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This study was approved by the Ethics Committee of the Hebei General Hospital (Approval No.: 2025-LW-0265). Due to the retrospective nature of the study, ethics approval was obtained after the data collection period, which was permitted by the committee for non-interventional studies using anonymized clinical data. The requirement for written informed consent was waived.
Participant selection
Potential participants were recruited from the Department of Nephrology at Hebei General Hospital between July 2022 and May 2024. All patients were screened according to predefined inclusion and exclusion criteria.
The inclusion criteria were as follows: diagnosis of diabetic nephropathy (DN) at Mogensen stage III23, defined as persistent albuminuria with a urinary albumin-to-creatinine ratio (UAlb/UCR) of 30–300 mg/g in at least two of three spot urine samples collected over 3–6 months, without hematuria or rapidly declining renal function suggestive of non-diabetic kidney disease; estimated glomerular filtration rate (eGFR) ≥45 mL/min/1.73 m2 calculated using the CKD-EPI equation; first-time diagnosis of DN with no prior vitamin D, probucol, or immunosuppressant therapy for DN; age <75 years; and clinically stable condition without acute diabetic complications such as diabetic ketoacidosis or hyperglycemic hyperosmolar state, or active serious infections within 4 weeks before enrollment. Kidney biopsy was not required for enrollment; however, patients with suspected non-diabetic kidney disease, including active urine sediment, rapidly progressive glomerulonephritis, or systemic autoimmune disease, were excluded.
The exclusion criteria included serious systemic diseases such as malignancies, severe cardiovascular disease, or autoimmune diseases; known allergy to vitamin D, probucol, valsartan, atorvastatin, or insulin; pregnancy or lactation; and recent use of medications within 4 weeks that could interfere with study outcomes, including immunosuppressants or corticosteroids.
Group allocation
The screening physician remained blinded to final group allocation until eligibility confirmation.
Baseline demographic and clinical characteristics, including age, sex, duration of diabetes and DN, and history of hypertension or hyperlipidemia, were recorded.
This study used a retrospective, non-randomized controlled design. To minimize selection bias and address potential confounding factors, including temporal changes and physician-related variability, group allocation was based on chronological admission order rather than physician or patient preference.
Patients admitted between July 2022, and December 2023 received standard triple therapy consisting of valsartan, atorvastatin, and probucol and were assigned to the control group (n = 56). Patients admitted between January 2024 and May 2024 who received quadruple therapy (valsartan, atorvastatin, probucol, and vitamin D) were assigned to the observation group (n = 94).
The larger number of patients in the observation group reflected increased clinical application of adjunctive vitamin D therapy at the study center beginning in early 2024, following emerging evidence. Baseline demographic and clinical characteristics were comparable between groups (Table 1; P>0.05), and no significant changes in diagnostic criteria, laboratory methods, or physician team composition occurred during the study period.
Treatment regimen administration
All participants received a standardized daily subcutaneous insulin infusion regimen for glycemic control throughout the 8-month study period. Insulin dosage was adjusted individually according to daily capillary blood glucose monitoring. Patients in the control group received valsartan (80 mg once daily on an empty stomach), atorvastatin (20 mg once daily), and probucol (250 mg twice daily) for 8 months. Patients in the observation group received the same valsartan, atorvastatin, and probucol regimen with identical dosages and schedules, together with daily oral vitamin D supplementation.
Vitamin D dosage was determined according to baseline serum 25-hydroxyvitamin D [25(OH)D] levels measured at enrollment using electrochemiluminescence immunoassay (ECLIA). Vitamin D sufficiency was defined as 25(OH)D ≥30 ng/mL, insufficiency as 20 ng/mL ≤25(OH)D <30 ng/mL, and deficiency as 25(OH)D <20 ng/mL. Patients receiving supplementation to prevent deficiency received 600–800 IU/day, whereas patients with established vitamin D insufficiency or deficiency received 2000 IU/day.
Safety monitoring and stopping criteria
To ensure participant safety during the 8-month treatment period, serum calcium, phosphorus, and parathyroid hormone (PTH) levels were measured at baseline and every 4 weeks to monitor vitamin D-induced hypercalcemia and calcium-phosphorus imbalance. A 12-lead electrocardiogram (ECG) was performed at baseline and after 8 months to assess QTc interval prolongation associated with probucol. In patients with baseline QTc ≥450 ms in men or ≥460 ms in women, the ECG was repeated at month 1.
Renal function parameters, including serum creatinine and eGFR, together with serum electrolytes, were monitored biweekly during the first month and monthly thereafter. Participants were instructed to report new symptoms immediately. Treatment discontinuation criteria included corrected serum calcium >10.5 mg/dL (2.62 mmol/L) confirmed by repeat testing within 48 h; QTc interval >500 ms or increase >60 ms from baseline on two consecutive ECGs; doubling of serum creatinine from baseline or development of acute kidney injury according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria; and any grade ≥3 adverse event according to Common Terminology Criteria for Adverse Events (CTCAE) considered possibly, probably, or definitely related to vitamin D or probucol. Serious adverse events were reported to the hospital ethics committee within 24 h.
