Visual Abstract
Keywords: complement, IgA nephropathy, immune complexes, outcome, progression of renal failures
Abstract
Background
IgA nephropathy is the most common primary GN worldwide, with dominant deposition of IgA and co-deposits of complement component 3 (C3). Phenotypes and progression of IgA nephropathy varies among different ethnic populations, while patients with IgA nephropathy from Asia showed more severe clinical phenotypes, active kidney lesions, and rapid progression. Our previous genome-wide association study identified complement factor H (CFH) variant rs6677604, tightly linked with the deletion of CFH-related protein 3 and CFH-related protein 1 genes (ΔCFHR3-1), as IgA nephropathy susceptible variant, and additionally revealed its effect on complement regulation in IgA nephropathy.
Methods
To further explore the effect of rs6677604 on IgA nephropathy progression, here we enrolled a Chinese IgA nephropathy cohort of 1781 patients with regular follow-up for analysis. The rs6677604 genotype was measured, and the genotype-phenotype correlation was analyzed using the t test, the chi-squared test, or the nonparametric test, and the association between rs6677604 genotype or mesangial C3 deposition and IgA nephropathy prognosis was analyzed using Kaplan–Meier analysis and Cox regression.
Results
We found that patients with rs6677604-GG genotype had a stronger intensity of mesangial C3 deposition than those with the rs6677604-AA/AG genotype. Patients with IgA nephropathy who had stronger intensity of C3 deposition manifested with more severe clinical and pathological manifestations, including lower eGFR and higher Oxford-M/S/T/C (mesangial hypercellularity, endocapillary cellularity, segmental sclerosis, interstitial fibrosis/tubular atrophy, and crescent) scores. In the survival analysis, stronger intensity of mesangial C3 deposition, but not rs6677604-GG genotypes, was associated with poor long-term kidney outcome in IgA nephropathy.
Conclusions
We found that in Chinese patients with IgA nephropathy, variant rs6677604 was associated with mesangial C3 deposition, and mesangial C3 deposition, but not rs6677604, was associated with IgA nephropathy severity and progression.
Introduction
IgA nephropathy is the most common primary GN worldwide, with highly variable clinical and pathological features, as well as extremely diverse disease courses, in which some patients maintain stable kidney function while up to 40% of patients progress to kidney failure in 20–30 years.1–3 Patients with IgA nephropathy from various ethnic groups differ greatly in clinical-pathological presentations, rate of progression, and response to treatment. Patients with IgA nephropathy from Asia were found to exhibit more severe clinical phenotypes, active kidney lesions, and rapid progression. These might be, at least partly, due to the difference in the indication or timing of the kidney biopsy between Asian patients and White patients.4
Although the pathogenies of IgA nephropathy remain unclear, in recent years, more and more evidence unveiled the involvement of complement activation in IgA nephropathy development and progression. Deposition of many complement components, such as C5b-9, C3, properdin, factor B, C4d, L-ficolin, mannose-binding lectin, and collectin-11, were found in glomerular mesangial areas of patients with IgA nephropathy.5–7 In addition, elevated urinary sC5b-9 and factor H were reported in IgA nephropathy.8–10 Eculizumab, an antibody targeting C5 and inhibiting complement activation, showed favorable therapeutic effect in some cases of IgA nephropathy.11,12 These findings suggest the devastating effect of complement activation in IgA nephropathy.13
The complement system comprises more than 30 proteins, including a plethora of complement component proteins and regulatory proteins. Our previous genome-wide association studies (GWAS) identified 1q32 as susceptible locus for IgA nephropathy. Within this locus, rs6677604, located in the regulators of complement activation gene cluster, was the top single-nucleotide polymorphism (SNP).14,15 The allele frequency of rs6677604 varies greatly among different ethnic groups16 because minor allele frequency (MAF) of rs6677604 is 0.048 in East Asian population, 0.187 in European population, and 0.389 in African population. The variant rs6677604, an intronic variant at complement factor H (CFH) gene, is in strong linkage disequilibrium with the deletion of two other complement regulatory proteins coding genes, CFH-related protein 3 and CFH-related protein 1 (ΔCFHR3-1), and strongly associated with plasma factor H concentration.17 Our previous study reported that rs6677604 was associated with the degree of complement activation in IgA nephropathy.18 In addition, the increased circulating level of factor H–related protein 1 was reported to be associated with poor prognosis in IgA nephropathy in two European cohorts.19,20 However, another study found that ΔCFHR3-1 did not influence IgA nephropathy progression in their monocentric cohort of White patients with IgA nephropathy.21 Currently, we still lack information about the association between rs6677604 and IgA nephropathy progression in Asian population.
