Clinical and Pathological Significance of Mesangial C1q Deposition in Kidney Transplant Recipients with Recurrent IgA Nephropathy and Patients with Native IgA Nephropathy Free

IgA nephropathy (IgAN) progresses to end-stage kidney disease (ESKD) at a rate of 15–20% in 10 years and 20–40% in 20 years [1, 2]. Kidney transplantation is an effective treatment for IgAN patients who have reached ESKD. However, recurrent IgAN occurs in approximately 30% of kidney transplant recipients (KTRs) with primary IgAN in their native kidneys, and recent studies have suggested that disease recurrence is associated with graft failure [3‒5]. A long-term observational study revealed that half of recipients with recurrent IgAN lost their allograft at 10 years after the onset of recurrence [6]. Among them, approximately 60% lost their allograft due to disease recurrence, followed by chronic allograft nephropathy.

Of the three complement-activation pathways, the alternative and lectin pathways are the main ones involved in the pathogenesis of IgAN [7]. Little is known about the significance of mesangial C1q deposition, which is the first subcomponent of the C1 complex in the classical pathway. Recently, mesangial C1q deposition was found to be associated with treatment failure in native IgAN patients [8] and to predict poor kidney outcomes in both native IgAN [9, 10] and posttransplantation recurrent IgAN [11] patients, highlighting the importance of the classical pathway in the pathogenesis of IgAN. Therefore, we performed a retrospective case-control study to investigate the relationships of mesangial C1q deposition with clinicopathological features and kidney outcomes in both KTRs with recurrent IgAN and patients with native IgAN.

This was a retrospective, single center, 1:2 matched case-control study of KTRs with biopsy-proven recurrent IgAN (Recurrent group) and adult patients with biopsy-proven native IgAN (Native group). Out of 48 recipients with primary IgAN who underwent kidney transplantation between 2000 and 2021 at the Jikei University School of Medicine in Japan, we enrolled 18 KTRs who had been diagnosed with recurrent IgAN. Some cases were involved in previous studies using the same database [12‒14]. We selected 36 age-, sex-, and kidney biopsy era-matched native IgAN patients for the control group. The inclusion criteria were (1) age ≥20 years at the time of kidney biopsy and (2) first biopsy-proven diagnosis of IgAN. Secondary IgAN such as systemic lupus erythematosus and IgA vasculitis were excluded. The levels of autoimmune antibodies, such as ANA and anti-dsDNA antibodies, were negative, and serum C3 and C4 levels were within normal limits in all patients enrolled in this study. We calculated estimated glomerular filtration rate (eGFR) using the Japanese-specific formula [15]. The clinical data of eGFR were collected from the time of kidney biopsy until June 2022.

Graft biopsies included protocol and episode biopsies. Protocol biopsies were performed at 3 months and 1, 3, 5, and 10 years after transplantation, and episode biopsies were performed in patients who met the following criteria: persistent proteinuria >0.5 g/day, decreased kidney function, or an increase of serum creatinine levels of 1.5-fold the baseline or by >0.3 mg/dL. All biopsies were evaluated using the Banff 2019 classification with light microscopy, immunofluorescence results, and electron microscopy. Diagnosis of IgAN recurrence in kidney allografts or IgAN in native kidneys was determined according to segmental or diffuse granular deposits of IgA in the mesangial area, using the 2016 Oxford classification. Immunofluorescence analyses were re-performed on frozen sections of all biopsy tissues from both the Recurrent group and Native group using fluorescein isothiocyanate-labeled rabbit anti-human IgG, IgA, IgM, C3, and C1q antibodies. C1q deposition was defined as diffuse mesangial or deposition along the capillary loop. C1q-negative staining was defined as segmental deposition or absence of staining.

Categorical variables were compared between groups using the χ² test. Continuous variables with normal distributions were compared using the unpaired t test, and the results are presented as the mean and standard deviation. Continuous variables with non-normal distributions were compared using the Mann-Whitney test, and the results are presented as the median and interquartile range. Statistical significance was defined as a p value <0.05. Data were analyzed using Stata 17.0 (Stata Corp LP, College Station, TX, USA).

