Treatment effects of Chinese Medicine (Yi-Qi-Qing-Jie Herbal Compound) combined With immunosuppression therapies in patients of IgA Nephropathy with high-risk of ESRD (TCM-WINE): study protocol for a randomized controlled trial

Background: IgA nephropathy (IgAN) is the most common glomerular disease worldwide. It has a high incidence in Asians and is more likely to progress to end-stage renal disease (ESRD). However, for high-risk IgAN clinically characterized by massive proteinuria and renal dysfunction, there has been no international consensus on treatment options. Compared with other developed countries, Chinese IgAN patients are often found to have severe kidney function loss at the initial diagnosis. Yi-Qi-Qing-Jie Formula Granule (a compound recipe of Chinese medicinal herbs, YQF) has shown potential renal protection in our previous clinical studies. To further confirm the efficacy and safety of YQF in the treatment of high-risk IgAN, we design a prospective double-blind randomized placebo-controlled trial. Methods/Design: The TCM-WINE study is a single-center, prospective, double-blind placebo-randomized controlled trial. We plan to randomize 60 participants with biopsy-proven IgAN to YQF combined group (YQF compound, combined with prednisolone, and cyclophosphamide if necessary) and immunosuppression group (placebo-YQF, combined with prednisolone, and cyclophosphamide if necessary). The two groups will enter 48-week in-trial treatment phase and receive post-trial follow-up till study completion (3-year). All patients will receive optimal supportive care. The primary composite outcome is defined as the first occurrence of a 40% decrease in estimated glomerular filtration rate (eGFR) from the baseline lasting for 3 months, initiating continuous renal replacement treatment or death due to chronic kidney disease (CKD), during the 3-year study phase. Secondary endpoint events are defined as the mean annual eGFR decline rate (eGFR-Slope, ml/min per 1.73 m 2 per year) which is calculated by the eGFR regression curve for each eligible patient, and proteinuria remission (prescribed as proteinuria<0.5g/day) at week 24, 36, 48 during the in-trial phase. Safety monitoring and assessment will be undertaken during the study . Discussion: TCM-WINE study will

evaluate effects and safety of YQF combined therapy compared with immunosuppression monotherapy on basis of optimal supportive treatment in high-risk IgAN. The evidence from this study will provide a novel, effective and safe Chinese characteristics therapy for high-risk IgAN patients. Trial registration: Clinicaltrials.gov, identifier: NCT03418779.
Registered on 18 June 2018. https://clinicaltrials.gov/show/NCT03418779 Background IgA nephropathy (IgAN) is the most prevalent primary glomerular disease worldwide and accounts for 45% of the primary glomerular disease in China which significantly contributes to the global burden of chronic kidney disease (CKD) [1,2]. Patients with IgAN are often diagnosed as young adults, and about 30% of the patients develop end-stage renal disease (ESRD) in 10-20 years [1,3]. Proteinuria, decreased kidney function and hypertension at diagnosis are the independent risk factors for progression to ESRD.
Latest Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guideline for Glomerulonephritis recommends angiotensin-converting enzyme inhibitors (ACEI) or angiotensin-receptor blocker (ARB) with up-titration to achieve a maximally tolerated dose, as an initial therapy for progressive IgAN. For those patients with persistent proteinuria>1g per day and estimated glomerular filtration rate (eGFR) >50mL/min/1.73m 2 , a 6-month course of high-dose corticosteroid therapy is suggested despite 3-6 months of optimized supportive care (ACE inhibitor, ARB, or both and bloodpressure control). Intensive immunosuppression [corticosteroid with cyclophosphamide (CTX) or azathioprine] is reserved for patients with crescents in more than half of the glomeruli and a rapidly deteriorating renal function [8]. A meta-analysis has shown that IgAN patients with more serious pathological features may be more resistant to steroid therapy than slight ones according to the earlier outlined Oxford classification system [9].
However, the benefits of systemic immunosuppression have been questioned in recently completed trials. Conflicting results indicate the presence of severe adverse events (SAEs), which imply long-term immunosuppression must be balanced against benefits carefully [10][11][12]. Other general immunosuppressants, such as mycophenolate mofetil [13,14], calcineurin inhibitors [15], and targeted immunosuppression regimens, like rituximab [16], targeted release formation -budesonide [17], the selective enzyme spleen tyrosine kinase inhibitor fostamatinib which was recently completed in a Phase Ⅱ randomized controlled trial (RCT) (ClinicalTrials.gov, NCT02112838), are waiting for more high-quality trials to access their safety and efficacy. Above all, for high-risk IgAN, a safe, disease-specific therapy remains limited.
Chinese medicine is gradually accumulating evidence-base application in CKD, which has been widely approved for its positive role in the prevention and treatment of IgAN [18][19][20][21]. Yi-Qi-Qing-Jie Formula (YQF) was developed from the classical Chinese prescription Yupingfeng San Yinqiao San and Wuweixiaodu Yin, the main constituents including Astragali radix (HUANG QI), atractylodes rhizome (BAI ZHU), Radix Saposhnikoviae (FANG FENG), oldenlandia diffusa (BAI HUA SHE SHE CAO), Rhizoma Dioscoreae Nipponicae (CHUAN SHAN LONG), and raw rhubarb (DA HUANG). We have conducted an ambispective cohort study [22]. According to serum creatinine, 24h urine total protein (UTP), etc. before treatment, age, sex, and groups, using a propensity score, 34 high-risk IgAN patients with UTP>3g/24h and eGFR<60ml/min/1.73m 2 , who received YQF combined therapy (treatment group) were matched to 34 patients who received immunosuppression monotherapy (control group) from Peking University First Hospital nephrology department, on basis of reninangiotensin system blockade (RASB) ( Table 1). In conclusion, YQF combined therapy exhibited a potential renal protective effect during the mean follow-up period of 43 months. 5 patients (14.71%) reached ESRDTable 2 and Figure 1) and there were no SAEs associated with immunosuppressants. In Mitsuiki's study, which was similar to our treatment protocol, 6 patients (22%) treated with prednisolone and cyclophosphamide reached ESRD during the mean follow-up period of 66.5 months, and 2 patients (7.4%) suffered adverse effects of immunosuppression during treatment [23].
However, the study did not use a standardized clinical study design (cases included were non-contemporaneous), the sample size was small and the observation period was shorter.
We will conduct a randomized, prospective, double-blind (placebo) controlled trial to confirm that, compared with immunosuppressive therapy alone, YQF combined with immunosuppressive therapy will be superior in renal function protection and reducing the severe treatment-related adverse effects in patients with high-risk IgA nephropathy.