Given the considerable differences regarding allele frequency of rs6677604 and disease phenotype of IgA nephropathy in White and Asian population, in this study, we aimed at exploring the association of rs6677604 with IgA nephropathy in a large, well-characterized Chinese IgA nephropathy cohort to find some clues for pathogenesis of IgA nephropathy.
Methods
Ethics and Informed Consents
The study got approval from the Ethics Committee of Peking University First Hospital, and all patients involved in this study signed the informed consent.
Study Population
We first enrolled 1839 patients with primary IgA nephropathy who were diagnosed from 1994 to 2020 and followed up regularly at Peking University First Hospital for at least 12 months. The diagnosis of IgA nephropathy is based on kidney biopsy revealing dominant IgA deposition in the glomerular mesangial area and deposition of electron-dense materials in the mesangium by ultrastructural examination. Patients with secondary IgA nephropathy or IgA vasculitis were excluded after detailed examinations. Then, among these patients with IgA nephropathy, 58 were additionally excluded for the following reasons: (1) diagnosis of IgA nephropathy was verified by histochemical identification of IgA deposition rather than immunofluorescence, n=17; (2) AKI occurred at the time of IgA nephropathy diagnosis (it was difficult to determine their baseline eGFR and calculate the percentage decline of eGFR during follow-up), n=10; and (3) DNA samples were unavailable, n=31. Finally, 1781 patients with IgA nephropathy were enrolled in this study.
Baseline Data Collection and Pathology Assessment
The clinical and histological information including sex, age, circulating IgA and C3, BP, 24-hour urine total protein, serum creatinine level, as well as glomerular IgA and C3 deposition intensities were measured and collected from medical records at the time of kidney biopsy. The modified GFR-estimating equation for the Chinese was used for calculating eGFR.22 Intensities of mesangial IgA and C3 were scored as being − (negative), + (weak), ++ (moderate), +++ (strong), or ++++ (powerful). The Oxford classification (MEST-C) score was used to assess the histological kidney injury by qualified pathologists who were blind to the design of this study.23
Follow-Up and End Point
Patients with IgA nephropathy were regularly followed. During follow-up, proteinuria and serum creatinine levels were monitored. Time-averaged proteinuria was calculated by giving the weight of interval follow-up time to one–time point proteinuria values and averaging all proteinuria values over the duration of follow-up, as previously reported.24 The composite end point used in this study was defined as 40% decline in eGFR, kidney failure (eGFR <15 ml/min per 1.73 m2 or KRT), or death during follow-up, whichever occurred first.
Genotyping of CFH Variant rs6677604
The genotyping of CFH variant rs6677604 was performed using TaqMan Assay. In brief, TaqMan genotyping master mix, Taqman genotyping probes (ID: C_2530309_10), and genomic DNA were mixed according to the manufacturer's instructions. Then genotyping assay was conducted by using the ABI Prism 7500 Sequence Detection System (Applied Biosystems) with 30 cycles (95°C for 15 seconds and 60°C for 1 minute).
Statistical Analysis
Statistical analyses were performed with the statistical software Statistical Program for Social Sciences (IBM SPSS version 26.0, IBM, Armonk, NY). GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA) was used for the presentations of graphs.
Quantitative data with normal distribution were presented as mean±SD, and median and quartile (25%–75%) were used for describing non-normal distributed data. Frequencies were used to describe categorical variables. The t test, the Mann–Whitney U test, or the Kruskal–Wallis test were performed for comparing continuous variables. For categorical variables, the chi-squared or the Fisher exact test was used. The Kaplan–Meier method was used to perform survival curves, and survival analyses were conducted using the unadjusted and multivariable Cox regression models. We considered a statistically significant difference when P < 0.05.
Results
Baseline and Follow-Up Information of the Study Population
A total of 1781 patients were recruited in this study, with their characteristics shown in Table 1. Of these, the median age of patients with IgA nephropathy at the time of kidney biopsy was 34 years. The study comprised 51% male, 51% of patients with hypertension, 24% with gross hematuria, and 32% with prodromal infection. By the time of the kidney biopsy, the median 24-hour urine total protein was 1.22 g/d, and the median eGFR was 81 (54–105) ml/min per 1.73 m2. Of the entire population, patients who were prescribed renin-angiotensin system blockade and corticosteroids comprised 95% and 38%, respectively.
Table 1.