The baseline clinical and laboratory characteristics of the Recurrent and Native groups are listed in Table 1. The Recurrent group included 18 KTRs (61.1% male) with a mean age of 41 ± 11 years and a median (interquartile range) serum creatinine level of 1.56 (1.05–1.93) mg/dL. The median time from kidney transplantation to biopsy was 3.6 (2.9–5.8) years. Six KTRs had proteinuria, and 3 had both hematuria and proteinuria, according to allograft biopsy. The other KTRs had no hematuria or proteinuria according to protocol biopsies. The median level of proteinuria was 0.36 (0.06–0.98) g/day. The Native group included 36 patients (58.3% male) with a mean age of 40 ± 10 years and a median serum creatinine level of 0.96 (0.78–1.09) mg/dL. The median time from onset to biopsy was 4.1 (2.0–11.1) years. Eight patients had hematuria, 4 had proteinuria, 17 had both hematuria and proteinuria, 4 had macroscopic hematuria, and 3 had edema on kidney biopsy. The median level of proteinuria in the Native group was 0.78 (0.43–1.07) g/day, and the Native group had more patients with severe hematuria (+2 or +3) compared with the Recurrent group. The serum creatinine level was significantly higher in the Recurrent group than in the Native group. The mean serum IgA concentration was higher in the Native group than in the Recurrent group. We found no differences in systolic or diastolic blood pressure, the time since kidney biopsy, or the frequency of graft loss or ESKD between the groups.

Mesangial C1q deposition was significantly higher in the Recurrent group (11/18; 61.1%) than in the Native group (5/36; 13.9%) (p = 0.001).

Mesangial C1q deposition was positive in 11 KTRs (61.1%). The clinical characteristics of the C1q-positive and C1q-negative groups are shown in Table 2. The C1q-positive group had a mean age of 39 ± 6 years, and 63.6% of the patients were men, while the C1q-negative group had a mean age of 44 ± 15 years, and 57.1% of the patients were men. There were no significant group differences in the age at transplantation, time from transplantation to kidney biopsy, mode of biopsy (protocol or episode), systolic or diastolic blood pressure, serum creatinine level, proteinuria, degree of hematuria, or serum IgA concentration at allograft biopsy. No differences in the incidence of antibody-mediated rejection (ABMR) or graft loss during the observational period were observed. The incidence of a preceding infection, however, was significantly higher in the C1q-positive patients, who had cytomegalovirus (CMV) infection (3 patients), urinary tract infection (2 patients), and candidemia (1 patient) (54.5% vs. 0%, p = 0.01).

Five patients were positive for mesangial C1q deposition (13.9%). The clinical characteristics of the C1q-positive and C1q-negative groups are detailed in Table 3. The C1q-positive group had a mean age of 30 ± 7 years, and 40.0% of the patients were men, while the C1q-negative group had a mean age of 42 ± 9 years, and 61.3% of the patients were men. We found no significant group differences in onset age, time from onset to kidney biopsy, mode of onset, systolic and diastolic blood pressure, serum creatinine level, proteinuria, degree of hematuria, or serum IgA concentration on kidney biopsy. No patients and 2 patients (6.5%) in the C1q-positive and C1q-negative groups, respectively, developed ESKD.

The histopathological findings regarding the presence or absence of mesangial C1q deposition in the Recurrent and Native groups are summarized in Table 4,. Peripheral deposition was evaluated in each immunofluorescence staining as well as mesangial deposition. The immunofluorescence findings of the representative cases of mesangial C1q-positive patients in both Recurrent and Native groups are shown in Figure 1. We found no significant differences in the proportion of M, E, S, or T lesions (MEST-C classification), between the C1q-positive and C1q-negative patients in the two groups. However, in the Recurrent group, the C1q-positive patients tended to have a higher incidence of crescent formation compared with the C1q-negative patients (36.4% vs. 0%, p = 0.07). Mesangial IgG and/or IgM deposition also tended to be more frequent in the C1q-positive patients. The incidence of ABMR, borderline changes, acute T cell-mediated rejection (TCMR)/active chronic TCMR, and the cg score from the Banff 2019 classification were similar between the C1q-positive and C1q-negative patients in the Recurrent group. Among the C1q-positive patients in the Recurrent group, those with peripheral C1q deposition presented higher levels of proteinuria (0.98 g/day vs. 0.51 g/day, p = 0.02), and a higher incidence of endocapillary hypercellularity (E1 lesion) (60.0% vs. 0.0%, p = 0.03). All the patients who were able to be evaluated by electron microscopy, electron dense deposits were seen in the paramesangial area. In addition, we performed electron microscopic analysis in one of the peripheral C1q-positive recurrent IgAN patients with a preceding infection. In this case, subepithelial deposits (SDs) were found, as shown in Figure 2.