Methods/design
Study design This is a single-center, prospective, double-blind, placebo-randomized controlled trial. This clinical trial is reported according to the Standard Protocol Interventions: Recommendations for Interventional Trials (SPIRIT) guidelines [24]study schedule (SPIRIT figure) is outlined in Figure 2, checklist see Additional file 1].

Setting and participants
Sixty high-risk IgAN participants will be followed up until 50% (30 of them) have a composite endpoint or been followed for 3 years. The trial will be conducted at Guang'anmen Hospital, Beijing, China, and was approved by the Ethics Committee of

Objectives
The trial is aiming to access superiority in renal protection and reducing severe treatmentrelated adverse events using YQF combined therapy compared with immunosuppression monotherapy based on optimal supportive care in high-risk IgAN.

Rationale of high-risk IgAN
Several high-quality clinical trials mentioned above [11,12] have respectively interpreted the concept of "high-risk" clinically and pathologically while not specific. Drawn from inclusion criteria of above-mentioned trials, this study will define "high-risk" as a persistent heavy proteinuria (≥1g/d despite intensive optimal supportive care) with impaired renal function (eGFR 15-60 ml/min/1.73m 2 ).

Inclusion and exclusion criteria
The inclusion criteria are as follows: (1) in accordance with IgAN pathological diagnosis, renal biopsy within 6 months; (2) a persistent proteinuria≥1g/d despite at least 8 weeks of optimal supportive care (maximally tolerated RAS blocker which refers to no symptomatic hypotension, no hyperkalemia, and serum creatinine (SCr) increase not more than 30% of baseline, blood pressure control meeting targets, and diet management); (3) eGFR 15 to The exclusion criteria are as follows: (1) secondary IgAN; (2) comorbidity of other primary or secondary glomerular diseases; (3) comorbidity of severe primary diseases such as cardiovascular, hepatic, cerebral, hematopoietic system diseases and mental disorders; (4) allergy or intolerance to the experimental medication (e.g., RAS blockers, prednisolone, cyclophosphamide, YQF compound and its placebo compound) ; (5) contraindications of immunosuppression therapy: acute and chronic infectious diseases, malignancies, leukopenia, thrombocytopenia, gastrointestinal hemorrhage, ulcers of stomach or duodenum, post-transplantation; (6) pregnant or lactating women; (7) unwilling to participate in this study, failure to accept or tolerate Chinese medicine compound; (8) a history of alcohol or drug abuse; (9) poor compliance, loss to follow-up from the study; (10) participation in another clinical investigation.

