Loss of RTN3 phenocopies chronic kidney disease and results in activation of the IGF2-JAK2 pathway in proximal tubular epithelial cells

Abstract

Reticulon 3 (RTN3) is an endoplasmic reticulum protein that has previously been shown to play roles in neurodegenerative diseases, but little is known about its function in the kidneys. The aim of the present study was to clarify the roles of RTN3 in chronic kidney disease (CKD) and kidney fibrosis. In this study, RTN3 levels were measured in kidney tissues from healthy controls and CKD or kidney fibrosis patients. An RTN3-null mouse model was generated to explore the pathophysiological roles of RTN3 in the kidneys. The underlying mechanisms were studied in primary proximal tubular epithelial cells and HEK293 cells in vitro. The results showed that (1) a reduction in RTN3 in mice induces CKD and kidney fibrosis; (2) decreased RTN3 expression is found in patients with CKD; (3) RTN3 plays critical roles in regulating collagen biosynthesis and mitochondrial function; and (4) mechanistically, RTN3 regulates these phenotypes by interacting with GC-Rich Promoter Binding Protein 1 (GPBP1), which activates the IGF2-JAK2-STAT3 pathway. Our study indicates that RTN3 might play crucial roles in CKD and kidney fibrosis and that a reduction in RTN3 in the kidneys might be a risk factor for CKD and kidney fibrosis.

Fig. 1. RTN3-null mice presenting phenotypes of CKD and kidney fibrosis.

a HE, b picrosirius red, and c PAS staining analysis showing the glomeruli in WT (n = 5) and RTN3-null mice (n = 5) of different ages. d Statistical evaluation of glomerulosclerosis in WT and RTN3-null mice of different ages. e, f Masson staining illustrating the distribution of collagen in the kidney tissues of WT (n = 5) and RTN3-null mice (n = 5) of different ages.

Fig. 2. RTN3 levels are decreased in CKD patients.

a, b Western blot analysis showing the expression levels of RTN3 in normal individuals (n = 2) and CKD patients (n = 4). c IHC analysis showing RTN3 expression levels in kidney tissues from healthy control subjects (n = 4) or CKD patients (n = 24). The red dots represent samples with lower RTN3 levels. d PAS, Masson, and IHC staining showing the conditions of glomerulosclerosis, kidney fibrosis, and RTN3 expression among patients with CKD of different stages. e Statistical analysis of Masson staining and IHC in the healthy group, slight-glomerulosclerosis group, and severe-glomerulosclerosis group.

Fig. 3. The expression of collagen is increased in RTN3-null mouse kidneys.

a Significantly differentially expressed genes between WT and RTN3-null mouse kidneys revealed by RNA-seq data. b KEGG pathway enrichment analysis of differentially expressed genes. The red square indicates the ECM−receptor interaction pathway. c mRNA levels of collagen-related genes in WT and RTN3-null mouse kidneys at 13 months as assessed by real-time PCR. d, e Western blot analysis showing the expression of collagen type I/III, COL4A5, RTN3, and β-actin in WT and RTN3-null mouse kidneys at 13 months. f mRNA levels of collagen-related genes in HEK293 cells transfected with si-Control and si-RTN3. g, h TEM analysis showing the glomerular basement membranes of WT and RTN3-null mice at 13 months.

Fig. 4. RTN3-null mouse kidneys showing mitochondrial structure damage.

a TEM analysis showing the mitochondrial structures in renal tubular cells of WT and RTN3-null mice. b Real-time PCR results showing the mRNA levels of MFN2, FIS1, OPA1, and RTN3 in WT and RTN3-null mouse kidneys. Western blot analysis showing the protein levels of MFN2, FIS1, OPA1, RTN3 and β-actin in c, d WT and RTN3-null mouse kidneys and e, f WT and RTN3-null mouse primary cultured renal tubular epithelial cells. g mRNA levels and h, i protein levels of MFN2, FIS1, OPA1, RTN3, and β-actin in HEK293 cells transfected with si-Control and si-RTN3.

Fig. 5. RTN3 can regulate the IGF2-JAK2-STAT3 pathway by interacting with GPBP1.

Western blot analysis showing the levels of IGF2, JAK2, p-JAK2, STAT3, p-STAT3, RTN3, and β-actin in a WT and RTN3-null mouse kidneys and b HEK293 cells in the si-Control and si-RTN3 groups. c Coimmunoprecipitation (Co-IP) confirmed the interaction between RTN3 and GPBP1 in mouse renal tubular epithelial cells. d Immunofluorescence staining showing the subcellular localization of RTN3 and GPBP1 in WT and RTN3-null mouse primary cultured renal tubular epithelial cells. e Western blot analysis of the levels of GPBP1, GAPDH, and histone in the cytoplasm and nuclei of WT and RTN3-null mouse primary cultured renal tubular epithelial cells. f In vitro dual-luciferase reporter assay showing the protein interaction. g, h Western blot analysis showing the levels of GPBP1 and IGF2 in RTN3-null cells.

Fig. 6. The mechanism by which RTN3 regulates the IGF2-JAK2 signaling pathway in CKD and kidney fibrosis.

Potential mechanism by which decreased reticulon 3 (RTN3) expression induces CKD and kidney fibrosis.