Baseline clinical and pathological characteristics according to the rs6677604 genotypes
| Characteristics | Total/Median (IQR) | rs6677604 (AA or AG) | rs6677604 (GG) |
|---|---|---|---|
| Baseline | |||
| No. (%) | 1781 | 193 (11) | 1588 (89) |
| Sex (male/female, %) | 916/865 (51/49) | 106/87 (55/45) | 810/778 (51/49) |
| Age, yr | 34 (27–43) | 35 (27–46) | 33 (27–42) |
| Hypertension (yes/no, %) | 911/870 (51/49) | 97/96 (50/50) | 814/774 (51/49) |
| Prodromal infection (yes/no, %) | 568/1213 (32/68) | 47/146 (24/76) | 521/1067 (33/67) |
| Gross hematuria (yes/no, %) | 433/1348 (24/76) | 39/154 (20/80) | 394/1194 (25/75) |
| Systolic BP, mm Hg | 122 (115–135) | 122 (111–131) | 122 (115–135) |
| Diastolic BP, mm Hg | 80 (70–85) | 80 (70–86) | 80 (70–85) |
| 24-h urine total proteina, g/d | 1.21 (0.64–2.38) | 1.29 (0.74–2.63) | 1.20 (0.63–2.37) |
| eGFR, ml/min per 1.73 m2 | 81 (54–105) | 79 (51–103) | 81 (55–105) |
| IgAb, g/L | 3.21 (2.45–3.98) | 3.2 (2.47–4.14) | 3.21 (2.45–3.98) |
| C3c, g/L | 0.98 (0.84–1.13) | 0.99 (0.88–1.15) | 0.98 (0.84–1.13) |
| IgA deposition (+/++/+++/++++) | 26/264/1282/209 (1/15/72/12) | 1/32/144/16 (1/16/75/8) | 25/232/1138/193 (1/15/72/12) |
| C3 deposition (−/+/++/+++ to ++++) | 118/207/544/912 (7/12/30/51) | 19/27/66/81 (10/14/34/42) | 99/180/478/831 (6/11/30/53) |
| Oxford classificationd, % | |||
| M0/M1 | 1039/711 (59/41) | 110/80 (58/42) | 929/631 (60/40) |
| E0/E1 | 1216/534 (70/30) | 125/65 (66/34) | 1091/469 (70/30) |
| S0/S1 | 592/1158 (34/66) | 71/119 (37/63) | 521/1039 (33/67) |
| T0/T1/T2 | 1189/397/164 (68/23/9) | 123/50/17 (65/26/9) | 1066/347/147 (68/22/10) |
| C0/C1/C2 | 690/871/189 (39/50/11) | 73/88/29 (39/46/15) | 617/783/160 (40/50/10) |
| Follow-up | |||
| Length of follow-up, mo | 282 | 282 | 282 |
| Mean, mo (SD) | 66 (48) | 66 (47) | 66 (48) |
| Median, mo (IQR) | 52 (28–91) | 54 (29–93) | 51 (28–90) |
| Time-average proteinuriae, g/d | 0.82 (0.44–1.45) | 0.75 (0.41–1.31) | 0.82 (0.44–1.46) |
| RAAS blocker (yes/no, %) | 1683/98 (95/5) | 183/10 (95/5) | 1500/88 (95/5) |
| Corticosteroid (yes/no, %) | 683/1098 (38/62) | 85/108 (44/56) | 598/990 (38/62) |
| Corticosteroid+immunosuppressants (yes/no, %) | 928/853 (52/48) | 105/88 (54/46) | 823/765 (52/48) |
| Composite end pointf (yes/no, %) | 360/1421 (20/80) | 32/161 (17/83) | 328/1260 (21/79) |
Oxford classification: mesangial hypercellularity (M), segmental sclerosis (S), interstitial fibrosis/tubular atrophy (T), and crescents (C). IQR, interquartile range; RAAS, renin-angiotensin-aldosterone system blockers.
Twenty-four-hour urine total protein was collected from 1773 patients.
The level of circulating IgA was collected from 1627 patients.
The level of circulating C3 was collected from 1584 patients.
Oxford classification scores were measured in 1750 patients whose number of glomeruli in the biopsy specimens was more than eight.
Time-average proteinuria was analyzed in 1688 patients.
The composite end point was defined as 40% eGFR decline or kidney failure or death.
Among these enrolled patients with IgA nephropathy, 31 patients with <8 glomeruli in the biopsy specimens were excluded for the Oxford classification score. Within the remaining 1750 patients, M1, E1, S1, T1, T2, C1, and C2 were found in 41%, 31%, 66%, 23%, 9%, 50%, and 11% of patients, respectively.