Biological sample collection and processing
Blood and urine samples were collected before treatment initiation and after completion of the 8-month treatment period. All samples were collected in the morning after an overnight fast of 8–12 h. Approximately 20 mL of venous blood was collected using standard phlebotomy techniques into serum-separator and EDTA tubes. Blood samples were allowed to clot at room temperature for 30 min and centrifuged at 1000 × g for 15 min at 4 °C. Clear serum supernatants were carefully aspirated without disturbing the clot or buffy coat, aliquoted into cryovials for different assays, labeled with participant ID, date, and sample type, and stored at −80 °C until analysis. Serum samples could be stored at −80 °C for up to 12 months.
For 24 h urinary protein excretion (24 h UPE), participants discarded the first morning urine sample at 7:00 AM on day 1 and subsequently collected all urine, including the first morning urine sample at 7:00 AM on day 2, into a provided collection container. Participants were instructed to refrigerate the collection container during the collection period. The total urine volume was recorded, thoroughly mixed, and aliquoted for laboratory analysis. Samples were stored at 4 °C when analyzed within 24 h or at −20 °C for later analysis.
For urinary albumin-to-creatinine ratio (UAlb/UCR), first-morning midstream urine samples were collected in sterile containers and processed similarly. The same procedures were repeated after completion of the 8-month treatment period.
Biochemical and clinical parameter analysis
Frozen serum samples were thawed on ice or at 4 °C before serum creatinine (SCr) analysis. SCr was measured using an enzymatic colorimetric assay on an automated biochemical analyzer, according to the manufacturer's instructions. Daily calibration was performed using multi-level calibrators, and two levels of commercial quality-control materials were included in each batch. Samples were analyzed in duplicate, and assays with a coefficient of variation >5% were repeated. The lower detection limit for SCr was 2.0 µmol/L.
Protein concentration in 24 h urine aliquots was measured using the pyrogallol red-molybdate method. Samples were analyzed in duplicate, and quality-control materials were included in each run. Samples above the calibration range (>3.0 g/L) were diluted with normal saline and reassessed. The 24 h UPE was calculated using urinary protein concentration and total urine volume.
Urinary albumin and creatinine concentrations in first-morning urine samples were measured using the biochemical analyzer, and the UAlb/UCR ratio was subsequently calculated.
Fasting plasma glucose (FPG) and 2 h postprandial glucose (2h-PPG) were measured using a validated blood glucose meter and capillary finger-prick blood samples, according to the manufacturer's instructions.
Glycated hemoglobin (HbA1c) was analyzed using high-performance liquid chromatography with a dedicated HbA1c analysis system. Participants were instructed to avoid lipid-lowering medications for 7 days and high-fat meals or alcohol for 24 h before post-treatment blood collection. Serum total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using enzymatic assays. Two-level quality-control materials were analyzed daily. Lipid parameters were initially measured in singlicate, and samples with a coefficient of variation >10% were reassessed in duplicate. Detection limits were 0.1 mmol/L for TC, 0.05 mmol/L for TG, 0.08 mmol/L for HDL-C, and 0.10 mmol/L for LDL-C. LDL-C values were determined by direct assay or by the Friedewald equation when triglyceride levels were <400 mg/dL.
Immune function and inflammatory marker analysis
Serum immunoglobulin G (IgG) concentrations were measured using a commercial human IgG ELISA kit. The assay detection range was 1.37–1000 ng/mL, the sensitivity was 0.52 ng/mL, the intra-assay coefficient of variation was <6%, and the inter-assay coefficient of variation was <9%. All samples were analyzed in duplicate, and high and low-quality-control materials supplied with the kit were included on each plate. Plates with QC values outside 2 standard deviations from target values were rejected and repeated. Samples above the upper standard limit were diluted 1:2 and reassessed.
All reagents and serum samples were equilibrated to room temperature before analysis. Serum samples were diluted according to kit instructions, typically at 1:100,000 dilution. Standards, diluted samples, and blanks (100 µL) were added to appropriate wells of pre-coated microplates and incubated at 37 °C for 90 min. Wells were washed three times before incubation with biotinylated detection antibody for 60 min at 37 °C. Following additional washing, horseradish peroxidase conjugate was added and incubated for 30 min in the dark. After washing, the substrate solution was added, and the mixture was incubated in the dark for 15 min before the stop solution was added. Optical density was measured at 450 nm with reference wavelengths of 570 nm or 630 nm. Standard curves were generated to calculate IgG concentrations.
