Disparities between IgG4-related disease with and without kidney involvement: a case-control study based on 450 patients

Background We aimed to compare the demographic, clinical and laboratory characteristics between IgG4-related kidney disease (IgG4-RKD+) and extrarenal IgG4-related disease (IgG4-RKD-) in a large Chinese cohort, as well as describing the radiological and pathological features of IgG4-RKD+. Methods We retrospectively analyzed the medical records of 450 IgG4-RD patients at Peking University People’s Hospital from January 2004 to January 2020. Results Among the 450 IgG4-RD patients, 66 were diagnosed with IgG4-RKD+. Compared with IgG4-RKD-patients, IgG4-RKD+ patients had older age at onset and at diagnosis (59 vs 54, P=0.007; 61 vs 56, P=0.002, respectively). Male to female ratio of IgG4-RKD+ patients is signicantly higher (2.5:1 vs 1.2:1, P=0.015). In the IgG4-RKD+ group, the most commonly involved organs were salivary gland (54.6%), lymph nodes (47.0%) and pancreas (40.9%). More than three organs involved (OR=6.212), retroperitoneal brosis involvement (RPF) (OR=12.460) and low serum C3 level (OR=0.202) were risk factors for the kidney disease in IgG4-RD patients. It was found that renal function was impaired in more than 50% of IgG4-RKD+ patients. The most common imaging nding is multiple, often bilateral, hypodense lesions, followed by ureteric obstruction and hydronephrosis related to RPF, and thickening of the renal pelvic wall. Conclusions or more organs, retroperitoneal brosis and low serum C3 level were risk indicate differences

To minimize the impact of irreversible renal damage resulting from the disease itself or unnecessary surgical intervention, early recognition and treatment are of pivotal importance. In this study, demographic, clinical, and laboratory disparities in the 66 IgG4-RKD + and 384 IgG4RKD-patients were investigated. To our best knowledge, this is the largest study to compare the two phenotypes of patients Page 3/18 with or without kidney involvement related to IgG4-RD. Determining the characteristics of these two phenotypes may help identify risk factors and their potential differences in pathogenesis.

Patients
From January 2004 to January 2020, four hundred and fty IgG4-RD patients who were presented at Peking University People's Hospital with complete clinical data were enrolled in this study. Based on the 2011 comprehensive IgG4-RD diagnostic criteria [9], there were 216 (48.0%), 35 (7.8%) and 199 (44.2%) cases diagnosed with de nite, probable and possible IgG4-RD, respectively (Table 1). 0.001** IgG4-RKD + vs IgG4-RKD-, **P < 0.05 Patients were screened for IgG4-RKD + in case of unexplained urinary abnormalities, renal imaging, or histology suggestive of IgG4 RKD + or if they had renal involvement with conditions known to be associated with IgG4-RD. Patients who had any de nite etiology of renal impairment were excluded from the study [11,12]. Renal pathological ndings were available for 6 patients: dense lymphoplasmacytic in ltration with more than 10 in ltrating IgG4-positive plasma cells per high-power eld and an IgG4/IgG plasma cell ratio of more than 40% with brosis. In total, there were 66 patients diagnosed with IgG4-

RKD+.
Gender, age, allergic diseases, clinical manifestations, organ involvement, radiological and pathological ndings were recorded. According to the criteria from the European Academy of Allergy and Clinical Immunology (EAACI), allergic disease was diagnosed by experienced specialists. This study was approved by the Medical Ethics Committee of Peking University People's Hospital (Beijing, China).
All patients underwent radiology examinations consisting of Computed Tomography (CT), or Magnetic resonance imaging (MRI), and some patients also received 18F-uorodeoxyglucose PET-CT.
We xed all tissue biopsy samples in formalin and embedded in para n, then stained them with hematoxylin and eosin (H&E) and immunocytochemistry (IHC). IHC was performed using the avidin-biotin complex-peroxidase method with monoclonal antibody to human IgG4 (Zymed, Carlsbad, CA; dilution 1:50 or 1:100 depending on staining laboratory) on sections from para n-embedded tissue.
The renal biopsy was examined speci cally for features suggestive of IgG4-RKD+, including presence of TIN, lymphoplasmacytic in ltration, as well as tubulointerstitial presentations on light microscopy, glomerular light microscopy abnormalities, glomerular or tubular basement membrane (TBM) deposits on IF or EM. IgG4 staining was done and the number of positive plasma cells/hpf were calculated.