During follow-up, 360 patients reached the composite end point (318 reached 40% eGFR decline, 198 reached kidney failure, and five died during follow-up).
Distribution of the rs6677604 Genotype
In our enrolled patients with IgA nephropathy, five patients presented with rs6677604-AA genotype (Supplemental Table 1), while rs6677604-AG was detected in 188 patients (11%). The rest of the 1588 patients carried the rs6677604-GG genotype (89%). The allele frequency of the minor allele rs6677604-A was 6% in our present IgA nephropathy population, which was in accordance with previous reports.25
Genotype-Phenotype Association of CFH Variant rs6677604 in IgA Nephropathy
We analyzed the association between rs6677604 with clinical and pathological information in patients with IgA nephropathy to evaluate genotype-phenotype correlations. We noticed that patients with rs6677604-AA/AG had a lower proportion of prodromal infection compared with those with rs6677604-GG (24% versus 33%, P = 0.017). Regarding other clinical and biological parameters, including BP, gross hematuria, urine protein levels, eGFR, as well as circulating IgA and C3 levels, no significant differences were observed between the rs6677604-GG and rs6677604-AA/AG genotypes.
For histopathologic characteristics, we observed that patients with rs6677604-GG had a significantly stronger C3 deposition intensity than those with rs6677604-AA/AG (−, +, ++, and +++ to ++++: 10%, 14%, 34%, and 42%, respectively, in rs6677604-AA/AG versus 6%, 11%, 30%, and 53%, respectively, in rs6677604-GG, P = 0.03). However, no other significant associations of MEST-C scores were noted with rs6677604 genotypes (Table 1).
Association of Mesangial C3 Deposits Intensity with Clinical and Pathological Characteristics in IgA Nephropathy
Because the intensity of mesangial C3 deposits was the only associated pathological feature of CFH variant rs6677604, next we further explored the correlation of mesangial C3 deposition with IgA nephropathy phenotypes. Patients with stronger C3 deposition intensities (+++ to ++++) had significantly increased circulating IgA levels (3.29 [2.58–4.07] g/L versus 3.11 [2.29–3.91] g/L, P < 0.001), stronger intensity of mesangial IgA deposition (+/++/+++/++++, 0.3%/4%/79%/17% versus 3%/26%/65%/6%, P < 0.001), higher Oxford-M (M0/M1, 53%/47% versus 66%/34%, P < 0.001), S (S0/S1, 29%/71% versus 39%/61%, P < 0.001), T (T0/T1/T2: 64%/24%/12% versus 72%/21%/7%, P = 0.001), and C scores (C0/C1/C2: 35%/55%/10% versus 44%/45%/11%, P < 0.001), as well as lower eGFR (86 [60–107] ml/min per 1.73 m2 versus 77 [51–103] ml/min per 1.73 m2, P < 0.001) and 24-hour urine total protein (1.12 [0.6–2.18] g/d versus 1.33 [0.7–2.65] g/d, P < 0.001) compared with patients with the milder intensity of C3 deposits (− to ++). In accordance with more C3 deposits in the glomeruli, decreased circulating C3 levels were found in these patients compared with those with fewer C3 deposits (1.03 [0.88–1.19] g/L versus 0.93 [0.80–1.08] g/L, P < 0.001) (Table 2).
Table 2.