Lymphocyte subsets were analyzed by flow cytometry on a BD FACSCanto II equipped with 488 nm and 640 nm lasers. Forward scatter voltage was set to 300 V, side scatter to 350 V, and photomultiplier tube voltages for FITC, APC, and PE channels to 400 V, 550 V, and 480 V, respectively. Doublets were excluded using FSC-A versus FSC-H gating.
Viability staining was performed using 7-aminoactinomycin D (7-AAD), and only 7-AAD-negative events were analyzed. Whole blood samples were distributed into unstained, isotype-control, and test tubes. Anti-human CD3-FITC, CD4-APC, and CD8-PE antibodies were added to test tubes, whereas matched isotype-control antibodies were added to control tubes. Samples were incubated in the dark at room temperature for 30 min. Compensation controls were prepared with BD CompBeads, and automatic compensation was performed in FACSDiva software. Red blood cells were lysed with RBC lysis buffer, then washed and resuspended in staining buffer containing 7-AAD.
Daily cytometer setup and tracking beads were used for quality control. Gating hierarchy included doublet exclusion, lymphocyte selection, viability gating, CD3+ gating, and CD4/CD8 subset identification. At least 50,000 live lymphocyte events were acquired per sample, with total acquired events ranging from 100,000–150,000. Data were analyzed using FlowJo software, and percentages of CD3+, CD4+, and CD8+ cells, together with CD4+/CD8+ ratios, were calculated.
Serum hs-CRP, IL-6, TNF-α, and MCP-1 concentrations were measured using commercial ELISA kits obtained. Detection ranges and sensitivities were as follows: hs-CRP (0.07–12.5 ng/mL; sensitivity 0.022 ng/mL), IL-6 (0.7–300 pg/mL; sensitivity 0.35 pg/mL), TNF-α (1.6–100 pg/mL; sensitivity 0.8 pg/mL), and MCP-1 (5–1000 pg/mL; sensitivity 2.5 pg/mL). All assays were performed in duplicate. Low and high-quality control materials were included on every plate. Intra-assay coefficients of variation were <8%, and inter-assay coefficients of variation were <10%. Values below detection limits were recorded as half the lower limit of quantification, whereas values above the highest standard were diluted and reassessed. Samples underwent only one freeze-thaw cycle before analysis. Cytokine ELISA procedures followed protocols identical to those described for IgG ELISA, according to manufacturer instructions regarding dilution factors, incubation conditions, and temperatures.
Clinical outcome assessment and data management
At the end of the 8-month treatment period, treating physicians, blinded to group allocation, evaluated participants' clinical status. Patients were classified as discharged when all predefined clinical improvement criteria for diabetic nephropathy were met for at least two consecutive weeks: ≥30% reduction in 24 h UPE compared with baseline, stabilization or improvement in eGFR defined as decline <5 mL/min/1.73 m2 from baseline, and absence of acute diabetic complications or other conditions requiring continued inpatient care. Participants who did not meet all criteria were classified as not discharged. Clinical response rates were subsequently calculated for each group.
All collected demographic, laboratory, and clinical data were entered into an electronic database using double data entry by two independent researchers. Range checks and consistency checks were performed to ensure data accuracy. Primary endpoints included changes from baseline to 8 months in renal-function parameters, including 24 h UPE and UAlb/UCR, and immune-function parameters, including CD3+ T-cell percentage and CD4+/CD8+ ratio. Secondary endpoints included changes from baseline to 8 months in glycemic parameters (FPG, 2h-PPG, HbA1c), lipid profile (TC, TG, LDL-C, HDL-C), inflammatory markers (hs-CRP, IL-6, TNF-α, MCP-1), and clinical response rate. All outcome measures were assessed at baseline, within 48 h before treatment initiation, and post-treatment, within 1 week after completion of the 8-month treatment period.
Statistical analysis
All statistical analyses were performed using SPSS software. Continuous variables with normal distribution were expressed as mean ± standard deviation, whereas categorical variables were expressed as frequency and percentage. Independent-samples t-tests were used for between-group comparisons, and chi-square tests were used for categorical variables. Statistical significance was defined as P<0.05. Given the exploratory nature of the study and the large number of outcomes assessed, secondary outcomes were not adjusted for multiplicity. Therefore, P-values were considered descriptive rather than confirmatory. Because of the retrospective non-randomized study design, all comparisons between the control and observation groups were unadjusted for potential confounders, and residual confounding may have remained. All 150 enrolled patients (56 in the control group and 94 in the observation group) completed the full 8-month treatment and follow-up period. No patients were lost to follow-up, withdrawn, or excluded after enrollment. No missing data were identified for baseline characteristics or outcome measures, including renal function, glucose and lipid profiles, immune markers, and inflammatory factors. Therefore, no imputation procedures were required.