Gut Microbiome and Sex as Risk Factors for Kidney Stones After Bariatric Surgery

Experimental Design: We will determine sex differences in 24-hour urine supersaturation (SS) with respect to calcium oxalate (CaOx) in Roux-en-Y Gastric Bypass (RYGB) and sleeve gastrectomy patients and the correlation of microbial species and their metabolic pathways with urine oxalate. This will be determined under the conditions of an isocaloric, standardized sodium (200 mmol/day), calcium, potassium, macronutrient distribution. We will recruit subjects from the Bariatric Surgery Clinic at The University of Chicago Medical Center.

Location: The study will take place at participants' homes (Periods 1, 4) and in the University of Chicago Medicine (UCM) Clinical Research Center. (Periods 2, 3, 5).

Study period:

The study will consist of 5 separate periods. Periods 1 and 2 will be before surgery and periods 3-5 will occur after surgery. Periods 1 and 4 will last three days and periods 2, 3, and 5 will last four days, three of which are at home and a fourth (half-day) in the Clinical Research Center (CRC).

Study period 1 will occur prior to RYGB or specific gravity (SG) in the participant's home. Each participant will provide a three-day diet record and complete a 1.5-hour phone call with the CRC dietician and one 24-hour urine collection (on day three). Urine collection kits will be couriered directly to home and couriered back to the Kidney Stone Lab after completion.

Study period 2 will be completed in the CRC and will occur prior to RYGB or SG. The fixed study diet will be shipped by courier along with a stool collection kit. Each participant will eat the study diet on days one, two, and three of this study period. On the morning of day four, subjects present to the CRC in a fasting state and bring stool collections to the CRC. Each participant will collect three 45-minute urines with a blood sample at the midpoint of each collection. Study participants then consume a fixed breakfast and collect six 45-minute urines with blood samples in the post-prandial period.

Study period 3 will be completed in the CRC and will occur between 2 and 4 weeks post-RYGB or SG. The 2 to 4 week timing of this period was selected because the biological changes in anatomy and neuroendocrine signaling would have occurred by 2 weeks post-surgery and patients are typically advanced from a diet of soft foods to regular foods by 2 weeks. Subjects will not participate in period 3 until after clinical clearance by the surgeon for diet advancement. It is also expected that by this time any effect of anesthesia or peri-operative antibiotics and medications is minimal. The protocol of study period 3 is identical to that of period 2 with three days of study diet and CRC on day four with fasting and fed blood and urine collections (see period 2 for specifics).

Study period 4 will occur at home between 9 and 12 months post-RYGB or SG in the participant's home. The timing of this period is selected because patients have typically undergone the majority of weight loss and had time to adopt new self-chosen home diets. Urine changes expected to occur in kidney stone formers are also expected to be detectable at this time. Participants will provide a three-day diet record and complete a 1.5-hour phone call with the CRC dietician and one 24-hour urine collection (on day three). Urine collection kits will be couriered directly to home and couriered back to the Kidney Stone Lab after completion.

Study period 5 will occur in the CRC at 12 months (+/- 1 month) post-RYGB or SG. The timing of this period is selected because patients have typically undergone the majority of their weight loss and most have had resolution of metabolic syndrome. The protocol of study period 3 is identical to that of periods 2 and 3 (see period 2 for specifics).

Study Diet: Subjects will eat a diet containing 65 mmol sodium, 1740-2940mg potassium, 800-1200 mg calcium, and meals will be isocaloric with controlled macronutrient distribution. Food will be prepared in the CRC kitchen and shipped to patients. Intake of water is ad lib, other fluids only as prescribed. The diet will be prepared in consultation with dieticians, including dieticians with expertise in bariatric surgery. Food will be shipped to participants on day 1. Participants will continue to take prescribed medications and vitamin supplements per the bariatric surgery clinic.

Home diet record and nutritional data collection (study period 1, 4): Each participant will be provided a food log and a handout on portion estimates prior to home diet record. A three-day diet record will be recorded by the participant. Within one to three days of completing the diet record each participant will have a 1.5-hour phone call with the CRC dietician. The dietician will review the record for completeness and clarification, and then enter the data into the Nutritional Data System for Research (NDSR). The NDSR is a well-established, computer based, dietary analysis software program that was developed at the University of Minnesota and provides quantitative data on both micro- and macronutrients from the dietary recall. The food database in the NDSR includes over 18,000 foods and uses the United States Department of Agriculture laboratory as their primary source for nutrient values and composition. The UCM CRC has an existing contract with NDSR to support use of this software for this study.

Urine and Serum Measurements: Two 24-hour urine collections will be performed at home (with instructions provided). 24-hour urine volume, Ca, oxalate, citrate, uric acid, Na, K, Cl, Mg, sulfate, ammonium, and creatinine will be measured. Urine measurements will be performed at the University of Chicago Kidney Stone Research Group laboratory. Serum 25-vitamin D and parathyroid hormone levels will be measured through the CRC in the University of Chicago laboratory. Serum sodium, potassium, chloride, bicarbonate, calcium, phosphorus, magnesium, creatinine, and glucagon-like peptide 1 (GLP-1) will be done at the University of Chicago Kidney Stone laboratory. Serum peptide YY and insulin will be assayed at the University of Chicago Diabetes Research and Training Center laboratory. Serum glucagon will be assayed at LabCorp. Glucose will be measured using the gold standard YSI methodology at the CRC. Serum and urine will be frozen at -80 degrees C and stored at the Kidney Stone laboratory for potential later assays.

Aim 1, Urine Statistical Analysis: Urine CaOx SS will be calculated using EQUIL2 (a computer program used for the calculation of urinary saturation). We will compare the CaOx SS of men vs. women using a t-test that accounts for any differences in variances between the two groups. We will perform linear regression models by sex to determine predictors of CaOx SS. We will use Spearman's correlation tests to find independent correlations between microbial species and/or metabolic pathways with CaOx SS. Generalized linear models will be generated for any modules that are associated with high CaOx SS.

Aim 2, Stool Microbial Species and Statistical Analysis: Shotgun sequencing will be performed for all stool samples. Shotgun sequencing will be performed for all stool samples. TruSeq DNA Sample prep kit (Illumina) and reagents from KAPA Library Preparation kit (Kapa biosystems) will be used to generate libraries. Libraries will be sequenced on Illumina MiSeq platform using 2x250 nucleotide paired-end sequencing. Sequencing reads will be converted into relative abundances of microbial metabolic modules using HUMAnN and mapped to KEGG Relative abundances will be calculated using the Metagenomic Phylogenetic Analysis (MetaPhlAn) pipeline.