Baseline clinical and pathological characteristics according to the C3 deposition
| Characteristics | Total/Median (IQR) | C3 (− to ++) | C3 (+++ to ++++) |
|---|---|---|---|
| Baseline | |||
| No. (%) | 1781 | 869 (49) | 912 (51) |
| Sex (men/women, %) | 916/865 (51/49) | 106/87 (55/45) | 810/778 (51/49) |
| Age, yr | 34 (27–43) | 33 (26–44) | 34 (28–42) |
| Hypertension (yes/no, %) | 911/870 (51/49) | 436/433 (50/50) | 475/437 (52/48) |
| Prodromal infection (yes/no, %) | 568/1213 (32/68) | 296/573 (34/66) | 272/640 (30/70) |
| Gross hematuria (yes/no, %) | 433/1348 (2476) | 218/651 (25/75) | 215/697 (24/76) |
| Systolic BP, mm Hg | 122 (115–135) | 120 (114–131) | 124 (115–135) |
| Diastolic BP, mm Hg | 80 (70–85) | 80 (70–85) | 80 (70–86) |
| 24-hour urine total proteina, g/d | 1.21 (0.64–2.38) | 1.33 (0.7–2.65) | 1.12 (0.6–2.18) |
| eGFR, ml/min per 1.73 m2 | 81 (54–105) | 86 (60–107) | 77 (51–103) |
| IgAb, g/L | 3.21 (2.45–3.98) | 3.11 (2.29–3.91) | 3.29 (2.58–4.07) |
| C3c, g/L | 0.98 (0.84–1.13) | 1.03 (0.88–1.19) | 0.93 (0.80–1.08) |
| IgA deposition (+/++/+++/++++) | 26/264/1282/209 (1/15/72/12) | 23/230/566/50 (3/26/65/6) | 3/34/716/159 (0.3/4/79/17) |
| Oxford classificationd (%) | |||
| M0/M1 | 1039/711 (59/41) | 567/285 (66/34) | 472/426 (53/47) |
| E0/E1 | 1216/534 (70/30) | 575/277 (67/33) | 641/257 (71/29) |
| S0/S1 | 592/1158 (34/66) | 330/522 (39/61) | 262/636 (29/71) |
| T0/T1/T2 | 1189/397/164 (68/23/9) | 610/182/60 (72/21/7) | 579/215/104 (64/24/12) |
| C0/C1/C2 | 690/871/189 (39/50/11) | 374/381/97 (44/45/11) | 316/490/92 (35/55/10) |
| Follow-up | |||
| Length of follow-up, mo | 282 | 282 | 282 |
| Mean, mo (SD) | 66 (48) | 69 (49) | 62 (46) |
| Median, mo (IQR) | 52 (28–91) | 58 (28–97) | 48.35 (28–82) |
| Time-average proteinuriae, g/d | 0.82 (0.44–1.45) | 0.81 (0.40–1.46) | 0.82 (0.49–1.44) |
| RAAS blocker (yes/no, %) | 1683/98 (95/5) | 810/59 (93/7) | 873/39 (96/4) |
| Corticosteroid (yes/no, %) | 683/1098 (38/62) | 352/517 (41/59) | 331/581 (36/64) |
| Corticosteroid+immunosuppressants (yes/no, %) | 928/853 (52/48) | 448/421 (52/48) | 480/432 (53/47) |
| Composite end pointf (yes/no, %) | 360/1421 (20/80) | 168/701 (19/81) | 192/720 (21/79) |
Oxford classification: mesangial hypercellularity (M), segmental sclerosis (S), interstitial fibrosis/tubular atrophy (T), and crescents (C). IQR, interquartile range; RAAS, renin-angiotensin-aldosterone system.
Twenty-four-hour urine total protein was collected from 1773 patients.
The level of circulating IgA was collected from 1627 patients.
The level of circulating C3 was collected from 1584 patients.
Oxford classification scores were measured in 1750 patients whose number of glomeruli in the biopsy specimens was more than eight.
Time-average proteinuria was analyzed in 1688 patients.
The composite end point was defined as 40% eGFR decline or kidney failure or death.
Association of CFH Variant rs6677604 and Mesangial C3 Deposits Intensity with the Progression of IgA Nephropathy
Furthermore, we investigated the prognostic value of CFH variant rs6677604 and its related phenotype of mesangial C3 deposits to IgA nephropathy progression. During the follow-up, the proportion of using renin-angiotensin-aldosterone system blockers in the rs6677604-GG group was 95% and that in the rs6677604 AA/AG group was 95% (P = 0.84). The proportion of corticosteroids therapy in patients with IgA nephropathy with rs6677604-GG genotype and rs6677604-AA/AG genotype was 38% versus 44%, respectively (P = 0.09). In addition, 52% of patients with IgA nephropathy with rs6677604-GG received corticosteroids combined with immunodepression therapy and that in the rs6677604 AA/AG group was 54% (P = 0.50). The patients with rs6677604-GG and rs6677604-AA/AG had a time-average proteinuria of 0.82 (0.44–1.46) g/d versus 0.75 (0.41–1.31) g/d (P = 0.23), respectively (Table 1). In addition, we also performed the survival analyses among the two groups by using the composite end point, and there was no significant difference in the kidney survival rate and Cox regression models between patients with IgA nephropathy with rs6677604-AA/AG and rs6677604-GG (Figure 1A, Supplemental Table 2).
Figure 1.
The Kaplan–Meier curves of rs6677604 and C3 deposition on the kidney outcome in patients with IgA nephropathy. There was no significant difference in the kidney survival rate between patients with rs6677604-AA/AG and rs6677604-GG (A). The Kaplan–Meier curves showed that the kidney survival rate was lower in patients with IgA nephropathy with stronger C3 deposition compared with those with milder C3 deposition (B).
