Research design and setting
This double-blind, randomized, placebo-controlled trial was carried out on the Urology and Nephrology Heart, Mansoura College, Egypt, between Might and November 2025. The research protocol was authorised by the Medical Analysis Ethics Committee of Mansoura School of Medication (approval code: MD.24.06.864, dated July 14, 2024) and prospectively registered at ClinicalTrials.gov (NCT06863194) on 13 Might 2025, previous to the enrollment of the primary participant. The research was carried out in accordance with the Declaration of Helsinki, and knowledgeable consent was obtained from all individuals.
No adjustments have been made after trial graduation to the research protocol, trial design, eligibility standards, interventions, knowledge assortment, or prespecified main and secondary outcomes. Further submit hoc analyses have been carried out utilizing the beforehand collected knowledge. These included baseline-adjusted ANCOVA, parsimonious covariate-adjusted fashions, an exploratory reconstruction of urinary creatinine excretion, a BMI-change sensitivity evaluation, and exploratory sex-stratified analyses. These analyses weren’t prespecified and are introduced as supplementary or exploratory findings.
The complete research protocol (approval code: MD.24.06.864) and statistical evaluation plan can be found upon affordable request from the corresponding writer. A abstract of the protocol can be registered at ClinicalTrials.gov (NCT06863194).”
Individuals
Grownup sufferers (≥ 18 years) with CKD levels 3 or 4, outlined by eGFR of 15–59 mL/min/1.73 m2, have been recruited from outpatient nephrology clinics. Exclusion standards have been current (inside 4 weeks) use of probiotics or antibiotics, lively malignancy, recognized structural gastrointestinal illness, or a historical past of gastrointestinal surgical procedure, being pregnant, and autoimmune illness requiring immunosuppressive remedy.
Pattern measurement was calculated utilizing G*Energy software program (model 3.1.9.4) based mostly on impact sizes reported by de Araújo et al.13. For a two-sample t-test with an alpha error chance of 0.05, an influence of 0.80, and an impact measurement d = 0.67, the required pattern measurement was 72 sufferers (~ 36 per group).
Randomization and blinding
Eligible sufferers have been randomly assigned in a 1:1 ratio to obtain both probiotic supplementation or a placebo utilizing computer-generated randomization sequences. Allocation concealment was ensured by sequentially numbered, sealed, opaque envelopes maintained by an impartial pharmacist. Each individuals and all analysis personnel remained blinded to therapy allocation all through the research. Probiotic and placebo capsules have been similar in look to take care of blinding integrity.
Interventions
Individuals within the intervention group acquired oral probiotic supplementation containing 101⁰ colony-forming items of Lactobacillus plantarum per capsule (Lactogemikan; Pescado Prescription drugs, Egypt), administered as soon as each day for six months. The management group acquired matching placebo capsules containing microcrystalline cellulose on the identical schedule. Each teams continued normal medical administration for CKD, together with standardized dietary counseling, as per institutional protocols. Dietary consumption was not formally quantified by meals frequency questionnaires. All individuals with hypertension acquired a secure angiotensin-converting-enzyme inhibitor or angiotensin-receptor blocker routine that remained unchanged throughout follow-up. Remedy adherence was assessed by affected person interviews and capsule counts at follow-up visits. The research flowchart is introduced in Fig. 1.
Research flowchart. Move diagram illustrating participant recruitment, allocation, follow-up, and evaluation. Of 120 people assessed for eligibility, 48 have been excluded based mostly on varied standards, leading to 72 individuals randomized. These individuals have been allotted to both the Probiotics group (n = 37) or the placebo group (n = 35). All allotted individuals acquired their assigned intervention, accomplished follow-up, and have been included within the ultimate evaluation. No individuals have been misplaced to follow-up, discontinued the intervention, or have been excluded from evaluation in both group.
Affected person and public involvement
No sufferers or public have been concerned within the design, conduct, or reporting of this research.
Consequence measures
Individuals have been assessed at baseline and at 6 months, equivalent to the top of the intervention. The first final result was the change in eGFR from baseline to six months, whereas secondary outcomes included adjustments in serum creatinine, 24-h proteinuria, urinary indoxyl sulfate, serum albumin, whole ldl cholesterol, and hemoglobin; CKD development was outlined as a ≥ 25% decline in eGFR or doubling of serum creatinine, and extra assessments and analyses included six-month eGFR adjusted for baseline eGFR, 24-h creatinine clearance, reconstructed 24-h urinary creatinine excretion, physique mass index (BMI), physique weight, blood urea nitrogen (BUN), and treatment-effect estimates in keeping with CKD stage, diabetes standing, and intercourse.
Hostile occasions have been systematically monitored and recorded at every follow-up go to by affected person interviews and evaluate of medical data. Harms have been outlined as any untoward medical incidence in a participant in the course of the research interval, no matter its causal relationship with the intervention. The severity and relatedness of all reported adversarial occasions to the research intervention have been assessed by the research investigators.
