Focal segmental glomerulosclerosis, excluding atypical lesion, is a predictor of renal outcome in patients with membranous nephropathy: a retrospective analysis of 716 cases

We included patients with a diagnosis of IMN, who are biopsied between January 1, 2007 and December 31, 2017 in our hospital, excluding patients with diagnoses such as lupus nephritis, MN related to hepatitis B virus, malignancy, metal poisoning, or other diseases associated with secondary MN. The enrolled patients were treated with angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers, corticosteroids, cyclophosphamide, cyclosporine, tacrolimus, azathioprine, Tripterygium wilfordii [16], and mycophenolate mofetil. All patients were ≥ 14 years of age at the time of renal biopsy. All renal biopsies were evaluated by light and immunofluorescence microscopy, ultrastructural evaluation was performed if necessary. Follow-up started at the time of biopsy and either continued until December 2018 or ended at the time of death or development of end-stage renal disease (ESRD).

For the enrolled patients, age, sex, duration of disease before biopsy, levels of serum creatinine and albumin, 24-h proteinuria content, systolic and diastolic blood pressures were recorded at the time of biopsy.

The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to estimate glomerular filtration rate (eGFR) [17]. The CKD was classified based on the KDIGO 2012 Clinical Practice Guideline [18]. Hypertension was defined as blood pressure exceeding 140 over 90 mmHg or currently receiving antihypertensive therapy.

Renal biopsy specimens including at least eight glomeruli were analysed in this study. Renal tissue specimens were examined by two pathologists with no knowledge of the patients’ clinical condition to establish the diagnosis by standard pathologic methods alone. The pathological features under light microscopy, stage, global sclerosis, segmental sclerosis, tubulointerstitial fibrosis, and arteriosclerosis were collected. FSL was graded as present or absent, including FSGS and aFSL. AFSL was defined as follows: 1) pure synechia (Fig. 1a), and synechia at the tip pole, and/or accompanied by proliferation of matrix (Fig 1b) also was also classified in aFSL in present study; 2) Hyperplasia of podocytes (Fig. 1c, arrow), which may be accompanied by segmental thickening of the GBM or proliferation of mesangial matrix; 3) Proliferation of the extracellular matrix of the segmental tuft (Fig. 1d, f), or thickening of the GBM (Fig. 1e); often accompanied by segmental endothelial cell hyperplasia (Fig. 1 e, f); and 4) absence of typical FSGS, with no accumulation of inframembranous hyaline, no collapsing tuft, and without foam cells occluding the lumina. FSGS lesions were categorized according to the Columbia FSGS classification system [9], but synechia at the tip pole (and/or accompanied with proliferation of matrix), was classified as aFSL in the present study, in order to coincide with the definition of other locations..

Based on this classification, the subjects were divided into three groups: patients without focal segmental lesion (FSL⁻), with FSGS (FSGS⁺), and with aFSL,but not co-occurring with FSGS (aFSL⁺). Tubulointerstitial fibrosis was defined as increased extracellular matrix separating tubules in the cortical area and was recorded according to the area of fibrosis. The score for degree of vascular sclerosis was based on the most severe lesion seen in either arterioles or arteries [11].

The primary end point was the composite of ESRD (defined by persistent eGFR< 15 ml/min per 1.73m², start of chronic dialysis, or preemptive kidney transplantation) or a 50% reduction in eGFR over follow-up. Secondary outcomes included all-cause death, and ESRD.

Normally distributed variables were expressed as the mean ± standard deviation and compared using one-way analysis of variance. Nonparametric continuous variables were expressed as medians (interquartile ranges) and compared using the Kruskal–Wallis test. Categorical variables were expressed as percentages and compared using the χ²-test or Fisher exact test. The cumulative probabilities of event-free survival for outcomes were determined using the Kaplan-Meier method and log-rank tests. The independent risk factors for outcomes were analysed with the Cox proportional hazards model. All P-values were two-tailed and values< 0.05 were considered to indicate statistical significance. The confidence interval included 95% of predicted values. Statistical analyses were performed using SPSS statistical software (version 23.0, SPSS, Chicago, IL).

A total of 716 patients with IMN were included in this study. The mean age at diagnosis was 49 ± 14 years and the median follow-up was 20(interquartile range:8–35) months. The median serum creatinine level was 77(interquartile range:62–91) μmol/L. There were 68 patients with an eGFR of < 60 mL/min per 1.73 m² and 388 with an eGFR of > 90 mL/min per 1.73 m².The median proteinuria value was 4.8 g/day (interquartile range: 2.7–7.5 g/day),and 150 patients had proteinuria of > 8.0 g/day.

A comparison of demographic and baseline clinicopathological data among the FSGS⁺, aFSL⁺, and FSL⁻ IMN patients is summarized in Table 1. FSGS was present in 174 patients, while aFSL was noted in 161 patients. Compared with FSL⁻ and aFSL⁺ patients, patients with combined FSGS lesions had higher systolic blood pressure, diastolic blood pressure, and serum creatinine level and a lower GFR. Compared with the FSL⁻ group, both the FSGS⁺ and aFSL⁺ groups had significantly lower albumin, and higher 24-h urine protein.