Statistical analysis
Using Stata software (V.15.0; Stata, College Station, TX, USA), data were analyzed by descriptive methods, with standard summary statistics including mean (S.D.), median, interquartile range (IQR), and proportions. We performed the Student's t test for differences for continuous, normally distributed data; continuous, non-normally distributed data were analyzed by the Mann-Whitney test. Categorical variables were processed by X 2 or Fisher's exact tests. Logistic regression analysis with enter method was performed to compare the IgG4-RKD + and IgG4-RKD-patients. Factors with P < 0.2 in the univariate analysis were included in the multivariate analysis. P < 0.05 was considered to be statistically signi cant.

Laboratory Parameters
Disparities in laboratory features between the IgG4-RKD + and IgG4-RKD-groups were indicated in Table 1. No signi cant differences were found in levels of serum CRP, IgG4 and IgE, eosinophil count, serum C3, and serum C4. Higher level of ESR was found in the IgG4-RKD + group (27.5 vs 16 for Hb, P = 0.015). However, Hb were signi cantly lower in the IgG4-RKD + group (121.5 vs 135.5, P = 0.001).
Renal damage indexes of the IgG4-RKD+ group As presented in Table 2, it was found that renal function was impaired with the reduced eGFR (53%), elevated blood urea nitrogen (37.9%) and elevated serum creatine (30.3%). The median eGFR was 78.5 mL/min/1.73 m 2 (IQR, 58.5, 100.2). The level of serum creatinine varied from 64 to 122 umol/L at baseline. The median blood urea nitrogen was 6.9 mmol/L (IQR, 5.   Risk Factors associated with IgG4-RKD+ As shown in   (Fig. 2).

Pathological ndings
In total, 6 patients with IgG4-RKD underwent renal biopsy; all of the 6 (100%) patients had TIN. TIN in 1 case (16.6%) was associated with glomerular disease. Membranous nephropathy was the cause of glomerular disease in this case.
All the patients with TIN had a lymphoplasmocytic (LPC) in ltrate with brosis. The LPC in ltration was diffuse in 3 patients and patchy in the other 3 patients. In all of the 6 patients with TIN, IgG4 staining demonstrated > 10 IgG4 + plasma cells per high-power eld (in the most concentrated area), and all these patients ful lled the Raissian criteria for IgG4-TIN [13].

