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. 2021 Dec;43(1):510-519.
doi: 10.1080/0886022X.2021.1896548.

The role of discoidin domain receptor 2 in the renal dysfunction of alport syndrome mouse model

Yuya Sannomiya  1 , Shota Kaseda  1   2 , Misato Kamura  1   2 , Hiroshi Yamamoto  3 , Hiroyuki Yamada  3 , Masataka Inamoto  3 , Jun Kuwazuru  1 , Saki Niino  1 , Tsuyoshi Shuto  1   4 , Mary Ann Suico  1   4 , Hirofumi Kai  1   2   4
Affiliations

The role of discoidin domain receptor 2 in the renal dysfunction of alport syndrome mouse model

Yuya Sannomiya et al. Ren Fail. 2021 Dec.

Abstract

Alport syndrome (AS) is a hereditary glomerular nephritis caused by mutation in one of the type IV collagen genes α3/α4/α5 that encode the heterotrimer COL4A3/4/5. Failure to form a heterotrimer due to mutation leads to the dysfunction of the glomerular basement membrane, and end-stage renal disease. Previous reports have suggested the involvement of the receptor tyrosine kinase discoidin domain receptor (DDR) 1 in the progression of AS pathology. However, due to the similarity between DDR1 and DDR2, the role of DDR2 in AS pathology is unclear. Here, we investigated the involvement of DDR2 in AS using the X-linked AS mouse model. Mice were treated subcutaneously with saline or antisense oligonucleotide (ASO; 5 mg/kg or 15 mg/kg per week) for 8 weeks. Renal function parameters and renal histology were analyzed, and the gene expressions of inflammatory cytokines were determined in renal tissues. The expression level of DDR2 was highly elevated in kidney tissues of AS mice. Knockdown of Ddr2 using Ddr2-specific ASO decreased the Ddr2 expression. However, the DDR2 ASO treatment did not improve the proteinuria or decrease the BUN level. DDR2 ASO also did not significantly ameliorate the renal injury, inflammation and fibrosis in AS mice. These results showed that Ddr2 knockdown by ASO had no notable effect on the progression of AS indicating that DDR2 may not be critically involved in AS pathology. This finding may provide useful information and further understanding of the role of DDRs in AS.

Keywords: Alport syndrome; Type IV collagen; discoidin domain receptor 2 (DDR2); fibrosis; inflammatory cytokines; proteinuria.

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Conflict of interest statement

The authors report no conflict of interest.

Figures

Figure 1.
Figure 1.
Expression of DDR2 in kidneys of Alport mice. (A,B) Total RNA was isolated from kidney tissues of 8-, 16-, and 24-week-old wild-type (WT) or Alport syndrome mice. Quantitative RT-PCR was performed to evaluate the expression of Ddr2 and Ddr1. The data were normalized to Gapdh. Bars indicate the mean ± S.E. (n = 4–6). ***p<.001 vs WT, assessed by Student’s t test. (C) Immunoblots of protein lysates from whole kidneys of WT and Alport mice were probed with the indicated antibodies. (D) The DDR2 expression was quantified by multi gauge software and normalized with Vinculin (loading control). Bars indicate the mean ± S.E. (n = 3). *p<.05, ***p<.001 vs WT, assessed by Student’s t test.
Figure 2.
Figure 2.
DDR2 ASO inhibits DDR2 expression specifically in Alport mice. (A) Scheme of experimental plan for CON ASO and DDR2 ASO injection. (B,C) Total RNA was isolated from kidney tissues of 24-week-old mice. Quantitative RT-PCR was performed to evaluate the expression of Ddr2 and Ddr1. The data were normalized to Gapdh. Bars indicate the mean ± S.E. (n = 5–6). ***p<.001 vs WT; #p<.05, ###p<.001 vs CON ASO, assessed by Dunnett’s test. n.s., not significant. (D,E) Immunoblotting of protein lysates from whole kidney of WT, CON ASO- and DDR2 ASO-injected mice. (F,G) Blots of DDR2 were quantified by multi gauge software and normalized with Vinculin (loading control). Bars indicate the mean ± S.E. (n = 4). **p<.01 vs WT; ##p<.01 vs CON ASO, assessed by Dunnett’s test.
Figure 3.
Figure 3.
DDR2 ASO did not improve kidney function in Alport mice. (A) Urinary protein and creatinine were measured by Bradford and Jaffe’s method, respectively. Proteinuria score was calculated based on urinary protein and creatinine concentrations. *p<.05, **p<.01 vs WT, assessed by Dunnett’s test. (B) Serum creatinine level, and (C) blood urea nitrogen (BUN) were measured in 24-week-old WT, CON ASO- and DDR2 ASO-treated mice. Bars indicate the mean ± S.E. (n = 5–6). *p<.05, **p<.01 vs WT, assessed by Dunnett’s test. (D,E) Total RNA was isolated from kidney tissues of 24-week-old mice. Quantitative RT-PCR was performed to evaluate the expression of the indicated renal injury markers. The data were normalized to Gapdh. Bars indicate the mean ± S.E. (n = 5–6). **p<.01, ***p<.001 vs WT, assessed by Dunnett’s test. n.s., not significant. (F) Images of PAS-stained renal sections of 24-week-old mice are shown. (G) Glomerulosclerosis score was quantified from PAS-stained sections. (H) PAM staining of renal sections of 24-week-old mice was performed. Lower panels, enlarged view of the area boxed by the red square in upper panel. Red arrows indicate the GBM. Scale bars in F and H, 50 µM.
Figure 4.
Figure 4.
DDR2 ASO did not affect inflammatory and fibrosis gene expression in Alport mice. Total RNA was isolated from kidney tissues of 24-week-old mice. Quantitative RT-PCR was performed to evaluate the expression of the indicated (A-D) cytokines and (E-G) renal fibrosis markers. The data were normalized to Gapdh. Bars indicate the mean ± S.E. (n = 5–6). *p<.05, **p<.01, ***p<.001 vs WT; #p<.05, ##p<.01 vs CON ASO, assessed by Dunnett’s test. (H) Masson-Trichrome staining of renal section of 24-week-old mice was performed. Scale bar, 100 µM. (I) Immunoblots of protein lysates from whole kidneys of WT and AS mice were probed with phosphorylated ERK (p-ERK) or ERK antibodies. (J,K) The p-ERK expression was quantified by multi gauge software and normalized with basal ERK. Bars indicate the mean ± S.E. (n = 4). #p<.05 vs CON ASO, assessed by Dunnett’s test.
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Grants and funding

Funding was provided by the Japan Society for the Promotion Science (JSPS) under Grant Nos. JP26460098 and JP17K08309, and JSPS Program for Advancing Strategic International Networks to Accelerate the Circulation of Talented Researchers under Grant No. S2803.

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