The objective of this trial is to evaluate whether the effect of losartan potassium combined with KLX is superior to that of losartan potassium in DKD treatment and to obtain validated evidence for the application of KLX in the treatment of DKD.
This will be a double-blind randomized controlled trial to investigate the efficacy of KLX combined with losartan potassium compared with a placebo. The study will be conducted in eighteen centers in China: Guang’anmen Hospital of the China Academy of Chinese Medical Sciences, Dongzhimen Hospital of Beijing University of Chinese Medicine, the First Affiliated Hospital of Chengdu Medical College, Mianyang Central Hospital, the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Dazhou Central Hospital, Dezhou People’s Hospital, Linfen Central Hospital, the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, the First People’s Hospital of Luoyang, Shenzhen Hospital of Southern Medical University, Shanghai Shuguang Hospital, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, the Second Affiliated Hospital of Wannan Medical College, Nanyang City Central Hospital, the First Affiliated Hospital of Henan University of Chinese Medicine, Xinxiang Central Hospital, and Heze Municipal Hospital. Patients with DKD who want to participate in the study will undergo a standardized baseline evaluation before treatment including detailed history evaluation, physical examination, and laboratory testing. The enrolled participants will be randomly allocated to the losartan potassium plus KLX treatment or losartan potassium plus placebo control groups. The participants will be administered KLX or placebo in addition to losartan potassium for 24 weeks. Data will be collected during three visits to assess the efficacy of KLX and will be recorded on case report forms (CRFs). A flowchart of the study design is shown in Figure 1, and the time points of assessment are shown in Table 1. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines  were followed during the development of the protocol of this study.
Study settings and recruitment
Assuming a 20% dropout rate, a total of 252 patients will be recruited from the eighteen centers mentioned above using posters, the hospitals’ websites, and networks from December 2019 to December 2020. Research assistants will manage the recruitment, and endocrinologists will diagnose the participants. On the consent form, participants will be asked for permission to apply their data in case of withdrawal. Participants will also be asked to agree to share the research data with people from the hospitals participating in the study or from regulatory authorities where relevant. Biological specimens are not stored in this study. The trial adopted clinical trial insurance, and affected participants will receive appropriate coverage if serious harm occurs following the trial.
Subjects will be deemed eligible participants if they meet all of the listed inclusion criteria and none of the listed exclusion criteria.
- Diagnoses of type 2 diabetes based on the American Diabetes Association (ADA) criteria  and DKD defined by National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF K-DOQI) guidelines . Participants who meet any of the following criteria can be considered as DKD: a, large amounts of albuminuria; b, diabetic retinopathy with chronic kidney disease at any stage; c, microalbuminuria combined with a greater than 10-year course of type 1 diabetes.
- Diagnoses of TCM syndromes of qi and yin deficiency and blood stasis were constructed based on Diagnosis and Treatment of Diabetes and Its Complications with Integrated Chinese and Western Medicine as follows: Primary signs/symptoms include dry mouth and throat, and fatigue. Secondary signs/symptoms include a propensity to be hungry and eat more; shortness of breath, heat sensation in chest, palms and soles; fixed pain in the chest, lower back or limbs; and numbness of the limbs. Participants who suffered from the primary signs/symptoms and at least two of the secondary signs/symptoms combined with the tongue manifestation are considered to exhibit syndromes of qi and yin deficiency and blood stasis.
- A UACR≥30 mg/g;
- An estimated glomerular filtration rate (eGFR, according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation) ≥ 30 ml/min/1.73 m2 and ≤ 59 ml/min/1.73 m2;
- A fasting blood glucose level < 13.9 mmol/L and/or a 2 h postprandial blood glucose level < 16.6 mmol/L;
- A glycated hemoglobin (hemoglobin A1c, HbA1c) percentage ≤ 10%;
- An age between 35 and 75 years, male or female;
- Voluntary participation in this clinical study and provision of written informed consent.
- A diagnosis of type 1 diabetes;
- A lack of diagnosed diabetic retinopathy;
- A history of simple renal hematuria or proteinuria with hematuria; sudden onset of edema and mass proteinuria without abnormal renal function; significant renal tubular dysfunction; coexisting renal tubular abnormalities; primary glomerulonephritis or secondary nephritis except DKD; or acute or chronic infection of the urinary system;
- A hemoglobin (HGB) concentration < 90 g/L;
- An ALB concentration <30 g/L;
- A diagnosis of renal artery stenosis;
- A serum potassium concentration >5.5 mmol/L;
- A serum creatinine (Scr) concentration ≥ 3 mg/dL (256 μmol/L);
- A history of severe cardiovascular or cerebrovascular disease within three months before screening;
- Severe systemic primary disease or dysfunction;
- Severe liver dysfunction or ALT or AST levels higher than 2.5 times the normal range;
- Hypertension treated with more than 3 types of antihypertensive drugs, blood pressure that is poorly controlled after administration of drugs with systolic blood pressure (SBP)≥140 mmHg or diastolic blood pressure (DBP)≥90 mmHg, or hypotension with SBP≤90 mmHg or DBP≤60 mmHg (resting position);
- Chronic (for more than 3 weeks) or recurrent diarrhea (where diarrhea is defined as elimination of increased volumes of thin fecal matter with increased frequency (>3 times/day);
- A history of active bleeding in the 3 months before screening;
- Coagulation disorders;
- Drug allergies or allergies to KLX or the active ingredient in losartan potassium;
- A history of alcohol or drug abuse or psychiatric disorders;
- Pregnancy, lactation or plans to become pregnant during the trial;
- A history of enrollment in other clinical studies in the past 3 months;
- Other circumstances under which the investigators considered it inappropriate to participate in this clinical study.
