Human kidney biopsy tissues of DN patients (n = 8) and glomerular minor lesion (GML) patients (n = 5) were recruited. GML is identified as minor morphological lesions by renal biopsy without DM. The percentage of tubular atrophy and interstitial fibrosis (IFTA) were assessed by two pathologists. The Ethics Committee of the Second Xiangya Hospital approved the protocol for this study. All patients signed approved informed consent forms. Renal tissues from mice were routinely processed, embedded in paraffin, sectioned (3–4 μm) and then subjected to HE staining.
Renal sections from human and mice were deparaffinized and rehydrated before being subjected to antigen retrieval in a microwave oven. Immunohistochemistry (IHC) was performed using anti-ALPK1 (1:200, Immunoway), CD68 (ZM-0060, ZSGB-Bio), F4/80 (GB11027, Servicebio), α-SMA (ab5694, abcam) and FN (ab2413, abcam) antibody as primary antibodies followed by secondary antibodies for2h. Then the slides were developed using a DAB detection kit. The human and mouse tissue sections were examined by light microscopy. Image J software (National Institutes of Health, Bethesda, MD) was used to measure the average intensity of at least 20 randomly selected fields.
Renal sections from human and mice were deparaffinized and rehydrated before being subjected to antigen retrieval in a microwave oven. Immunohistochemistry (IHC) was performed using anti-ALPK1 (1:200, Immunoway), CD68 (ZM-0060, ZSGB-Bio), F4/80 (GB11027, Servicebio), α-SMA (ab5694, abcam) and FN (ab2413, abcam) antibody as primary antibodies followed by secondary antibodies for2h. Then the slides were developed using a DAB detection kit. The human and mouse tissue sections were examined by light microscopy. Image J software (National Institutes of Health, Bethesda, MD) was used to measure the average intensity of at least 20 randomly selected fields.