The MFI for IgG staining was calculated by measuring and averaging the IF intensities of 20 randomly selected glomeruli under the same conditions using the image analysis software. For nephrin staining, 20 randomly selected glomeruli were graded semi-quantitatively as follows: 0, severely decreased staining; 1, mildly decreased staining; 2, intense staining. The nephrin IF score was then calculated using the formula: (0 × N0 + 1 × N1 + 2 × N2)/(N0 + N1 + N2), where N is the number of the glomeruli for each staining grade.