The Effect of Oxalobacter formigenes Colonization in Patients with Calcium Oxalate Renal Stones in Comparison with Healthy People in Qom: A Case-Control Study

Background: Urinary stones are a major problem world, and their incidence has increased significantly in recent years. Objectives: This study aimed to develop a simple and rapid molecular method based on PCR and qPCR assays to detect Oxalobacter formigenes (which causes oxalate degradation in intestines) in fecal samples of healthy volunteers and patients with calcium oxalate nephrolithiasis, and determine the amount of urinary oxalate in the two groups. Methods: This study was performed on urine and fecal samples of 73 patients with kidney stones and 52 healthy individuals. After DNA extraction, PCR and qPCR assays were performed on two gene regions of Oxalobacter formigenes, OXC, and FRC. Also, urine oxalate was measured in the study population using biochemical methods. Results: We found that the presence of Oxalobacter formigenes could reduce the risk of kidney stones and calcium oxalate stones. In fact, both FRC and OXC genes were involved in the diagnosis of Oxalobacter formigenes; however, the results based on the FRC gene showed higher efficiency. In addition, the presence or absence of stones did not affect the amount of urinary excretion of oxalate, rather it is affected by diet. Conclusions: Molecular identification of Oxalobacter formigenes by PCR and qPCR assays allows rapid, specific, and reproducible detection in fecal samples, which also allows immediate processing of these samples in clinical conditions.

Microbial genetic and transcriptional contributions to oxalate degradation by the gut microbiota in health and disease

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. The exact oxalate-degrading taxa in the human microbiota and their ODP have not been described. We leverage multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota primarily uses the type II ODP, rather than type I. Furthermore, among the diverse ODP-encoding microbes, an oxalate autotroph, Oxalobacter formigenes, dominates this function transcriptionally. Patients with inflammatory bowel disease (IBD) frequently suffer from disrupted oxalate homeostasis and calcium oxalate nephrolithiasis. We show that the enteric oxalate level is elevated in IBD patients, with highest levels in Crohn’s disease (CD) patients with both ileal and colonic involvement consistent with known nephrolithiasis risk. We show that the microbiota ODP expression is reduced in IBD patients, which may contribute to the disrupted oxalate homeostasis. The specific changes in ODP expression by several important taxa suggest that they play distinct roles in IBD-induced nephrolithiasis risk. Lastly, we colonize mice that are maintained in the gnotobiotic facility with O. formigenes, using either a laboratory isolate or an isolate we cultured from human stools, and observed a significant reduction in host fecal and urine oxalate levels, supporting our in silico prediction of the importance of the microbiome, particularly O. formigenes in host oxalate homeostasis.

Risk Factors for Kidney Stone Formation following Bariatric Surgery

Kidney stones are painful, common, and increasing in incidence. Obesity and bariatric surgery rates are also on the rise in the United States. Although bariatric surgery is associated with improvements in metabolic outcomes, malabsorptive bariatric surgery procedures are also associated with increased risk of kidney stones. Restrictive bariatric surgeries have not been associated with kidney-stone risk. Higher risk of kidney stones after malabsorptive procedures is associated with postsurgical changes in urine composition, including high urine oxalate, low urine citrate, and low urine volume. Certain dietary recommendations after surgery may help mitigate these urine changes and reduce risk of kidney stones. Understanding risk of kidney stones after surgery is essential to improving patient outcomes after bariatric surgery.

End Points for Clinical Trials in Primary Hyperoxaluria

Patients with primary hyperoxaluria experience kidney stones from a young age and can develop progressive oxalate nephropathy. Progression to kidney failure often develops over a number of years, and is associated with systemic oxalosis, intensive dialysis, and often combined kidney and liver transplantation. There are no therapies approved by the Food and Drug Association. Thus, the Kidney Health Initiative, in partnership with the Oxalosis and Hyperoxaluria Foundation, initiated a project to identify end points for clinical trials. A workgroup of physicians, scientists, patients with primary hyperoxaluria, industry, and United States regulators critically examined the published literature for clinical outcomes and potential surrogate end points that could be used to evaluate new treatments. Kidney stones, change in eGFR, urine oxalate, and plasma oxalate were the strongest candidate end points. Kidney stones affect how patients with primary hyperoxaluria feel and function, but standards for measurement and monitoring are lacking. Primary hyperoxaluria registry data suggest that eGFR decline in most patients is gradual, but can be unpredictable. Epidemiologic data show a strong relationship between urine oxalate and long-term kidney function loss. Urine oxalate is reasonably likely to predict clinical benefit, due to its causal role in stone formation and kidney damage in CKD stages 1–3a, and plasma oxalate is likely associated with risk of systemic oxalosis in CKD 3b–5. Change in slope of eGFR could be considered the equivalent of a clinically meaningful end point in support of traditional approval. A substantial change in urine oxalate as a surrogate end point could support traditional approval in patients with primary hyperoxaluria type 1 and CKD stages 1–3a. A substantial change in markedly elevated plasma oxalate could support accelerated approval in patients with primary hyperoxaluria and CKD stages 3b–5. Primary hyperoxaluria type 1 accounts for the preponderance of available data, thus heavily influences the conclusions. Addressing gaps in data will further facilitate testing of promising new treatments, accelerating improved outcomes for patients with primary hyperoxaluria.

Activation of the PKA signaling pathway stimulates oxalate transport by human intestinal Caco2-BBE cells

Most kidney stones are composed of calcium oxalate, and small increases in urine oxalate enhance the stone risk. The mammalian intestine plays a crucial role in oxalate homeostasis, and we had recently reported that Oxalobacter-derived factors stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells through PKA activation. We therefore evaluated whether intestinal oxalate transport is directly regulated by activation of the PKA signaling pathway. To this end, PKA was activated with forskolin and IBMX (F/I). F/I significantly stimulated (3.7-fold) [14C]oxalate transport by C2 cells [≥49% of which is mediated by the oxalate transporter SLC26A6 (A6)], an effect completely blocked by the PKA inhibitor H89, indicating that it is PKA dependent. PKA stimulation of intestinal oxalate transport is not cell line specific, since F/I similarly stimulated oxalate transport by the human intestinal T84 cells. F/I significantly increased (2.5-fold) A6 surface protein expression by use of immunocytochemistry. Assessing [14C]oxalate transport as a function of increasing [14C]oxalate concentration in the flux medium showed that the observed stimulation is due to a F/I-induced increase (1.8-fold) in Vmax and reduction (2-fold) in Km. siRNA knockdown studies showed that significant components of the observed stimulation are mediated by A6 and SLC26A2 (A2). Besides enhancing A6 surface protein expression, it is also possible that the observed stimulation is due to PKA-induced enhanced A6 and/or A2 transport activity in view of the reduced Km. We conclude that PKA activation positively regulates oxalate transport by intestinal epithelial cells and that PKA agonists might therapeutically impact hyperoxalemia, hyperoxaluria, and related kidney stones.