Randomization, allocation concealment, and blinding
The participants will have an individual trial identification number and be randomly assigned to either the losartan potassium plus KLX group or losartan potassium plus placebo group in a 1:1 ratio. Randomization will be performed using Statistics Analysis System (SAS) software by an independent statistical agency. The Data Management Center of Guang’anmen Hospital will be responsible for drug blinding and randomization concealment. The statistician will not participate in the randomization and will analyze the collected data without having access to allocation information. Research assistants will enroll patients at the 18 participating medical centers sequentially on the basis of screening order. Both the participants and the investigators will be kept blinded to the allocations until the trial is completed. In case of a severe adverse event, an administrator at the Data Management Center of Guang’anmen Hospital will unblind the patient information as an emergency measure and provide appropriate treatment.
All investigators will be trained before the start of the study according to an investigators’ brochure. The enrolled patients with DKD will be asked to take losartan potassium tablets (50 mg per day) as the basic treatment and will also be administered KLX or placebo. The subjects in the treatment group will take four capsules of KLX orally thrice a day for 24 weeks. The participants in the control group will take the same amount of placebo for 24 weeks. Data from all the participants will be collected in four periods: on day 0 (V2), at 4 weeks ±3 days (V3), at 12 weeks ±5 days (V4), and at 24 weeks ±5 days (V5).
KLX consists of six natural herbs: Astragalus membranaceus, Ligustrum lucidum, leech, rhubarb, Radix Pseudostellariae and the fruit of the Chinese wolfberry. The placebo is identical in appearance and properties to KLX capsule without any active ingredient. Both KLX and the placebo will be manufactured by Chengdu Kanghong Pharmaceutical Co., Ltd., at a dose of 500 mg. The placebo, which will be identical in appearance and properties to KLX, will contain no active ingredients. The placebo will be verified by the quality control department and will meet the standards of placebo preparation. The interventions should stop for a given participant if the creatinine doubles or ESRD is detected. The participants will be asked to refrain from taking any other treatment for diabetic nephropathy, including ACEI or ARB drugs other than losartan potassium tablets and calcium hydroxybenzenesulfonate as well as Chinese medicine that affect evaluation of qi and yin deficiency and blood stasis syndrome during the trial.
The primary endpoint is the decline in the eGFR (ml/min/1.73 m2/year).
The secondary outcomes will include the incidence of Scr doubling, the incidence of ESRD, the proportion of subjects with a progressive decline in eGFR of >30% from baseline, the percent change in 24-h UTP, the change in UACR from baseline and the total effective rate of TCM syndrome scale scores at every visit and follow-up point, including the 4th, 12th, and 24th weeks as well as the 6th and 12th months.\
To assess the safety of KLX treatment in patients with DKD, vital signs and some laboratory parameters will be measured and electrocardiography will be performed during the intervention period of the trial. In detail, the vital signs will include body temperature, blood pressure, respiratory rate and pulse rate. The biological indicators we will monitor will include routine blood indices (red blood cell count (RBCs), hemoglobin(HGB), white blood cell count (WBCs), and platelet count (PLTs)), routine urine and urine sediment parameters (assessed under microscopy), 24-h UTP levels, UACRs, liver function indices (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL), and γ-glutamyl transferase (GGT)), renal function indices (eGFR and Scr), serum electrolytes (K+, Na+, and Cl-), blood lipid profile indices (total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)), coagulatory function indices (prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB)), HbA1c levels, and fasting blood glucose levels. To reduce risk, vital signs and some laboratory parameters of the participants will be monitored during the intervention period of the trial. Clinal experts will evaluate the participants’ condition at every visit. Moreover, detailed information about any adverse events will be recorded, including the severity, rate of incidence, and correlation with the treatment. Adverse events will be monitored and treated until properly resolved.
The investigators are responsible for informing and supervise the participants to take the products strictly according to regulations. The details about medication should be reported and recorded at every visit in the case report forms. Those with drugs doses less than 80% or greater than 120% of the prescribed amount will be withdrawn from the trial and considered as noncompliance. Moreover, the specific withdrawal reasons should be recorded in CRFs as well.
All included participants will be re-evaluated at 6 and 12 months through phone calls or as outpatients.