Laboratory procedures
Venous blood samples have been collected after an in a single day quick at baseline and follow-up visits. Serum creatinine was measured utilizing an enzymatic methodology with IDMS-traceable calibration on a Roche Cobas automated analyzer. eGFR was calculated utilizing the CKD-EPI 2021 equation. Different biochemical parameters have been measured utilizing normal automated strategies on the laboratory of Urology and Nephrology Heart, Mansoura College, Egypt.
For twenty-four-h urine assortment, sufferers have been instructed to discard the primary morning void and gather all subsequent urine over the following 24 h. Samples have been saved at 4 °C throughout assortment and analyzed inside 2 h.
Twenty-four-hour urinary creatinine excretion was reconstructed from the accessible CrCl and serum creatinine values as urinary creatinine excretion (mg/day) = CrCl (mL/min) × serum creatinine (mg/dL) × 14.4. BUN, BMI, and physique weight have been recorded at each visits. Twenty-four-hour urinary urea excretion was not measured; BUN was subsequently examined as an exploratory serum marker associated to urea metabolism and catabolism, however not as a direct quantitative measure of protein consumption. Cystatin C was not measured as a result of assay kits have been unavailable at our heart and the related procurement prices exceed the accessible funding for this research.
Urinary indoxyl sulfate was quantified utilizing a commercially accessible ELISA equipment (Human IS ELISA Package, Catalog ELK9248; detection vary 31.25–2000 ng/mL, sensitivity 12.1 ng/mL). First morning midstream urine samples have been collected, centrifuged at 2000 × g for 10 min, and both assayed instantly or saved at −20 °C. All samples have been analyzed in duplicate.
The ELISA process was adopted in keeping with the producer’s directions. Briefly, 100 μL of requirements (31.25–2000 ng/mL) or samples have been added to microtiter wells and incubated at 37 °C for 80 min. After washing 3 times, 100 μL of biotinylated antibody was added and incubated at 37 °C for 50 min. Following three washes, 100 μL of streptavidin-HRP was added and incubated at 37 °C for 50 min. After 5 washes, 90 μL of TMB substrate was added, and the combination was incubated at 37 °C for 20 min. The response was stopped with 50 μL of cease answer, and absorbance was measured at 450 nm inside 15 min. Customary curves have been constructed utilizing energy operate becoming, with R2 > 0.97 required for acceptance. High quality management included duplicate measurements with a coefficient of variation < 15%.
Statistical evaluation
Statistical analyses have been carried out utilizing SPSS model 26. Normality was assessed utilizing the Shapiro–Wilk take a look at. Usually distributed knowledge is introduced as imply ± normal deviation; non-normally distributed knowledge as median with interquartile vary (IQR). Categorical variables are expressed as frequencies and percentages.
Baseline traits have been in contrast utilizing the Chi-square or Fisher’s precise take a look at for categorical variables, and the independent-samples t-test or Mann–Whitney U take a look at for steady variables. Inside-group adjustments have been analyzed utilizing paired t-tests or Wilcoxon signed-rank exams as applicable. Between-group change-score comparisons have been analyzed utilizing independent-samples t exams or Mann–Whitney U exams and have been retained as secondary analyses.
For the first between-group evaluation, ANCOVA was carried out with eGFR at six months because the dependent variable, therapy group as the principle issue, and baseline eGFR because the covariate. Adjusted group means, the adjusted probiotic–placebo distinction, 95% confidence interval (CI), p worth, and partial η2 have been estimated. Mannequin diagnostics included evaluation of residual normality, homoscedasticity, linearity, and multicollinearity.
Separate parsimonious sensitivity fashions have been fitted: baseline eGFR alone; baseline eGFR plus secure ACE inhibitor/angiotensin-receptor blocker use; baseline eGFR plus age and diabetes standing; and baseline eGFR plus baseline proteinuria. An additional sensitivity mannequin included change in BMI. Intercourse-stratified therapy estimates and a treatment-by-sex interplay take a look at have been added to the subgroup analyses. CKD-stage and diabetes-status interplay exams have been additionally retained. Spearman rank correlation assessed associations between adjustments in urinary indoxyl sulfate and adjustments in scientific variables.
All exams have been two-tailed, with p < 0.05 thought-about statistically vital. The research was designed to detect the prespecified impact measurement with 80% energy at an alpha degree of 0.05.
All 72 randomized individuals accomplished the 6-month intervention interval with no dropouts or lacking knowledge. The excessive retention fee was attributed to the great scientific companies and glorious affected person care supplied by the Urology and Nephrology Heart at Mansoura College, which ensured sturdy participant engagement and adherence to follow-up visits.