Then, patients with IgA nephropathy were divided into two groups with comparable patients' numbers, C3 +++/++++ (n=912) and C3 −/+/++ (n=869). In the two age-matched and sex-matched groups of C3 deposition, patients with stronger C3 deposition intensity had significantly higher proportion of renin-angiotensin-aldosterone system blockers use than patients with lower levels (96% versus 93%, P = 0.01). During follow-up, corticosteroids were prescribed to 52% of patients with milder C3 deposition and 49% of patients with stronger C3 deposition (P = 0.07). There was no statistically significant difference regarding the corticosteroid combined with immunodepression treatment between the stronger and milder C3 deposition group (53% versus 52%, P = 0.61). Although the lower baseline 24-hour urine total protein was found in the stronger C3 deposition group, the time-average proteinuria of these two groups showed no significant difference (Table 2).
We further constructed the survival curves according to C3 deposition. Patients with IgA nephropathy with stronger C3 deposition had significantly poor kidney survival than those with milder C3 deposition as defined by the log-rank test (P = 0.01, Figure 1B). Next, we constructed the Cox regression model. In the unadjusted Cox regression analysis, stronger C3 deposition was significantly associated with a higher risk of reaching the kidney composite end point (P = 0.01). The significant association remained after adjusted for age and sex (hazard ratio, 1.31; 95% confidence interval, 1.06 to 1.61; P = 0.01), as well as additional for rs6677604 genotype (hazard ratio, 1.30; 95% confidence interval, 1.05 to 1.60; P = 0.01). After full adjustment with age, sex, rs6677604 genotype, eGFR, 24-hour urine total protein, hypertension, and Oxford Classification, we failed to identify C3 deposition as an independent prognostic factor for kidney survival by multivariable Cox regression analysis (Table 3).
Table 3.
Cox regression model for the effect of C3 deposition on kidney outcome
| Variables in the Model | Model 0 HR (95% CI) | Model 1 HR (95% CI) | Model 2 HR (95% CI) | Model 3 HR (95% CI) | Model 4 HR (95% CI) |
|---|---|---|---|---|---|
| C3 deposition | 1.30 (1.06 to 1.60)a | 1.31 (1.06 to 1.61)a | 1.30 (1.05 to 1.60)a | 1.21 (0.98 to 1.50) | 1.11 (0.90 to 1.39) |
| Age | N/A | 1.00 (0.99 to 1.00) | 0.99 (0.99 to 1) | 0.97 (0.96 to 0.98)b | 0.98 (0.97 to 0.99)b |
| Sex | N/A | 1.02 (0.83 to 1.26) | 1.02 (0.83 to 1.25) | 1.09 (0.88 to 1.34) | 1.14 (0.92 to 1.41) |
| rs6677604 | N/A | N/A | 1.29 (0.89 to 1.85) | 1.40 (0.97 to 2.01) | 1.36 (0.94 to 1.98) |
| eGFR | N/A | N/A | N/A | 0.98 (0.98 to 0.98)b | 0.99 (0.98 to 0.99)b |
| 24-h urine total protein | N/A | N/A | N/A | 1.06 (1.02 to 1.10)c | 1.07 (1.02 to 1.12)c |
| Hypertension | N/A | N/A | N/A | 1.32 (1.05 to 1.65)a | 1.23 (0.97 to 1.54) |
| Oxford classification | |||||
| M1 | N/A | N/A | N/A | N/A | 1.07 (0.85 to 1.34) |
| E1 | N/A | N/A | N/A | N/A | 1.24 (0.99 to 1.56) |
| S1 | N/A | N/A | N/A | N/A | 1.72 (1.31 to 2.25)b |
| T1 | N/A | N/A | N/A | N/A | 1.75 (1.33 to 2.31)b |
| T2 | N/A | N/A | N/A | N/A | 3.46 (2.46 to 4.88)b |
| C1 | N/A | N/A | N/A | N/A | 1.15 (0.90 to 1.46) |
| C2 | N/A | N/A | N/A | N/A | 1.19 (0.84 to 1.68) |
The composite end point was defined as 40% eGFR decline, kidney failure, or death. HR, hazard ratio; CI, confidence interval; N/A, not available.
Represents P value < 0.05.
Represents P value < 0.01.
Represents P value < 0.001.