This study will be a trial of a new therapeutic regimen of KLX treatment. The sample size was calculated using software PASS based on a similar study on a Chinese herbal medicinal supplement conducted previously [20,21]. According to the results of our previous trial, the mean decline of eGFR in the expected population was assumed to be 1.38±3.21 and 2.61±3.16 ml/min/1.73 m2/half a year in losartan potassium plus KLX vs. losartan potassium plus placebo group, respectively. Therefore, we estimated that with a sample size of 105 patients assigned to each group (ratio 1:1) would be sufficient to achieve 90% power in detecting the differences of the primary endpoint based on a two-sided level of significance of 5%. Allowing for a 20% withdrawal rate, we plan to include 126 patients in this trial.
The independent researchers performing the biochemical tests, assessing the clinical outcomes, and analyzing the data will be kept blinded to the patients’ identities and grouping.
Continuous indicators are described as means, standard deviation, medians and interquartile ranges, while classification variables are reported as absolute values and percentages. The following aspects should be analyzed at the end of the trial: the actual number of participants in the two groups, dropped and excluded cases, baseline characteristics, compliance, efficacy and safety. To insure the comparability between groups, baseline comparisons, including the indicators of demographic characteristics, clinical characteristics, biochemical variables, medical history and medication history (Table 2 and Table 3), were performed according to the data features by independent t test, chi-squared test or Fisher exact test.
Full Analysis Set (FAS), Per Protocol Set (PPS) and Safety Set (SS) are to be used for data analysis. According to intention-to-treat principle, FAS is defined as analysis of all cases who met all the inclusion criteria and none of the exclusion criteria, were randomized and had at least one dose of the study drug. It included all enrolled and dropout cases except excluding participants. Multiple imputation analysis will be carried to explain the effect of missing data. Missing data of the primary outcomes will be carried forward with the principle of the last observation carried forward (LOCF). PPS includes the data from the subjects who follow the test protocol strictly, have good compliance, do not take the prohibited drugs during the test period and complete the CRF form. SS refers to the participants who have made at least one visit and actual data recorded for safety indicators.
To evaluate the effect of KLX, data were analyzed primarily using an intention-to-treat principle, and per-protocol analysis served as a supplement. For the primary outcomes, we will calculate the changes of eGFR from baseline. To compare the decline in eGFR between two groups, repeated-measures analysis of covariance model will be performed using baseline eGFR as covariates. Least-square means with SEMs and two-sided 95% CIs for differences between groups were estimated. For secondary outcomes statistical description and comparison between groups will be performed according to whether the variables are continuous or categorical data. To be specific, the difference in classification indicators among secondary outcomes, including incidence of Scr doubling, the incidence of ESRD and the proportion of subjects with a progressive decline in eGFR of >30% from baseline within 24 weeks, between groups will be compared using chi-square or Fisher exact test. While the difference in the total effective rate of TCM syndrome scale scores between the two groups will be tested as rank data using Wilcoxon test or CMH test. Comparison of the continuous variables, such as the percent change in 24-h UTP and the change in UACR will be performed applying independent t tests (or the Mann–Whitney U test). Safety evaluation is composed of comparison of parameters listed above as well as adverse events evaluation. Besides the detailed description, reason and explanation of the adverse events, the chi-square test or Fisher exact test will be used to compare the incidence of AEs between the two groups.
There will be no interim analysis of the data because there are no anticipated problems that are detrimental to the participants. All the data will be analyzed using Statistical Package for the Social Sciences (SPSS) version 17.0 (SPSS, Inc., Chicago, IL) software. All statistical tests will be two-sided, and significance will be considered for p-values <0.05.
Data collection and management.
All researchers in this study will be qualified physicians and will receive training in standard operating procedures for trial execution, biological sample collection, and handling. Based on the initial observation records, all center investigators will complete the CRFs in an accurate and timely manner. The sponsor and administrators of Guang’anmen Hospital will visit each center regularly to ensure the quality of data collection and to facilitate problem solving. All data collected will be properly classified and saved/archived under confidential conditions and only accessed by researchers of the trial team. The Institute of Data Management Center of Guang’anmen Hospital is responsible for inspecting whether the trial is performed in compliance with the relevant regulations, GCP, and trial protocols.
The study is investigator sponsored and will be monitored and audited by the administrators of Guang’anmen Hospital to ensure that the trial to be conducted follows Good Clinical Practice principles. The administrators of Guang’anmen Hospital are also responsible for all aspects of local organization. The trial management committee (TMC) is composed of investigators from different research centers and administrators of Guang’anmen Hospital. The TMC will meet every three months during the study. The committee will provide guidance on day to day support for the trial, identify potential recruits, monitor the trial procedures, and report information to the Trial Steering Committee (TSC). The TSC, which is independent from the TMC, will meet at least every 6 months to supervise the trial and offer guidance to ensure proper study conduct and progress. The Data Management Center and Ethics Committee of Guang’anmen Hospital will meet every 6 months to review conduct throughout the trial period. Supplementary Table 1 (S1) provides a description of the roles for the study groups that are responsible for trial management and monitoring
If any amendments to the study are needed, the amendment will be agreed upon by the TMC and approved by the Sponsor. Then, the administrators of Guang’anmen Hospital will notify the centers of the changes to the protocol. Any protocol changes will be fully documented using a breach report form, and the protocol will be subsequently updated in the clinical trial registry.