Solidified glomerulosclerosis, identified using single glomerular proteomics, predicts end-stage renal disease in Chinese patients with type 2 diabetes

Abstract

Few histological prognostic indicators for end-stage renal disease (ESRD) have been validated in diabetic patients. This biopsy-based study aimed to identify nephropathological risk factors for ESRD in Chinese patients with type 2 diabetes. Histological features of 322 Chinese type 2 diabetic patients with biopsy-confirmed diabetic nephropathy (DN) were retrospectively analysed. Cox proportional hazards analysis was used to estimate the hazard ratio (HR) for ESRD. Single glomerular proteomics and immunohistochemistry were used to identify differentially expressed proteins and enriched pathways in glomeruli. During the median follow-up period of 24 months, 144 (45%) patients progressed to ESRD. In multivariable models, the Renal Pathology Society classification failed to predict ESRD, although the solidified glomerulosclerosis (score 1: HR 1.65, 95% confidence interval [CI] 1.04-2.60; score 2: HR 2.48, 95% CI 1.40-4.37) and extracapillary hypercellularity (HR 2.68, 95% CI 1.55-4.62) were identified as independent risk factors. Additionally, single glomerular proteomics, combined with immunohistochemistry, revealed that complement C9 and apolipoprotein E were highly expressed in solidified glomerulosclerosis. Therefore, solidified glomerulosclerosis and extracapillary hypercellularity predict diabetic ESRD in Chinese patients. Single glomerular proteomics identified solidified glomerulosclerosis as a unique pathological change that may be associated with complement overactivation and abnormal lipid metabolism.

Figure 1

Kaplan–Meier survival curves for end-stage renal disease stratified by each pathological feature. (A) Renal Pathology Society glomerular classification. (B) Interstitial fibrosis and tubular atrophy. (C) Solidified glomerulosclerosis. (D) Extracapillary hypercellularity. RPS, Renal Pathology Society; IFTA, interstitial fibrosis and tubular atrophy; GS, glomerulosclerosis; EXHC, extracapillary hypercellularity.

Figure 2

Heatmaps of the proteins identified by laser microdissection and liquid chromatography-electrospray tandem mass spectrometry of solidified glomerulosclerosis and Kimmelstiel–Wilson nodules. The heatmaps are plotted with columns representing the samples and rows representing the pathways. (A) Heatmap of proteins that were similarly expressed in solidified glomerulosclerosis lesions and Kimmelstiel–Wilson nodules. The blue bar represents the cytoskeleton organisation pathway, the pink bar represents the extracellular matrix components and the green bar represents glycolysis/glucose metabolic process. (B) Heatmap of up- and downregulated proteins in solidified glomerulosclerosis lesions and Kimmelstiel–Wilson nodules. The red bar represents the complement activation pathway and the black bar represents the lipid metabolic pathway.

Figure 3

Distribution of complement C9 and apolipoprotein E in class I glomeruli in diabetic nephropathy, Kimmelstiel–Wilson (KW) nodules, solidified glomerulosclerosis (GS) and focal segmental glomerular sclerosis (FSGS). (A) Immunohistochemical staining for complement C9 and apolipoprotein E. (B) Quantification of complement C9 staining shows significant higher levels in KW nodules and solidified glomerulosclerosis (GS) than in class I glomeruli from a diabetic kidney or FSGS. (C) Quantification of apolipoprotein E staining shows significant higher levels in KW nodules and solidified GS than in class I glomeruli from a diabetic kidney or FSGS. Experiments were performed with samples from 10 participants for KW nodules, 10 for solidified GS, three for class I, and three for FSGS. Data are presented as mean ± SEM. * P < 0.05; ** P < 0.01. Bar = 50 µm.

Figure 4

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.