Renal injury following long-term exposure to carbon disulfide: analysis of a case series

Renal specimens were collected from nine patients with heavy proteinuria who underwent renal puncture biopsy between January 2013 and December 2014. Samples were analyzed at the Department of Pathology, Dong fang Hospital. The clinical history data included gender, age, course of disease (the number of days from proteinuria to renal puncture), and blood pressure (systolic pressure of 140 = mmHg and/or diastolic pressure of 90 mmHg were defined as hypertension). Laboratory indicators before renal penetration included: quantitative/24 h urine protein, microscopic haematuria, creatinine, serum creatinine (SCR, normal 53 ~ 124umol/L), urea nitrogen (BUN, normal tendency of 2.9 ~ 8.9 L), Fasting blood - glucose (FBG, normal 3.89 ~ 6.11 mmol/L), autoantibody (anti cardiolipin, ANA, anti dsDNA, anti SSA, SSB resistance, resistance to SM, ENA resistance, anti GBM antibody, c - ANCA and p - ANCA), immunoglobulin and complement (IgG, IgM and IgA, C3 and C4), five hepatitis B virus (HBsAg, HBeAg, anti - HBs, anti - HBe, anti - HBc).

All specimens of renal puncture were fixed in 4% neutral buffered formalin, embedded in paraffin and then cut in 5 μm sections. Samples were analyzed using light microscopy (one case was analyzed using transmission electron microscopy) and IHC.

Samples were stained with one of the following methods: HE staining, PAS staining, PAM – Masson staining and Congored staining. For IHC, samples were incubated with the following antibodies: IgG, IgA, IgM, C3d, C4d, C1q, fibrinogen (Fib), collagen type III, fibronectin, amyloid A and Ig kappa, Ig lambda, HBsAg and HBcAg dyeing predominate; C3d and C4d were acquired form Abcam company and Biomedica company, respectively; HBsAg, HBcAg and EliVision kit were bought from Fuzhou company; while all others antibodies were purchased from Dako company.

For antigen repair, the following antibodies were used IgG, IgA, IgM, C3d, C4d, C1q, Fib, Amyloid A, Ig common wealth, and Ig mind chains. Briefly, samples were treated with antibodies, mixed with 0.01 mol/L pH 6.0 citrate buffer at high-temperature and high-pressure repair of the antigen, plus 0.4% gastric enzyme (purchased from Anresco) for digestion for 5 min [4]_. After HBsAg and HBcAg staining was repaired with high-temperature high-pressure antigen, 0.05% 24-type protease was added for 7 min [5]_. Collagen type II (HWD1.1) and fibronectin in only 0.05% of 24 type protease digestion for 10 min.

The pathological diagnostic criteria were classified according to the WHO glomerular disease classification published in 1995 [6] and diabetic nephropathy classification principle of the international association of nephropathy from 2010 [7]. The tubulointerstistitial lesion (TIL) scores [8] were classified as: score 0, < 6% lesion; score 1, 6~25% lesion; score 2, 26%~ 50% lesion; score 3, > 50% lesion. The numbers of arteriosclerosis (SAS) were analyzed as follows: 0 points for non-sclerosis, 1 for 1–25% arteriosclerosis; two for 26% ~ 50% arteriosclerosis; three for > 50% arteriosclerosis; 1 point was added if the intima of the interlobular artery thickens beyond the mesenchymal. IHC semi-quantitative scoring [9] was analyzed according to the following criteria: positive range accounted for < 5% of total glomerular area; 1 for 6%~ 25% area; 2 for 26%~ 50% area; 3 for > 50% area. The color intensity was 1, 2 and 3. A positive score (0~9 points) was obtained when the score of positive range and color intensity was multiplied.

Clinical data of patients are shown in Table 1. Patients were all male, aged between 30 and 37 (median age 33; working age 11 to 17 years (mean 13.2 years)); course 1~150 days, median course 16 days. Large amounts of proteinuria and microscopic hematuria were found in nine cases; five cases had increased SCR and BUN, and 2 cases had hypertension. In addition, chronic renal failure (CRF) was observed in 4 patients. Psychiatric symptoms (anxiety, paranoia and personality changes) and neurological symptoms (lower limb pain, claudication and slow nerve conduction in extremities) were found in 3 cases. The fasting blood – glucose (FBG) of 9 patients were normal, which indicated that those patients were not diabetic. Complete set of autoantibodies, immunoglobulin, complement and serum HBsAg, HBeAg and anti-HBc were negative. Responses to treatment showed hormonal resistance in nine cases. Proteinuria persisted for 14 to 50 months of follow-up, and two cases developed ESRD.

Similar pathological changes were observed in nine patients; nevertheless, the degrees of lesions were different (Table 2, Fig. 1). All cases showed moderate to severe nodular mesangial hyperplasia; type “K - W nodule” was observed in four cases, fSour cases had proliferative extracapillary glomerulonephritis (GN) (2 cases had fibrous crescent GN and 2 cases cellular crescent GN), while there were no concomitant changes in one patient. Besides, six cases had diffuse basement membrane thickening, focal segmental sclerosis or bulbar sclerosis; two cases had diffuse glomerular sclerosis, and one case had focal segmental capillary hyperplasia. Moreover, all patients had renal tubular atrophy/interstitial fibrosis with less to moderate chronic inflammatory cell infiltration, as well as renal arteriosclerosis (Table 2).

In all patients, IgM were strongly expressed in segmental sclerosis and weakly expressed in the mesangial region; among these, 4 cases had a small amount of mesangial IgA deposition in focal segmental region. C3d was strongly expressed in diffuse mesangial region and weakly expressed in the vascular loop. C4d, C1q and Fib were all expressed in segmental sclerotic region; nevertheless, IgM and IgA, IgG, C4d, C1q and Fib were no specificity of sedimentary. Type III collagen and Fibronectin, Amyloid A and Ig kappa, Ig lambda predominate, both for HBsAg and HBcAg were negative (Table 2, Fig. 2).

Samples from one patient were further examined by electron microscopy. Three glomeruli were seen under electron microscopy; two were spherically sclerotic and one was segmentally sclerotic. No electron dense deposition was found in the mesangial cells and mesangial matrix hyperplasia. Capillary cavities were unobstructed, local focal stenosis, collapse and occlusion, basilar diffuse thickening, flexion, collapse and shrinkage, foot process fusion with microvilli degeneration, Bowman’s capsule adhesion, thickening of vesicle wall, renal tubular atrophy; basilar thickening, epithelial cells mild edema degeneration, and the number of transparent tube types were observed (Fig. 3). The moderate renal interstitial fibrosis, moderate lymphoid, mononuclear and foam cell infiltration were also observed.