Discussion
In this study, we analyzed the effects of IgA nephropathy susceptible variant rs6677604 and mesangial C3 deposition on IgA nephropathy in a large Chinese IgA nephropathy population. We found that although rs6677604 correlated with mesangial C3 deposition in IgA nephropathy, regarding long-term kidney outcome, mesangial C3 deposition and other factors, such as age, eGFR, 24-hour urine protein, hypertension, and Oxford classification, but not rs6677604, showed the significant association with IgA nephropathy progression.
Because MAF of rs6677604 is low in Asian population (about 5% in our IgA nephropathy cohort), we obtained a large population of 1781 patients with IgA nephropathy to provide higher power to evaluate the effects of variant rs6677604 on IgA nephropathy progression. Among our recruited Chinese patients with IgA nephropathy, 193 patients were rs6677604-AA/AG genotype, which was in line with the 222 patients with IgA nephropathy with rs6677604-AA/AG genotype in a previous study that recruited White patients with IgA nephropathy. During follow-up, 360 patients reached the end point, accounting for 20% in our IgA nephropathy cohort. However, we found no significant association of rs6677604 with IgA nephropathy progression, as similar to those previously reported in European cohorts.21
In our study, we found that patients with IgA nephropathy with rs6677604-GG genotype had stronger intensity of mesangial C3 deposition, while the stronger intensity of mesangial C3 deposition was associated with more severe clinical and pathological characteristics, including the baseline eGFR and Oxford classification scores, which reinforced the findings in previous reports.18,26 Regarding proteinuria, another important feature for IgA nephropathy severity, although patients with IgA nephropathy who had the stronger intensity of mesangial C3 deposition presented with lower baseline 24-hour urine total protein, their time-average proteinuria level during follow-up was comparable with those in patients with milder intensity of mesangial C3 deposition. Therefore, it seems that IgA deposition-induced complement activation in the glomerular mesangial area might not significantly influence proteinuria in IgA nephropathy.
Considering that C3 deposition was associated with multiple clinical and pathological features in our study, which were well established as risk factors for IgA nephropathy prognosis, we next explored the effect of C3 deposition on IgA nephropathy progression. In the unadjusted Cox regression model, we found that stronger intensity of C3 deposition was associated with poor long-term kidney outcome, and the association remained significant after adjusting with demographic characteristics (age and sex). However, when additionally adjusted with clinical (baseline 24-hour urine total protein, eGFR, hypertension) and pathological (MEST-C scores) features, C3 deposition lost its association with kidney outcome. These results confirmed the contribution of local complement activation on kidney injury, evidenced by pathological changes, and kidney function impairment. However, in the multivariable survival analysis, although proteinuria, eGFR, and Oxford scores affected IgA nephropathy progression as previously reported,27 the degree of local complement activation could not be used as an independent risk factor for IgA nephropathy progression. We supposed that C3 intensity might be not the perfect indicator for quantification of complement activation degree, and local complement activation, through its damage to kidney tissue, thereafter, presented an association with IgA nephropathy progression.
The complement system is the essential element of the innate immune system and serves as one of the first barriers to defense against pathogenic microorganisms. However, the complement system is a double-edged sword because the overactivation of the complement system results in tissue damage. Regardless of which pathways initiate complement activation, it will eventually start the terminal pathway and lead to the formation of membrane attack complex (MAC). It was reported that sublytic C5b-9 might induce mesangial proliferation,28 as well as activate the NLRP3 inflammasome, trigger the caspase-1 activation, and release the proinflammatory cytokines IL-1β and IL-18.29,30 In IgA nephropathy, C5b-9 were not only found to deposit in the mesangial areas31 but also elevated in urine.8,10 Therefore, complement activation–induced MAC formation might lead to sublytic injury to mesangial cells in IgA nephropathy. Moreover, complement activation will effectively cleave C3 and C5 into active C3a and C5a, which are anaphylatoxins and play important roles in inducing inflammation by attracting inflammatory cells.32 In patients with IgA nephropathy, urinary C3a and C5a levels, as well as several inflammatory factors, were increased and related to impaired kidney function and/or disease progression.33,34 Inflammatory cells residing in the local glomerular were frequently observed in IgA nephropathy,35 indicating active inflammation in the glomeruli.36,37 Taking these clues together, we speculated that in addition to the MAC, the inflammation induced by complement activation might also take part in kidney injury and progression of IgA nephropathy.
