Castrated autoimmune glomerulonephritis mouse model shows attenuated glomerular sclerosis with altered parietal epithelial cell phenotype

Abstract

Sex hormones help in maintaining proper immunity as well as renal homeostasis in mammals, and these multi-functional properties characterize the onset of sex-dependent diseases. To clarify the contribution of sex hormones to autoimmune disease-related renal pathogenesis, BXSB/MpJ-Yaa was investigated as a murine autoimmune glomerulonephritis model. BXSB/MpJ-Yaa and its wild-type, BXSB/MpJ-Yaa⁺ were castrated or sham-operated at three weeks and examined until six months of age. Both castrated strains showed significantly lower serum testosterone levels and body weights than sham-operated mice. Castration did not change the disease phenotypes in BXSB/MpJ-Yaa⁺. At three months, both sham-operated and castrated BXSB/MpJ-Yaa manifested splenomegaly, autoantibody production, and glomerulonephritis, and castrated BXSB/MpJ-Yaa tended to show heavier spleen weights than the sham-operated group. At six months, both the treated BXSB/MpJ-Yaa showed equivalent autoimmune disease conditions; however, castrated mice clearly showed milder glomerular sclerotic lesions than the sham-operated groups. Urinary albumin excretion in castrated BXSB/MpJ-Yaa was significantly milder than in sham-operated mice at four months, but those of both the treated BXSB/MpJ-Yaa were comparable at six months. The examined renal histopathological indices in parietal epithelial cells were remarkably altered by castration. Briefly, castration decreased the height of parietal epithelial cells and total parietal epithelial cell number in BXSB/MpJ-Yaa at six months. For immunostaining, parietal epithelial cells facing the injured glomeruli of BXSB/MpJ-Yaa expressed CD44, an activated parietal epithelial cell marker, and CD44-positive parietal epithelial cells showed nuclear localization of the androgen receptor and proliferation marker Ki67. CD44- or Ki67-positive parietal epithelial cells were significantly fewer in castrated group than in sham-operated BXSB/MpJ-Yaa at six months. Further, quantitative indices for CD44-positive parietal epithelial cell number and frequency in renal corpuscles positively correlated with glomerular sclerotic severity in BXSB/MpJ-Yaa. In conclusion, androgen seemed to have an effect on both systemic immunity and renal morpho-function; however, the effect on the latter could be more clearly observed in BXSB/MpJ-Yaa, as parietal epithelial cell activation resulted in glomerular sclerosis.

Keywords: Autoimmune disease; androgen; glomerular sclerosis; glomerulonephritis; kidney disease.

Figure 1.

Indices of male reproductive function and autoimmune disease condition in mice. (a) The serum level of testosterone. (b) Body weight. (c) Spleen weight. (d) The ratio of spleen weight to body weight. (e) The serum level of anti-double stranded DNA antibody. Each bar represents mean ± SE (n = 4–10). s: sham-operated; c: castrated. *: Significant differences in castrated groups against sham-operated group in the same strain at the same age (*: P < 0.05, **: P < 0.01, Mann–Whitney U-test). †: Significant differences in Yaa against BXSB with the same treatment at the same age (^†P < 0.05, ^††P < 0.01, Mann–Whitney U-test). ^#: Significant differences in six-month-old groups against three-month-old groups in the same strain with the same treatment (^#: P < 0.05, Mann-Whitney U-test).

Figure 2.

Histopathological analysis of glomeruli in mice. (a) Histology of glomeruli in sham-operated and castrated BXSB at three and six months. Sections are stained with periodic acid-Schiff-hematoxylin (PAS-H). (b and c) Histology of glomeruli in sham-operated and castrated Yaa at three months (b) and six months (c). Sections are stained with PAS-H, periodic acid methenamine-silver (PAM), and Masson’s trichrome (MT). At three months, glomerular hypertrophy, increase in mesangial matrix lesion, and wrinkling of the glomerular basement membrane were observed, but there were no treatment-related differences. These histological changes are clearer at six months compared with three months in both the treatment groups. Expansion of sclerotic lesion is more significant in sham-operated Yaa than in castrated Yaa. (d) Representative histology of glomeruli with global sclerotic lesions stained with PAS-H and PAM in sham-operated Yaa at six months. Increased parietal epithelial cells and accumulation of extracellular matrix are significant, resulting in the adhesion of glomerular tuft and Bowman’s capsule. Bars = 50 µm. (e) The area of glomeruli in sham-operated Yaa (sYaa) and castrated Yaa (cYaa) at six months. (f) The number of nuclei per unit area of a glomerulus in sYaa and cYaa at six months. (g) The area of sclerotic lesion in glomeruli in sYaa and cYaa at six months. (h) The fraction of sclerotic lesions in sYaa and cYaa at six months. Each bar represents mean ± SE (n = 8–10). Significant differences in castrated groups compared to the sham-operated group are indicated with an asterisk. *: P < 0.05, **: P < 0.01 (Mann–Whitney U-test). (A color version of this figure is available in the online journal.)