We investigated the annual rate of eGFR decline, as a measure of kidney outcomes, in the C1q-positive and C1q-negative patients in each group. The mean observational periods in the Recurrent group and in the Native group were 6.9 ± 5.6 years and 8.3 ± 5.9 years. The median annual rates of eGFR decline in the C1q-positive and C1q-negative patients were −1.20 (−3.87 to 0.72) and −3.11 (−6.53 to −0.94) mL/min/1.73 m²/year (p = 0.19) in the Recurrent group and −2.91 (−2.94 to −2.44) and −0.91 (−2.02 to −0.95) mL/min/1.73 m²/year (p = 0.14) in the Native group, respectively. We found no significant difference in the annual rate of eGFR decline according to the presence or absence of mesangial C1q deposition in either group.

This retrospective case-control study revealed that patients with posttransplantation recurrent IgAN had a significantly higher positivity rate of mesangial C1q deposition compared with those with native IgAN. Furthermore, the incidence of crescent formation was relatively higher in the C1q-positive patients in the Recurrent group, although the kidney outcome during the follow-up period did not differ between the C1q-positive and C1q-negative patients in either group.

According to previous studies, mesangial C1q deposition in native IgAN is relatively rare and occurs in 2.0–21.8% of patients [8‒10]. Few reports have assessed mesangial C1q deposition in patients with posttransplantation recurrent IgAN. However, Zhang et al. [11] evaluated 102 KTRs with recurrent IgAN and reported that mesangial C1q deposition was found in only 15.7% of the study sample. In contrast, our study indicated that mesangial C1q deposition in patients with recurrent IgAN was significantly more prevalent, affecting 61.1% of all patients. It is unclear why the mesangial C1q positivity rate in our sample was so high. This may be related to the small number of cases and differences in test sensitivity between institutions. Several studies have evaluated the importance of mesangial C1q in IgAN. For example, Tan et al. [10] conducted an observational study of 1,071 biopsy-proven native IgAN patients to investigate the relationships between mesangial C1q deposition and kidney outcomes. They reported that C1q-positive patients had a higher incidence of endocapillary hypercellularity (E1), tubular atrophy or interstitial fibrosis (T1 or T2), cellular or fibrocellular crescent formation (C1 or C2), IgG deposition, and IgM deposition compared with C1q-negative patients. They also showed that mesangial C1q deposition, in addition to the serum creatinine level, urinary protein level, and T1/T2 lesions, was an independent risk factor for kidney survival. Other studies have suggested that mesangial C1q deposition is related to IgG staining [9], IgM staining [16], tubular atrophy or interstitial fibrosis [9], and crescent formation [16]. However, in patients with posttransplantation recurrent IgAN, the relationships between C1q deposition and other pathologic features or kidney outcomes have not been identified. In this study, we found a higher rate of crescent formation in the C1q-positive patients with posttransplantation recurrent IgAN, suggesting that mesangial C1q deposition may be associated with crescent formation in posttransplantation recurrent IgAN as well as in native IgAN.

Contrary to the results of previous studies [9‒11], the kidney outcomes in our study did not differ between patients with and those without mesangial C1q deposition. This result could be explained by differences in the number of cases or the length of the observational period. Thus, since crescentic formation in kidney allograft is associated with a worse graft outcome [17] and C1q-positive recipients appear to have a higher rate of crescent formation, as shown in this study, it is possible that mesangial C1q deposition is related to poor kidney outcomes.