Based on our findings, we believed that kidney local complement activation accounted for kidney damage and progression in IgA nephropathy, which emphasized the importance of complement activation in IgA nephropathy. However, although variant rs6677604 was associated with kidney local complement activation, it showed little influence on pathological lesions and progression in IgA nephropathy. We had some thought-out explanations for this ambiguity. IgA nephropathy is a common complex disease with complicated pathogenesis. In IgA nephropathy, complement activation is widespread, as evidenced by approximately 90% patients with glomerular C3 deposition.7,38 Recent novel therapies targeting complement activation showed promising effects in IgA nephropathy.11,39,40 Glomerular complement activation in IgA nephropathy is also influenced by multiple factors and mechanisms. Previous studies found that poly-IgA immune complex could activate complement C3 in vitro experiments,41 and deposit on mesangial cells, stimulate intracellular Ca2+ release and activate complement,7,42,43 as we observed in the glomeruli of patients with IgA nephropathy. In this study, we found that the intensity of glomerular IgA and C3 deposits were positively correlated. These evidence suggested that mesangial deposited IgA1 was also an important factor for complement activation in IgA nephropathy. Moreover, in addition to rs6677604, which regulated the balance of factor H, factor H–related protein 1, and factor H–related protein 3, there are several other complement components and regulatory factors that take part in complement activation in IgA nephropathy, including factor H–related protein 5, mannose-binding lectin, L-ficolin, and collectin-11.7,19,44 We could not rule out the possibility that these factors also influenced the magnitude of complement activation in IgA nephropathy. We speculated that rs6677604, combined with mesangial deposited IgA, other complement proteins, and so on, worked together to determine the magnitude of complement activation in IgA nephropathy. Therefore, rs6677604 alone showed little influence on IgA progression.
Our study also had some limitations. First, the intensity of C3 deposition in glomeruli was assessed by the semiquantitative scoring, which had very limited sensitivity, and C3 intensity was collected from biopsy reports from different times and pathologists. These factors influenced the efficiency of subsequent survival analysis. Second, although we identified the contribution of kidney local complement activation to IgA nephropathy severity and progression, which confirmed the importance of complement activity in IgA nephropathy, we did not reveal a pathogenic mechanism. The evaluation of intensity of mesangial C3 deposits deeply relied on invasive kidney biopsy. Therefore, development of noninvasive biomarkers reflecting the magnitude of glomerular complement activation is needed in the future, but this is beyond the objective of our study. Third, given that IgA nephropathy is a chronic progressive kidney disease and the MAF of rs6677604 in the Asian population is low, re-evaluation of the effect of rs6677604 in a larger and longer follow-up IgA nephropathy cohort may still be worthwhile and meaningful in the future.
In conclusion, in the Chinese cohort of IgA nephropathy, we found that IgA nephropathy susceptible variant rs6677604 was associated with mesangial C3 deposition. In addition, mesangial C3 deposition, but not rs6677604, was associated with IgA nephropathy severity and progression.
Supplementary Material
Disclosures
P. Chen, Y. Kang, Y. Leng, Y. Li, L. Liu, J. Lv, B. Xu, X. Zhou, and L. Zhu report employment with Peking University First Hospital. H. Zhang reports employment with Peking University First Hospital and consultancy for Alebund, Calliditas, Chinook, Novartis, OMEROS, and Ostuka. The remaining author has nothing to disclose.
Funding
L. Zhu: National High-Level Hospital Clinical Research Funding (Interdisciplinary Clinical Research Project of Peking University First Hospital, 2022CR39), National Science Foundation of China (81970598, 82270740), National Key Research and Development Program of China (2020YFC2005003). S. Shi: National Science Foundation of China (82170710).
Author Contributions
Conceptualization: Li Zhu.
Data curation: Pei Chen, Yuqi Kang, Lijun Liu, Jicheng Lv, Sufang Shi, Hong Zhang, Xujie Zhou, Li Zhu.
Formal analysis: Yuqi Kang.
Funding acquisition: Li Zhu.
Investigation: Pei Chen, Yuqi Kang, Yueqi Leng, Yebei Li, Lijun Liu, Jicheng Lv, Sufang Shi, Boyang Xu, Hong Zhang, Xujie Zhou, Li Zhu.
Project administration: Li Zhu.
Resources: Hong Zhang.
Supervision: Li Zhu.
Visualization: Yuqi Kang.
Writing – original draft: Yuqi Kang, Li Zhu.
Writing – review & editing: Yuqi Kang, Li Zhu.
Data Sharing Statement
All data are included in the manuscript and/or supporting information.
Supplemental Material
This article contains the following supplemental material online at http://links.lww.com/CJN/B804.
Supplemental Table 1. Clinical and pathological characteristics of five patients with rs6677604-AA genotype.
Supplemental Table 2. Cox regression model for the effect of rs667704 variant on kidney outcome.
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Data Availability Statement
All data are included in the manuscript and/or supporting information.