs of PAS and Sirius red. Arrows point to the renal tubular injury in PAS-stained images and renal fibrosis in Sirius-red-stained images. (Original magnification 20×; Scale bar = 50 μm). Quantification of (g) PAS and (h) Sirius red. Data are presented as means ± SD. * p < 0.05, ** p < 0.01 between the two groups.

Figure 2

Uromodulin expression during cisplatin (CP)-induced AKI-to-CKD transition in rats. (a) Relative expression of uromodulin in kidney after normalization with GAPDH. (b) Urinary uromodulin level normalized by UCRE (n = 6). (c) Representative Western blots of uromodulin, CFH and α-SMA. The relative protein expression of (d) uromodulin, (e) CFH and (f) α-SMA to GAPDH at different time points (n = 3). Data are presented as means ± SD. * p < 0.05 and ** p < 0.01 between the two groups.

Figure 3

Transcriptomic analysis of kidneys from SD rats before and 4 days after the first cisplatin (CP) injection. (a) The top 20 enriched KEGG pathways of significant DEGs (total). (b) The enrichment of three complement activation pathways of significant DEGs (up). (c) Heatmap showing the complement-pathway-related DEGs differentially expressed across the two groups, n = 3. KEGG, Kyoto Encyclopedia of Genes and Genomes; DEGs, differentially expressed genes; Clu, clusterin; Itgax, integrin subunit alpha X; Itgb2, integrin subunit beta 2; Pros1, protein S (alpha).

Figure 4

CFH expression during cisplatin (CP)-induced AKI-to-CKD transition in rats. Relative expression of CFH mRNA in (a) liver and (c) kidney after normalization with GAPDH. (b) Quantification of serum CFH level by ELISA with a 500-fold dilution of serum. Data are presented as means ± SD. * p < 0.05 and ** p < 0.01 between the two groups.

Figure 5

Immunohistochemical staining of uromodulin, CFH and α-SMA in kidneys on a series of days. (a) Representative micrographs (40×, bar = 25 μm) and (b) quantification of positive staining on kidney sections on different days as indicated (n = 3). ** p < 0.01 between the two groups. CP, cisplatin.

Figure 6

Interaction between uromodulin and CFH in rat kidneys and complement activation during AKI-to-CKD transition. (a) Total protein lysates of normal rat kidneys were immunoprecipitated using an antibody against uromodulin and immunoblotted against CFH. (b) Representative micrographs (40×, bar = 25 μm) and (c) quantification of positive-stained C3 on kidney sections on different days as indicated (n = 3). * p < 0.05, ** p < 0.01 between the two groups. CP, cisplatin.