Randomization and masking
In the case that the subjects are eligible to participate and have signed informed consent, 30 patients in each group of the YQF group and the control group will be randomly

Interventions
Run-in period (pre-trial, 4 weeks) Eligible participants will enter a run-in period for 4 weeks, during which they will receive an optimized basic treatment, including living behavior management (smoking cessation, alcohol restriction, weight control, low-salt and proper protein diet), maximum tolerated dose of ACEI or ARB, blood pressure control to a target below 130/80mmHg, controlling glycated hemoglobin≤7% by using insulin or oral hypoglycemic agents in diabetics,

Sample size calculation
Based on our recent trial from above [22] of which the primary end point was eGFR-Slope after 12-month treatment, we define δ = (μ1-μ2)/σ, σ as the pooled standard deviation (sample size calculation, see Additional file 4), where μ is the treated mean, respectively.
We calculate δ = 0.994 which can be approximately equivalent as 1

Statistical analysis
Investigators will fill the Case Report Forms as making follow-up observations. Data analysis of treatment effect and safety follows the intention-to-treat principle. The endpoint events will be described by Kaplan-Meier method and the between-group difference will be compared using log-rank test. Chi-square test will be used to compare the rates, t-test or variance analysis will be used for comparison between data sets. The eGFR-slope will be calculated as the individual slopes obtained from individual linear regressions of eGFR during the follow-up period. All analyses were performed using SPSS software version 23.0 (IBM Analytics). Differences were considered to indicate statistically significant for 2-sided P < 0.05.

Quality control Data monitoring
The Clinical Pharmacological Research Base, Scientific Research Department and the Ethics Committee of Guang'anmen Hospital will review the study data regularly and monitor compliance with the protocol until completion. The Clinical Pharmacological Research Base will assess that participants receive good clinical care and that safety concerns are interpreted and addressed appropriately.
Intention-to-treat analysis will be used for subjects who dropout or withdraw from the study which includes every subject who is randomized according to randomized treatment assignment regardless of whether they receive the treatment of this group or not, should finally be included in the assigned group for statistical analysis of efficacy.
Last observation carried forward method will be applied in missing data of longitudinal observations.

Discussion
IgAN is considered to be an autoimmune disease, it is logical that immunosuppression therapy may be effective [10,26]. As the therapeutic benefits of intensive supportive care show, it's the cornerstone of treatment in IgAN. Recently completed high-profile RCTs with rigorous standardization and optimization of supportive management have focused on outcomes of additional immunosuppression. STOP-IgAN trial [11] showed that the additional immunosuppression therapy (oral corticosteroids plus CTX and azathioprine) did not exert substantial renal protection for those with eGFR 30-59 ml/min/1.73m 2 , including one death due to sepsis related to steroids. However, the trial excluded patients with 24hUTP >3.5g, eGFR <30 ml/min/1.73m 2 , or eGFR decreased more than 30% at the end of 6-month run-in phase which often underlies relatively high risk in disease progression and a better response to steroids. In TESTING study [12], after a 3-month run-in phase of optimal supportive treatment, recruiting participants with 24hUTP >1g and eGFR of 20-120 ml/min/1.73m 2 , was terminated because of two deaths of infection and excess SAEs in methylprednisolone group. Early results could not demonstrate definite treatment efficacy in steroid therapy though they have shown indication in short-term potential renal benefits. Both trials highlight the safety concerns about immunosuppression and necessary to seek for alternative, safe and effective approach for high-risk IgAN.
Traditional Chinese medicine has initiative proved promising effects regarding safety and renoprotection with some RCTs in chronic kidney diseases, for instance, Abelmoschus manihot [27], however, it is notable that this trial excluded patients with impaired renal function (eGFR <60 ml/min/1.73m 2 ) and nephrotic syndrome. Therefore, we conduct TCM-WINE, a prospective, double-blind, single-dummy, randomized controlled trial, aiming to evaluate safety and long-term outcomes with YQF herbal compound compared to immunosuppression monotherapy in treating high-risk IgAN. In conclusion, the results of TCM-WINE will provide clinical evidence for the efficacy and safety of YQF compound, moreover, this trial will serve as an important step toward new era of treatment in highrisk IgAN.