Of the 174 patients with FSGS lesions, 70 presented with tip lesions, 73 with NOS lesions, 23 with perihilar lesions, 3 with cellular lesions, and 5 with collapsing lesions; Of the 161 patients with atypical focal segmental lesions, 95 presented with synechia lesions,19 with segmental hyperplasia of podocytes (without synechia lesions), and 47 with segmental thickening of GBM, accompanied by proliferation of the mesangial matrix (without hyperplasia of podocytes or synechia lesions).

The percentage of obsolescent glomeruli was highest in the group of patients with FSGS (vs. FSL⁻ group, P < 0.001; vs. aFSL⁺ group, P = 0.001). A significant increase in the severity of vascular sclerosis lesions was found in biopsy specimens in group FSGS⁺ (vs. aFSL⁺, P = 0.002; vs. FSL⁻, P < 0.001). Furthermore, a significant increase in severe tubulointerstitial fibrosis was also found in biopsy specimens in group FSGS⁺(vs. aFSL⁺, P < 0.001;vs. FSL⁻, P < 0.001) and aFSL⁺(vs. FSL⁻, P < 0.001). Regarding the staging of membranous lesions, a later stage was observed in the FSGS⁺ group (P < 0.001) and in the aFSL⁺ group (P < 0.01) compared with the FSL- group (Table 1).

Two hundred eighty-nine (75.9%) patients in the FSL⁻ group, 136 (84.4%) in the aFSL⁺ group, and 151 (86.8%) in the FSGS⁺ group were treated with immunosuppressive therapy (Table 1). At the end of follow-up, 44 patients (6.1%) experienced a 50% decline in eGFR (including progression to ESRD), of which 27(3.8%) progressed to ESRD. Twenty-four patients (3.4%) died; causes of death included severe infection (12/25), pulmonary embolism (1/19), and cardio-cerebral vascular events (11/25).

The significance of each factor affecting the primary outcome is shown in Table 2. Among the clinical parameters, old age (≥60 years old), hypertension, increased serum creatinine (≥134umol/L), and eGFR (< 60 ml/min per 1.73 m²) were significant risk factors for primary outcome by univariate analysis. However, male sex, low level of albumin, and high level of 24-h urine protein were not identified as risk factors in our analysis. By univariate analysis, a high incidence of global glomerulosclerosis (present in > 5% of total glomeruli), and focal segmental lesions (including both atypical focal segmental lesions and typical FSGS lesions), the presence of FSGS lesions alone, tubulointerstitial fibrosis occupying ≥15% of the total specimen area, proliferation of mesangial area, and vascular sclerosis were significant risk factors for primary outcome (Table 2).

By multivariate analysis, in model 1, old age(≥60 years old), eGFR< 60 ml/min per 1.73 m², tubulointerstitial fibrosis area ≥ 15% were significant risk factors for progression to primary outcome, while focal segmental lesions (including both atypical focal segmental lesions and typical FSGS lesions) was not independent risk factor. However, in model 2, old age(≥60 years old) (HR, 2.870; 95% CI, 1.519–5.424; P = 0.001), eGFR< 60 ml/min per 1.73 m² (HR, 2.925; 95% CI, 1.441–5.936; P = 0.003;), FSGS lesions (HR, 2.471; 95% CI, 1.113–5.486; P = 0.026), tubulointerstitial fibrosis area ≥ 15%(HR, 2.553; 95% CI, 1.248–5.224; P = 0.010) were significant independent risk factors for progression to primary outcome (Table 3). Twenty-eight patients (16.1%) in the FSGS⁺ group reached the primary outcome, compared with 12 patients (3.1%) in the FSL⁻ group(P < 0.001) and 4 patients (2.5%)in the aFSL⁺ group(P < 0.01). Sixteen patients (9.2%) in the FSGS⁺ group reached the ESRD outcome compared with 9 patients (2.4%) in the FSL⁻ group(P < 0.001) and 2 patients (1.2%) in the aFSL⁺ group (P < 0.01). Nine patients (2.4%) in the FSL⁻ group reached the all-cause death outcome compared with 9 patients (5.2%) in the FSGS⁺ group and 6 patients (3.7%) in the aFSL⁺ group.

The IMN patients with FSGS had a significantly higher risk of progressing to the primary outcome and ESRD outcome compared with patients in the other groups (Fig. 2). The IMN patients with FSGS also had a significantly higher risk of progressing to the all-cause death outcome compared with the FSL⁻ groups, while the FSGS⁺ group and aFSL⁺ group had no significant difference (Fig. 3). At the 24-months follow-up point, the event-free survival for primary outcome in the FSGS⁺ groups was 84.2 ± 3.5%, compared with 99.4 ± 0.6% in the aFSL⁺ group and 97.2 ± 1.1% in the FSL⁻ group.

The patients with NOS, tip variant and collapsing variant had a higher ratio of progression to the primary outcome (Fig. 4a), and those with NOS, and tip variant had a higher ratio of progression to ESRD (Fig. 4b) and a numerically higher ratio of progression to all-cause death (Fig. 4c).