The objective of this trial is to evaluate whether the effect of losartan potassium combined with KLX is superior to that of losartan potassium alone in DKD treatment and to obtain validated evidence for application of KLX in treatment of DKD.
This will be a double-blind randomized controlled trial to investigate the efficacy of KLX combined with losartan potassium compared with losartan potassium combined 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 a detailed history evaluation, physical examination, and laboratory testing. The enrolled participants will be randomly allocated to the losartan potassium plus KLX treatment group or losartan potassium plus placebo control group. 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.
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 will be considered to have DKD: a, high levels of albuminuria; b, diabetic retinopathy with CKD at any stage; c, microalbuminuria combined with an over 10-year course of type 1 diabetes.
- Diagnosis of the TCM syndromes of qi and yin deficiency and blood stasis 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 the chest, palms and soles; fixed pain in the chest, lower back or limbs; and numbness of the limbs. Participants who suffer from the primary signs/symptoms and at least two of the secondary signs/symptoms combined with the tongue manifestation are considered to have qi and yin deficiency syndromes and blood stasis.
- A UACR ≥ 30 mg/g/
- An 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%.
- 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 alanine aminotransferase (ALT) or aspartate aminotransferase (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 be randomly assigned to either the losartan potassium plus KLX group or the 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 Institute of 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. The drug administrator 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 (AE), 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 the KLX capsule but does not contain 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 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), calcium hydroxybenzenesulfonate, and Chinese medicine that would affect evaluation of qi and yin deficiency and blood stasis syndrome during the trial.
The primary endpoint is a 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 within 24 weeks, the percent change in 24 h UTP, the change in UACR and the total effective rate of TCM syndrome scale scores.
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 (ALT, 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 condition of each participant at every visit. Moreover, detailed information about any AEs will be recorded (including the severity, rate of incidence, and correlation with the treatment), monitored, and treated until properly resolved.
The investigators are responsible for informing and supervising the participants in taking the products strictly according to regulations. The details regarding medication intake should be reported and recorded at every visit in the CRFs. Those who take a drug dose less than 80% or over 120% of the prescribed amount would withdraw from the trial and would be considered noncompliant when calculating the results. Moreover, the specific withdrawal reasons will also be recorded in the CRFs.
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 PASS software 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 in eGFR in the expected population is assumed to be 1.38 ± 3.21 and 2.61 ± 3.16 ml/min/1.73 m2/half a year in the losartan potassium plus KLX group vs. the losartan potassium plus placebo group, respectively. Therefore, we estimate that a sample size of 105 patients assigned to each group (ratio 1:1) would be sufficient to achieve 90% power in detecting differences in 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 the 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 demographic characteristics, clinical characteristics, biochemical variables, medical history and medication history (Table 2 and Table 3), will be carried out according to the data features using an independent t test, chi-square 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 the 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. This includes all enrolled and dropout cases except excluded participants. Multiple imputation analysis will be carried out to explain the effect of missing data. Missing data for the primary outcome will be carried forward with the principle of the last observation carried forward (LOCF). PPS includes 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. SS refers to the participants who have made at least one visit and for which actual data were recorded for safety indicators.
To evaluate the effect of KLX, the data will primarily be analyzed using an intention-to-treat principle, with per-protocol analysis as a supplement. For the primary outcome, we will calculate the changes in eGFR from baseline. To compare the decline in eGFR between the two groups, a repeated-measures analysis of covariance model will be performed with baseline eGFR as the covariate. Least-square means with SEMs and two-sided 95% CIs for differences between groups will be estimated. For secondary outcomes, statistical description and comparison between groups will be performed according to whether the variables are continuous or categorical data. Specifically, the differences in classification indicators among secondary outcomes: 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 will be compared between groups using a chi-square or Fisher exact test. The difference in total effective rate of TCM syndrome scale score between the two groups will be tested as rank data using a 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 by applying independent t tests (or a Mann–Whitney U test). Safety evaluation is composed of comparison of the parameters listed above as well as the AE evaluation. In addition to the detailed description, reason and explanation of the AEs, a chi-square test or Fisher exact test will be used to compare the incidence of AEs between the two groups.
All 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 a p value < 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 and 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 administrators of Guang’anmen Hospital will visit each center regularly to ensure the quality of data collection and to facilitate problem solving. All files will be properly classified and saved/archived under confidential conditions.