Impact of Humid-Heat on Gut-Tryptophan-Stone Pathway

• Objectives

To investigate differences in gut microbiota composition and tryptophan metabolite levels between kidney stone patients and healthy individuals, specifically focusing on:

• Comparing gut microbiota composition between stone patients and healthy controls, with emphasis on the relative abundance of Lactobacillus salivarius.
• Comparing serum levels of tryptophan metabolites-indole-3-carboxylic acid (ICA) and kynurenine (Kyn)-between groups.
• Exploring correlations between gut microbiota composition and tryptophan metabolite levels.
• Analyzing the impact of environmental conditions (season, temperature/humidity) on gut microbiota and metabolite levels.

• Trial Design This prospective case-control study compares gut microbiota composition and serum metabolite levels between kidney stone patients (case group) and stone-free healthy volunteers (control group), while exploring associations with environmental factors.

• Participants Case Group: Patients diagnosed with kidney stones. Control Group: Healthy volunteers without kidney stones.

• Group Allocation Case Group: Kidney stone patients. Control Group: Stone-free healthy volunteers. Participants are assigned based on clinical status (no randomization).

Stratified analyses will consider:

Environmental exposure (temperature/humidity data). Seasonal factors (summer vs. non-summer). Gut microbiota composition (L. salivarius abundance via 16S rRNA sequencing). Serum metabolite levels (ICA, Kyn).

• Endpoints

Primary Endpoints:

Gut microbiota differences (α/β diversity, L. salivarius abundance). Serum ICA and Kyn level differences.

Secondary Endpoints:

Tryptophan pathway metabolite changes (Trp, IAA, Kyn/Trp ratio, ICA/Trp ratio). Microbiota-metabolite correlations. Environmental impact analysis.

• Observational Parameters

Primary Parameters:

Gut microbiota structure (α/β diversity, L. salivarius abundance). Serum ICA/Kyn concentrations (ng/ml).

Secondary Parameters:

Tryptophan pathway metabolites (Trp, IAA, ratios). Environmental factors (temperature, humidity, season). Demographics (gender, age, BMI). Stone history (type, frequency, seasonality). Comorbidities (hypertension, diabetes, intestinal diseases).

• Randomization Not applicable (case-control design). Participants are assigned based on clinical diagnosis.

• Blinding No blinding during enrollment. Laboratory personnel are blinded to group allocation during 16S rRNA sequencing and metabolomic analyses. Samples are coded, and statisticians design analysis plans before data unblinding.

• Sample Size Calculation

Accounting for 10% attrition and multiple analyses, final recruitment targets:

200 cases and 100 controls (expected completions: 180 cases, 90 controls).

• Statistical Analysis Descriptive Statistics: Mean±SD for continuous variables; frequencies for categorical variables.

Group Comparisons: t-test/Mann-Whitney U (continuous); χ²/Fisher's exact test (categorical).

Correlations: Spearman/partial correlation analysis. Multivariate Analysis: Linear/logistic regression adjusting for confounders. Microbiome Analysis: QIIME2 (α/β diversity, LEfSe, ANCOM). Metabolomics: MetaboAnalyst (pathway enrichment). Software: R 4.3.0; P<0.05 deemed significant.

• Follow-up Plan

Screening Period (-7 days):

Informed consent. Demographics, medical history, physical exam, vital signs (blood pressure, pulse, temperature, respiration).

Case group: Collect routine renal function tests, electrolytes, and imaging data.

Sample Collection Phase:

Case Group:

Fecal sample (5g) for 16S rRNA sequencing. Venous blood (10ml) for LC-MS metabolomics. Residual surgical stones (if available).

Control Group:

Fecal sample (5g) and venous blood (10ml). All samples collected in a single visit. Follow-up via phone for health status confirmation.

Inclusion criteria

• Inclusion criteria for patients with kidney stones

• Age>=18 years;
• Diagnosed with kidney stones by ultrasound, CT or urography;
• Willing to provide stool samples and serum samples for study;
• No history of antimicrobial use in the past 3 months;
• Signed and dated informed consent indicating that the patient or his/her legal representative is fully informed of the study-related information and agrees to participate.
• Inclusion criteria for the healthy control group

• Age>=18 years;
• No history of kidney stones and family history;
• Imaging examination (such as abdominal ultrasound) showed no kidney stones;
• Willing to provide stool samples and serum samples for research;
• No history of antibiotic use in the past 3 months;
• Signed and dated informed consent indicating that the volunteer is fully informed about the study-related information and agrees to participate.

Exclusion criteria

• Use of antimicrobials or probiotics within the past 3 months;
• Presence of active urinary tract infection;
• Presence of other serious systemic diseases, such as hepatic or renal insufficiency, cardiac or pulmonary diseases, malignant tumors, and immunodeficiency states;
• Congenital urinary tract abnormalities;
• Previous history of kidney transplantation or urinary diversion surgery;
• Pregnant or lactating women;
• Presence of chronic intestinal diseases, such as inflammatory bowel disease, irritable bowel syndrome, etc.;
• Inability to provide samples or complete follow-up according to the research protocol;
• Participation in other clinical studies within the past 3 months;
• Other conditions deemed unsuitable for participation in this study by the researcher.