Interestingly, the C1q-positive patients with posttransplantation recurrent IgAN in our study had a higher rate of preceding infections, including CMV infection, urinary tract infection, and candidemia compared with the C1q-positive patients (66.7% vs. 0%, p = 0.01). Said et al. [18] analyzed 24 C1q-positive kidney allograft biopsies and found preceding infections in half of them. They concluded that the rate did not differ from that reported in KTRs from a previous study [19]. As native IgAN is considered to be closely related to the pathogenesis of mucosal immunity, such as upper respiratory tract infection and intestinal infection [20], preceding infections might contribute to the onset or exacerbation of posttransplantation recurrent IgAN. Thus, there are several studies that reported IgAN or IgA vasculitis accompanied by CMV infection [21‒23], yet the relationships between C1q deposition and infections have not been addressed in those studies. In our study, patients with peripheral C1q deposition in the Recurrent group had a higher incidence of endocapillary hypercellularity. Furthermore, electron microscopic analysis of a patient with both peripheral C1q deposition and a preceding infection showed SD. Haas et al. [24] have reported that the histopathological features of IgA-dominant infection-related glomerulonephritis are endocapillary hypercellularity and hump-shaped SD. Electron dense deposits in the mesangial area are the typical electron microscopic findings of IgAN, but some IgAN patients show additional SD. There has been reported that those with IgAN with SD (IgA-SD) tended to have the characteristics of IgA-dominant infection-related glomerulonephritis, which implicates the possible association of IgA-SD and infections [25]. Based on these findings, IgA-SD may be more common in recurrent IgAN and may be the possible explanation of a higher rate of preceding infections in the C1q-positive patients with posttransplantation recurrent IgAN.

Native IgAN was originally thought to involve mainly the alternative and lectin pathways, but recent studies have emphasized the importance of the classical pathway as well. In this study, we found that posttransplantation recurrent IgAN patients had a significantly higher rate of mesangial C1q positivity compared with patients with native IgAN and that the C1q-positive patients had a higher rate of crescent formation. These findings indicate that mesangial C1q deposition may trigger a sustained inflammatory response that induces endothelial proliferation and crescent formation, as reported previously [10]. Thus, C1q deposition may be an indicator of more advanced disease activity. Although the number of patients in this study was small, and we did not find any significant differences in C1q disposition, IgM deposition may be more common in C1q-positive patients with recurrent IgAN. This may be because the classical pathway is based on the binding and activation of C1q to the Fc portion of IgM via the formation of antigen-antibody complexes [26]. Since the complement activation of ABMR is activation of the classical pathway starting at C1q, when donor-specific anti-HLA antibodies bind to the donor antigen on the endothelial cell membrane [27], we also examined cg lesions of the Banff 2019 classification for possible association with ABMR (e.g., C1q binding donor-specific anti-HLA antibodies). However, no significant differences were found.

Although the detailed mechanisms remain unclear, the classical pathway may play an important role in recurrent IgAN after transplantation, in addition to the alternative and lectin pathways. The limitations of this study include the small number of cases, the insufficient observation period, and the lack of confirmation of the presence of C1q deposition at the time of native IgAN diagnosis in patients with posttransplantation recurrent IgAN.

Mesangial C1q deposition is significantly more common in posttransplantation recurrent IgAN than in native IgAN, and it may be an indicator of more advanced disease activity. Further investigation is warranted regarding C1q deposition in recurrent IgAN patients after transplantation and its clinical significance.

This study protocol was reviewed and approved by the Ethics Committee of The Jikei University School of Medicine (approval number 34-260[11412]), and the study was conducted in accordance with the World Medical Association Declaration of Helsinki. Written informed consent was not required because the data and frozen sections of renal biopsy tissue were collected retrospectively.

The authors have no conflicts of interest to declare.

This work was not supported by any funding sources.

Ayaka Hayashi, Mayuko Kawabe, and Izumi Yamamoto worked in the clinics, co-designed the study protocol, and co-drafted the manuscript. Ayaka Hayashi and Mayuko Kawabe collected and analyzed the data. Yutaro Ohki, Akimitsu Kobayashi, Hiroyuki Ueda, Yudo Tanno, Ichiro Okido, Nobuo Tsuboi, and Hiroyasu Yamamoto participated in the patient’s care and revised the manuscript. Fumihiko Urabe, Jun Miki, Hiroki Yamada, and Takahiro Kimura performed the kidney transplantation and revised the manuscript. Takashi Yokoo is the divisional directors and approved the final version.

All datasets presented in this study are included in the article material.