Oxalate is involved in the pathophysiology of kidney stones (KS) and chronic kidney disease (CKD), which, respectively, affect more than 9% and 15% of the adult US population. Higher urine oxalate (UOx) can lead to calcium oxalate KS and CKD incidence and progression. The gut microbiome plays a key role in human oxalate homeostasis, which differs between stone formers (SF) and non-stone formers (NSF). In SF, rates of oxalate absorption and endogenous oxalate synthesis are higher than in NSF. The determinants of UOx and their interplay are still unknown limiting our ability to develop therapeutics to treat hyperoxaluria. Our data demonstrate oxalate degradation in the human intestine is performed by multiple bacterial species (the oxalobiome) with variable contributions to global oxalate metabolism and that alteration of the oxalobiome function is associated with higher UOx in SF. In mice, we showed that the gut microbiota is an important determinant of UOx and colonization with an oxalate degrader (Oxalobacter formigenes) led to lower UOx. We hypothesize that the gut microbiome is a major determinant of UOx, and its manipulation can reduce UOx levels. The overarching goal of this proposal is to better understand how gut microbial metabolism of dietary oxalate is linked to UOx levels within a host and can serve as a potential therapeutic target. We propose to leverage the rich resources available for participants in the Nurses' Health Study II (n = 584 women; 218 SF and 366 NSF) and the Health Professionals Follow-up Study (n = 308 men; 58 SF and 250 NSF), including existing and to be completed sequencing data from fecal samples, existing genomic data, 24-hour UOx, validated dietary information, comorbidities, and medication use. In Aim 1, we plan to investigate the metaorganismal oxalate degrading function in determining UOx, and whether it differs in SF and NSF by performing additional sequencing and analyzing metagenomes and metatranscriptomes from fecal samples to quantify the oxalobiome function and then conduct comprehensive multi-layer microbiome data analysis around the oxalobiome. In Aim 2, we will identify the interwoven role of gut microbiome, host genetics, comorbidities, and dietary information in determining UOx. In Aim 3 we will test the role of the oxalobiome in UOx excretion and the efficacy of several probiotic combinations in reducing UOx. In these multidisciplinary, computational, translational, and animal-model based investigations, we aim to reveal a role for the gut oxalobiome in urinary oxalate excretion with the goal of developing therapeutic approaches for the treatment and prevention of the adverse effects of oxalate in humans.