Histopathological findings

Renal biopsy samples were prepared for light microscopy, immunofluorescence and electron microscopy using standard procedures at West China Hospital. For light microscopic examination, renal specimens were stained with haematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome and periodic acid-Schiff silver methenamine. Immunofluorescence staining was performed for IgG, IgM, IgA, C1q, C3, albumin, fibrinogen, and kappa and lambda light chains. The pathological features evaluated using light microscopy are listed in Supplementary Table ^S1. In addition to the RPS DN classification⁷, numerous common pathological lesions were evaluated, including EXHC (Supplementary Figure ^S4), segmental sclerosis, KW lesion, periglomerular fibrosis and glomerular capsular adhesion, mesangial expansion, capillary microaneurysm, capsular drop, fibrin cap and atubular glomerulus^13,16,56. Atubular glomerulus was defined as a glomerulus attached to a short atrophic segment of proximal tubule, with a tip lesion at the glomerulotubular junction⁵⁶. Global glomerulosclerosis was classified as solidified, ischemic obsolescent or not otherwise specified. They were defined as follows:

Solidified glomerulosclerosis was defined as glomeruli in which the entire tuft was solidified with increasing accumulation of mesangial matrix that expands the glomerular tuft, with the absence of collagenous changes in the capsular space (Fig. 4A)^15,16.

Light photomicrographs of the different patterns of global glomerulosclerosis in diabetic nephropathy. (A) Solidified glomerulosclerosis is characterised by expansion of the mesangial matrix of the glomerular tuft, which fills Bowman’s capsule (periodic acid-Schiff [PAS], × 400). (B) Obsolescent glomerulosclerosis is characterised by wrinkling of the glomerular basement membrane, with subsequent glomerular tuft retraction toward the vascular pole, and gradual accumulation of collagenous connective tissue (stars), beginning at the vascular pole adjacent to the glomerular stalk. Severe arteriolar hyalinosis (arrowhead) can also be observed (PAS, × 400). (C) An example of not otherwise specified glomerulosclerosis, showing disappearing glomerulosclerosis (dashed line), and absence or partial disappearance of Bowman’s capsule, such that there is a continuum between the sclerotic glomerulus and the fibrotic interstitium (PAS, × 400). (D) Another example of not otherwise specified glomerulosclerosis, showing a shrunken glomerulus surrounded by inflamed interstitium (PAS, × 400). Bar = 50 µm.

Ischemic obsolescent glomerulosclerosis was characterized by a retracted glomerular tuft and surrounded by a hypocellular homogeneous collagen matrix beginning at the vascular pole adjacent to the glomerular stalk (Fig. 4B)^16,19.

Not otherwise specified glomerulosclerosis included disappearing glomerulosclerosis and non-specific defined glomerulosclerosis. Disappearing glomerulosclerosis was defined by the absence or partial disappearance of Bowman’s capsule (Fig. 4C)¹⁶. Sclerotic glomeruli that could not be defined as solidified, ischemic obsolescent, or disappearing were assigned to the not otherwise specified glomerulosclerosis category (Fig. 4D).

IFTA, interstitial inflammation, arteriosclerosis and arteriolar hyalinosis were assessed and scored according to the RPS DN classification⁷. Epithelial tubular degeneration⁵⁷, protein casts and red blood cell casts were further reviewed. Renal specimens were examined by two nephropathologists (L.L. and H.X.) and any scoring discrepancies were resolved by discussion. The inter-observer agreement for the scoring of global glomerulosclerosis was 93% and for the identification of EXHC was 97%. The pathologists were blinded to the clinical data and renal outcomes.