This study was an open-labeled randomized controlled clinical study conducted in accordance with the ethical standards of Tanta University Research Ethical Committee, with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was prospectively registered on clinicaltrial.gov and its identification code is NCT04224428. The study record is available at https://classic.clinicaltrials.gov/ct2/show/NCT04224428.

Eligible patients were randomly assigned to the fexofenadine group and control groups. The two groups were randomized with a simple randomization method based on the hospital visit days. Patients were recruited from the hospital four days per week; 2 days for the fexofenadine group and 2 days for the control group. Patients in the fexofenadine group received ramipril plus fexofenadine 60 mg daily and patients in the control group received ramipril only for six months.

The recruitment phase started in January 2020 at Internal Medicine Department, Tanta University Hospital, Tanta, Egypt. Inclusion criteria were age ≥ 18 years, confirmed diagnosis of Type 2 diabetes mellitus at least six months prior to screening, and stage 2 or 3 diabetic nephropathies (persistent micro- or macroalbuminuria with urinary albumin creatinine ratio (UACR) > 30 mg/g) despite treatment with ramipril 10 mg daily for at least 8 weeks prior to recruitment. Exclusion criteria were Type 1 diabetes mellitus, severe renal impairment (eGFR < 30 mL/min/1.73 m2), pregnancy or lactation, chronic heart failure, malignancy, inflammatory or autoimmune disease, and history of kidney disease other than diabetic nephropathy.

Personal data were obtained from each recruited patient at the screening visit including age, gender, height, weight, and body mass index (BMI). Urine samples were collected at baseline and after six months to assess UACR, cyclophilin A, MCP-1, 8-OHdG, and PCX using enzyme-linked immunosorbent assay (ELISA) kits. Analytes were performed using human cyclophilin A ELISA Kit with catalogue No. 201-12-0673, human monocyte chemoattractant protein-1 (MCP-1) ELISA Kit with catalogue No. 201-12-0125, human 8-hydroxy-2′ deoxyguanosine (8-OHdG) ELISA Kit with catalogue No. 201-12-1437, and human podocalyxin (PCX) ELISA Kit with catalogue No. 201-12-1835.The previous kits were purchased from Sunred Biological Technology Company, Shanghai, China. Blood samples were also collected at baseline and after six months to assess fasting blood glucose (FBG), glycosylated hemoglobin (Hemoglobin A1C), and serum creatinine using standard colorimetric methods.

The estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiological Collaboration (CKD-EPI) equation as it is more accurate at higher levels of renal function and better to be used for clinical assessment of DKD [20]. Patients had regular visits every month for medication refills and to report any encountered side effects. Every month, medications administered by all patients were reviewed to exclude drugs that induce albuminuria or interact with fexofenadine.

The primary endpoint was the change in UACR, and eGFR after six months. Changes in other measured biomarkers were considered secondary outcomes.

Statistical analysis was carried out using SPSS statistical package version 28.0, May 2021, IBM corporation software group, USA. A chi-square test was used to compare categorical clinical variables between groups. The Shapiro–Wilk test was applied to the measured parameters before running a parametric statistical analysis. The normality test revealed normally distributed data. Analysis of baseline characteristics and biomarkers were analyzed using an unpaired student t-test for parametric data. Whereas a student t-test was used to compare the percent change of variables of the two groups. Correlation analysis was done using Pearson correlation where correlation coefficients were interpreted as weak (< 0.4); moderate (0.4– < 0.7), or strong relationship (> 0.7) [23]. To evaluate the association between the measured biomarkers and UACR, a linear regression test was performed.

Considering that the primary objective of this trial was to compare UACR between the two groups, we calculated the minimum number of patients needed to detect the 20% change in UACR. The lower limit of 20% UACR reduction was chosen as a cut-off point representing clinical relevance which is not likely to be subjected to variance error. The assumed mean of the control group was 275 mg/g, and the expected mean difference between the control and treatment groups was 55 mg/g. We assumed 80% power, a two-sided type I error rate of 0.05, an allocation ratio (r = 1), and a standard deviation equal to 70 mg/g. After applying a 15% dropout rate, 30 participants were needed in each arm of the trial [21].