Figure 3.

Analysis of podocyte function in six-month-old Yaa. (a) Immunofluorescence of podocyte function molecules (podocin, nephrin, synaptopodin) in Yaa at six months. Nephrin-, podocin- and synaptopodin-immunopositive areas are faint at the center of the glomerulus of Yaa in both the treatment groups, but linear positive reactions at the peripheral area of glomeruli are partially eliminated in only sham-operated groups. Bars = 50 µm. (b) Relative mRNA expression of podocyte function molecules in kidneys of sham-operated Yaa (sYaa) and castrated Yaa (cYaa). The expression levels were normalized to the levels of Actb. (c) uACR in sYaa and cYaa from three to six months. (d and e) The serum levels of serum blood urea nitrogen (BUN, d) and serum creatinine (Cre, e) in sYaa and cYaa at six months. Each bar represents mean ± SE (n = 8–10). Significant differences in castrated groups compared to the sham-operated group are indicated with an asterisk. *: P < 0.05, **: P < 0.01 (Mann–Whitney U-test). (A color version of this figure is available in the online journal.)

Figure 4.

Analysis of androgen receptor in six-month-old Yaa. (a) Immunohistochemistry of androgen receptor (AR) in outer medulla, cortex, and glomerulus of Yaa kidneys at six months. Positive reaction is detected in nucleus of proximal tubules in medulla and cortex, and parietal endothelial cells (PECs). Dotted line represents the boundary between outer stripe (OS) and inner stripe (IS) in outer medulla. Arrowheads represent AR-positive PECs. Both the treatment groups show same localization, but reaction strengths in PECs tend to be lower in castrated Yaa. Bars = 100 µm. (b) Relative mRNA expression of Ar in kidneys of sham-operated Yaa (sYaa) and castrated Yaa (cYaa). The expression levels were normalized to the levels of Actb. Each bar represents mean ± SE (n = 8–10). (A color version of this figure is available in the online journal.)

Figure 5.

Histopathological analysis of parietal epithelial cells in six-month-old Yaa. (a) Immunohistochemistry of CD44 in Yaa kidneys at six months. Arrowheads represent CD44-positive parietal epithelial cells (PECs). In renal corpuscles with uninjured glomeruli, CD44-positive PECs are not observed in both the treatment groups. PECs in renal corpuscles with injured glomeruli show positive reaction on their cell membrane. Treatment-related change of CD44 localization is not observed. Bars = 50 µm. (b) The number of CD44-positive PECs per unit length of Bowman’s capsule in sham-operated Yaa (sYaa) and castrated Yaa (cYaa) at six months. (c) The ratio of renal corpscles with CD44-positive PECs in sYaa and cYaa at six months. (d) Immunofluorescence of androgen receptor (red, AR) and CD44 (green) in sYaa at six months. Dotted line represents renal corpuscles. Arrowheads represent both markers-positive PECs. Some of the CD44-positive PECs stratify, and some of their nuclei are positive for AR. Bar = 50 µm. (e) Immunofluorescence of Ki67 (red) and CD44 (green) in Yaa at six months. Dotted line represents renal corpuscles. Arrowhead represents both markers-positive PECs. Some CD44-positive PECs stratify, and some of their nuclei are positive for Ki67 in sham-operated Yaa. Bars = 50 µm. (f) The number of Ki67-positive PECs in a renal capsule in sYaa and cYaa at six months. (g) The total number of PECs per unit length of Bowman’s capsule in sYaa and cYaa at six months. Each bar represents mean ± SE (n = 8–10). Significant differences in castrated groups compared to the sham-operated group are indicated with an asterisk. *: P < 0.05, **: P < 0.01 (Mann–Whitney U-test). (A color version of this figure is available in the online journal.)