Discussion
In this study, we compared the demographic, clinical, and laboratory characteristics of 66 IgG4-RKD+ patients and 384 IgG4-RKD-patients, as well as describing the radiological and pathological ndings in patients with IgG4-RKD+. To our best knowledge, this is the largest case-control study of IgG4-RKD+ and IgG4-RKD-phenotypes.
IgG4-RD is manifested by typical clinical features, including tumor-like lesions, dense in ltration with IgG4-positive plasma cells, and extensive brosis of multiple organs. There is a great variability of disease manifestations for IgG4-RD, and the identi cation of different IgG4-RD subgroups is crucial, as a consequence of signi cant disparities in the characteristics of IgG4-RD regarding different organs [6,14]. In our study, the frequency of kidney involvement in IgG4-RD patients was 14.7%, which is lower than that of Japan (23.7%) and Mexico (24.6%) [6,15], but similar to that of UK study [11]. The heterogeneity of IgG4-RKD+ de nition and ethnicity in various studies may explain for this diversity. The following organs were found more commonly involved in IgG4-RKD+ patients, including salivary gland, lymph nodes and pancreas. In addition, multi-organ involvement was common in IgG4-RKD+ patients. Therefore, it is necessary to perform a general checkup to obtain a comprehensive view of the patients especially for patients with IgG4-RKD+.
Involvement of three or more organs, retroperitoneal brosis and low serum C3 level were risk factors for the kidney disease in IgG4-RD patients. IgG4-RD is a multi-organ immune-mediated condition that could in uence almost any organ system in the body. More involved organs may suggest the higher activity of disease. In most cases, IgG4-RKD+ is diagnosed in the context of known extrarenal IgG4-RD or active status of IgG4-RD. With progressive renal decline or detection of characteristic radiological features when evaluating extrarenal IgG4-RD, Kidney involvement became evident [13,16], which may explain for the association between involvement of 3 or more organs and IgG4-RKD+. Additionally, renal involvement may appear as an intrinsic kidney disease (IgG4-RKD+) or as a consequence of ureteric obstruction from retroperitoneal brosis (IgG4-RPF). IgG4-RPF often concentrated in the periaortic region and ureters can be entrapped, leading to hydronephrosis and renal injury. Therefore, it is reasonable that retroperitoneal brosis is a risk factor for IgG4-RKD+.
There are also some interpretations for the association of low serum C3 levels and IgG4-RKD+. The rst descriptions of IgG4 TIN was previously described as "idiopathic hypocomplementemic tubulointerstitial nephritis with extensive tubulointerstitial deposits [17]. Only about 16-34% of IgG4-RD patients have low serum complement levels, despite that hypocomplementemia is a feature of IgG4-RD. Nevertheless, more than 50% of patients with active IgG4-TIN have low concentration of complement [13,16]. Therefore, hypocomplementemia is considered an important serological feature of the disease [18]. In our study, hypocomplemenemia was found in 57.7% of IgG4-RKD+ patients, which is consistent with previous studies. Wang Rong et al found that complement activation may promote the development of IgG4-RKD+ [19]. Therefore, this study added insights into the hypocomplementemia in IgG4-RKD+ patients. Low serum C3 level was found a risk factor for the development of IgG4-RKD+. Hypocomplementaemia is not characteristic feature of most IgG4-RD patients, which often suggests the existence of IgG4-RKD+, thus scrutiny is necessary.
In previously reported studies, kidney function in IgG4-RKD+ patients varies from normal to renal failure, and the development of renal dysfunction also varies from relatively acute to slowly progressive [13,16,18,20,21]. In our cohort, the renal function was impaired manifesting as the reduced eGFR, elevated blood urea nitrogen, elevated serum creatine and abnormal speci c renal tubular function test. It could be attributed to IgG4-related TIN in the patients, or to the glomerular disease. IgG4-related TIN occurred in the vast majority of IgG4-RKD+ patients, and MGN was reported less than that. Consistent with the rates in previous studies, IgG4-related TIN occurred in all the 6 IgG4-RKD+ patients who had received renal biopsy.
In the urinalysis in IgG4-related TIN, we could nd typically mild to moderate proteinuria, as well as occasionally the presence of white blood cells [16], which was also consistent with our result. For IgG4-RD patients, it is necessary to carry out urine routine test and renal function test (both glomerular and tubular function), in order to timely detect glomerular and renal tubular lesions.
Main abnormalities on renal imaging were revealed in a total of 56 (84.8%) of IgG4-RKD+ patients: multiple low-density nodules, hydronephrosis and thickening of renal pelvic wall. Similar with previous studies, there were also some other imaging manifestations in our cohort, including diffuse patchy involvement of the bilateral kidneys and rim-like lesion of the kidney [11,22]. CT was the most common mode of renal imaging, including PET-CT, which is increasingly used. PET-CT could contribute to excluding malignancy with little radiative damage. Moreover, PET-CT is helpful for discovering the involvement of some silent lesions, however, its cost should also be taken into account.
One of the limitations of this study is its retrospective nature, indicating that some affected organs may be neglected, although most patients have undergone general examinations, including FDG-PET. Moreover, only a small number of IgG4-RKD+ cases were diagnosed based on renal biopsy. Compared to biopsy from super cial tissue, there would be higher risk of iatrogenic trauma when patients receive deep kidney biopsy, thus some patients would not accept it.

Conclusion
In summary, we have speci ed demographic, clinical, and laboratory differences between IgG4-RKD+ patients and IgG4-RKD. IgG4-RKD+ patients had older age at onset and older age at diagnosis. The male to female ratio of IgG4-RKD+ patients is signi cantly higher. The most commonly involved organs of IgG4-RKD+ patients were salivary gland, lymph nodes and pancreas. Involvement of three or more organs, retroperitoneal brosis and low serum C3 level were risk factors for the kidney disease in IgG4-RD patients. These ndings indicate potential differences in pathogenesis of these two phenotypes with or without kidney involvement.

Declarations
Ethics approval and consent to participate According to national regulations and in accordance with the Declaration of Helsinki, data protection authority and medical research ethical committee gave their approval. Written informed consent was obtained from all participants.

Consent for publication Not applicable.
Availability of data and materials The datasets generated and/or analyzed during the current study are not publicly available for ethical reasons, as well as privacy reasons but are available from the corresponding author on reasonable request.
Competing interests The authors declare that they have no competing interests.   Multivariate analysis of logistic regression.