Histological Analyses of Mouse Kidney

Histological analyses were performed as described previously^{54 (link),55 (link)}. Briefly, mouse kidneys were fixed in 4% paraformaldehyde in PBS, incubated overnight at 4 °C, and embedded in paraffin. Sections (4 μm thick) were stained with periodic acid–Schiff (PAS) and PSR. The glomerular area was measured by tracing the outline of the glomerular tuft of at least 50 glomeruli in the cortical fields of PAS-stained specimens. Fibrotic areas were measured digitally using a fluorescence microscope (BZ‐X800; Keyence, Osaka, Japan) in the cortical fields of PSR-stained specimens. Immunohistochemistry was performed as described previously^{56 (link)}. Sections were incubated with anti‐F4/80 antibodies (1:100; ab111101; Abcam, Cambridge, MA, USA). The TUNEL assay was conducted using an in situ apoptosis detection kit (MK-500; Takara Biomedicals, Tokyo, Japan). Interstitial TUNEL-positive cells were counted in 10 randomly selected cortical fields (magnification: × 200). All measurements were blinded.