• To summarize the treatment patterns in daily practice for IMN patients in China, and to evaluate the effectiveness in terms of clinical remission among IMN patients who receive different treatment regimens, including CHM, conventional drugs, as well as the combination of both therapies.

• To identify the potential biomarkers for IMN population and/or subgroup population with specific characteristics.

This is a multicenter prospective hospital-based cohort study, and has been registered with the Chinese clinical trial registry. (ChiCTR2000033680).

In order to collect a representative sample, the nephrology departments in 11 tertiary-care hospitals across multiple regions in China will recruit and enroll patients continuously. The 11 study sites include Guangdong Provincial Hospital of Chinese Medicine, Guangdong Provincial Hospital of Integrative Medicine, The First People’s Hospital of Zhaoqing, Liuzhou Hospital of Chinese Medicine, and Yue Bei People’s Hospital in south China; Hubei Provincial Hospital of Traditional Chinese Medicine in southwest China; Jiangsu Provincial Hospital of Chinese Medicine and the First Affiliated Hospital of Anhui University of Chinese Medicine in east China; Heilongjiang Institute of Traditional Chinese Medicine and First Affiliated Hospital of Heilongjiang University of Chinese Medicine in northeast China; Shanxi Traditional Chinese Medicine Hospital in northwest China.

Eligible candidates for the study are IMN patients of any risk levels to capture a broad spectrum of clinical presentations. Inclusion criteria include Chinese adults aged from 18 to 85 years old; diagnosis of biopsy-proven IMN or antibodies against phospholipase A2 receptor (PLA2R)-positive patients who are not associated with other diseases which cause secondary MN, such as systemic lupus erythematous, infections with hepatitis B virus, hepatitis C virus, or syphilis, sarcoidosis, and cancer. Patients with the following criteria will be excluded: patients with a GFR less than 15 ml/min/1.73 m²; patients with comorbidities including malignant cancers, decompensated cirrhosis, hematologic diseases, and mental diseases.

This observational study is designed to evaluate the effectiveness of CHM in a real-word hospital setting. Therefore, there are neither treatment protocols nor interference with therapeutic schedules for the included participants. But the information of therapeutic regimens will be collected to identify the exposure of CHM. Exposure of CHM includes either oral or intravenous administration proprietary CHM products and oral administration CHM formulas. Low, moderate, and high level of CHM exposure will be defined as the cumulative exposure time of CHM accounts for 25, 50, and 75% of individual follow-up time. The individual follow-up time will be from the baseline visit to the last visit or the end of the study.

This study is designed to be a long-term longitudinally extensible cohort. The study aims to include 2000 Chinese IMN adults. The recruitment is estimated to take 42 months, from July 2020 until December 2023. The patients who meet the inclusion criteria will be enrolled continuously. Both incident patients and prevalent patients will be recruited by physicians in nephrology clinics and inpatient departments in 11 participating sites. Incident patients are individuals who are newly diagnosed and first initiated on treatments. Prevalent patients are individuals who have already been diagnosed as IMN and have been receiving treatments before the study. Meanwhile, the electronic medical record in 11 participating sites will be retrospectively screened to recruit the confirmed cases.

Individuals who are eligible and consent to participate will be invited to attend a baseline visit. Participants will be followed for up to at least 2 years. Participants who experience the primary endpoint (remission) will be continued to be followed up until the end of the study or death. The schedule of follow-up visits is presented in Figure 1. Follow-up visits will occur quarterly in the first year after enrollment and then semi-annually from the second year. The baseline, semi-annual, and annual follow-up visits in the first year and annual visits from the second year are required in-person visits. All other visits can be in-person or remote via telephone call or smartphone applications. In order to retain participants and minimize the number of dropouts and withdraws as well as the missing data, a reasonably flexible visit time window within a range of 4 weeks forward or backward from the scheduled visit day will be acceptable.

The data elements of the personal domain (demographics, medical history, Chinese medicine specific information including symptoms and tongue images, etc.), the exposure domain (treatment prescriptions of CHM and conventional drugs), and the outcome domain will be recorded. Each participant will have a unique number, and their personally identifiable information will not be collected. Both spot urine specimen and 24 h urine specimen will be collected to assay for red blood cells, white blood cells, urinary protein-to-creatinine ratio, and 24 h total urinary protein. Fasting blood draw will be tested for complete blood cells (red blood cell, hemoglobin, white blood cell, neutrophil, leukomonocyte, and platelets), metabolic panel (blood glucose, potassium, albumin, aspartate aminotransferase, alanine aminotransferase, uric acid, creatinine, total cholesterol, triglycerides, and low-density lipoprotein), and PLA2R. GFR will be estimated using the 2009 Chronic Kidney disease Epidemiology Collaboration (CKD-EPI) equation based on serum creatinine (Levey et al., 2009). Recently, a new equation based on both serum creatinine and cystatin C, which is also proposed by CKD-EPI, will be used to estimate GFR as well (Inker et al., 2021). Patient report outcome measures including the Kidney disease Quality of Life-36 which will be used to assess quality of life. At the baseline and each annual visit, a blood sample and a urine sample will be collected for cryopreservation, which will be used for detection of disease specific biomarkers in further research phases. Additionally, if participants are newly diagnosed by kidney biopsy, the kidney biopsy tissue will be cryopreserved as well. The scheduled time points for data collection are summarised in Figure 1.

The data of the core Chinese medicine syndrome of dampness in IMN will be collected by using the validated Dampness Syndrome Scale (Lu et al., 2021) and the tongue image acquisition system. The scale consists of 30 items. There are five options for each item from 0 to 4 which indicate no symptoms, mild, moderate, heavy, and very heavy symptoms. The total score ranges from 0 to 120. More than 15 items with at least one point can be defined as dampness syndrome. The higher the score, the more severe the dampness syndrome. Tongue images will be captured by using a smartphone tongue image acquisition application with a standard color correction chart (Figure 2) and quantified the tongue characteristic by using a computerized tongue image analysis system (Shanghai Daosh Medical Technology Co., Ltd.). (ISO, 2019; ISO/TS, 2020).

All data will be collected and recorded using electronic data collection (EDC). A systematic training is required for the personnel in all participating sites before the commencement of recruitment.

The primary outcome is clinical composite remission at 24 months after enrollment. Composite remission is defined as either complete remission (CR) or partial remission (PR). Secondary outcomes are CR, PR, time to remission (CR, PR, and CR or PR), no response, relapse, urine protein excretion, urinary protein creatinine ratio, serum albumin, annual change of GFR (GFR slope), PLA2R, adverse events, and composite endpoint of 40% reduction of GFR, doubling of serum creatinine, end-stage renal disease (ESRD), and death. The outcome variables and definitions are presented in Table 1.

The composite remission rate in Chinese adults with IMN under treatment of immunosuppressive agents in clinical practice in a single center in China was 73.7% (Jiang et al., 2021). An analysis of our retrospective unpublished data from three hospitals in China showed that composite remission rate of IMN patients received CHM alone or combination therapies in clinical practice was 67.0% (75/112). Under the assumption of 10% of loss to follow-up and two-sided alpha level of 0.05, enrolment of 2000 patients would provide 80% power to detect the 2-year composite remission rate.

Attempting to ensure equivalence in baseline characteristics of participants between the CHM group and the conventional therapy group, a propensity score method will be performed. Variables involving age, sex, proteinuria, eGFR, serum albumin, uric acid, PLA2R antibody level, and pathological type will be included into the logistic regression model to calculate a propensity score for each participant. Participants who are exposed to CHM will be matched to those who receive conventional drugs alone based on the propensity score, using the greedy matching with a standard calliper width of 0.2 and without case replacement. Covariate balance of the matched cohort will be assessed using the mean standardized differences. When a |d| > 0.10, it is considered as an imbalance. Inverse probability of treatment weighting based on the propensity score will also be performed to evaluate the robustness of the matching results.

Baseline demographic variables will be presented as means, medians, or percentages, and using Chi-square test, independent t test, or Mann-Whitney U test, as appropriate. Outcomes of composite remission, CR, PR, no response, death, relapse, and composite endpoint will be analysed by using Chi-square test and logistic regression. Odd ratios and 95% confidence intervals will be estimated. Time to remission will be analysed by using Cox regression model, adjusting for gender, age, baseline proteinuria, GFR, PLA2R antibody level, uric acid, Chinese medicine syndrome, and propensity score.

Subgroup analysis will be performed on age, gender, diagnosis criteria (i.e., biopsy proven or anti-PLA2R positive), PLA2R positive or negative, blood lipid levels, different risk levels of progressive loss of kidney function, “incident patient” and “prevalent patient”, Chinese medicine syndromes, different assay methods for serum creatinine and proteinuria, different levels of CHM exposure, treatment regimens (CHM alone, conventional medication alone and the combination), conventional medications, and CHM with and without immunosuppressive effect. For the analyses of Chinese medicine syndromes, besides Chi-square test between groups of patients with and without dampness syndrome, a trend test will be performed to assess the dose-response relationship between dampness syndrome and clinical outcomes. CHM with immunosuppressive effect will be defined as either herbal formulas or proprietary CHM products which contain either Tripterygium wilfordii Hook. f, Tripterygium hypoglaucum (Levl.) Hutch and Sinomenium acutum (Thumb.) Rehd. et Wils (Menispermaceae, SA) or their compounds (Zhao et al., 2012; Chen et al., 2014; Pei et al., 2016; Lv et al., 2019; Zheng et al., 2019).

GFR will be estimated by 2009 CKD-EPI creatinine equation and 2021 CKD-EPI creatinine-cystatin C equation. As the 2021 equation has not yet been validated in the Chinese population, analyses involved eGFR will use the estimates calculated by the 2009 equation, and sensitivity analyses based on the 2021 equation will be performed. To eliminate the acute effect, incident patients who experience a serum creatinine increase of more than 30% within 12 weeks after initiated treatments will be excluded in analyses on endpoints in terms of ESRD, 40% eGFR reduction, and eGFR slope.

Additionally, in order to assess the risk of performance bias, the change of prescription pattern before and after the commencement of the study will be analyzed.

All analysis will be tested with a two-sided alpha level of 0.05. For the missing data, the reasons why the data is missing will be analyzed, and multiple imputation will be used.

This study has been approved by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (BF 2020-094-01) on June 3, 2020. A copy of ethical approval documents will be provided to the local research ethics boards of each participating site. The study team will follow the study standard operating procedures to ensure that all participants’ consent will be informed and voluntary. The participants have the right to withdraw at any time, and they will be given a copy of signed informed consent forms.

As this is an observational study, there are no financial and other competing interests for principal investigators for the whole study and each participating site, as well as the funding agencies.

Important protocol modifications, such as, changes to eligibility criteria, outcomes, and participating sites, will be submitted to the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine for approval. An updated version will upload into the Chinese clinical trial registry after the approval.

Results of the study will be published in both national and international peer-reviewed journals and presented at scientific conferences. Investigators will inform the participants as well as other IMN patients of the findings via health education.