Daniel Massana Roquero, PhD, was one of the recipients of the 2024 Urology Care Foundation™ Research Scholar Awards. These awards provide $40,000 annually for mentored research training for clinical and postdoctoral fellows or early-career faculty. The Endourological Society sponsored Dr Massana Roquero’s award.

Kidney stone disease is highly prevalent and most commonly caused by calcium stones. In most cases, calcium stones are present in patients without any apparent metabolic disorder or systemic disease.¹ It has long been thought that calcium stones originate from an imbalance in the urine salts, leading to supersaturation and precipitation in the form of crystals. While still seen as a contributing factor, research indicates that mineral supersaturation is not more prevalent in stone formers than in control groups.^2,3 In our experience, we have found that approximately 35% of calcium stones freshly collected from stone surgeries harbored a wide range of gram-positive and gram-negative uropathogens. This prompted us to consider the connection between bacteria and calcium stones from a different perspective. Assessing whether the connection between bacteria and calcium stones is causative or coincidental is essential to identify risk factors for stone formation and guide prevention and therapy. In the last decade, the association of bacteria with calcium stones has been under heavy scrutiny by several research groups, which have shown that various types of bacteria can induce or aggravate calcium stone disease.^4,5

In this proposal, we aim to study the influence of bacteria in calcium stone, with a particular focus on the role that bacterial biofilms play in contributing to mineralization and overall stone growth. Bacteria can adhere to solid substrates, such as stones or calcium oxalate crystals, by encasing themselves in a self-produced polymeric matrix, forming biofilms. We hypothesize that biofilms may play a role in inducing calcium crystal aggregation and encrustation and also promote the mineralization of calcium salts in their interface with the urine microenvironment. In addition, we hypothesize that calcium stones with significant bacterial burdens have specific chemical constituents and structural domains that are not found in abiotic, pure metabolic calcium stones.

Our hypotheses find strong support in naturally occurring bacterial-induced calcium stones within aqueous ecosystems. These formations, known as microbialites, arise from the intricate interplay between microbial activities, specifically bacterial metabolisms, and the heightened saturation of ions in water.^6,7 Furthermore, numerous recent studies have illustrated that bacteria can stimulate the biomineralization of calcium carbonates and calcium phosphates.^8,9 Our objective is to gather robust evidence of these occurrences through an in vitro model simulating supersaturated urine and by performing a thorough biochemical exam of human-derived stones ex vivo.

Employing a rigorous in vitro model simulating hypercalciuria, we will examine the influence of patient-derived uropathogen biofilms on the processes of crystal aggregation, encrustation, and mineralization by optical and electron microscopy. Further, we aim to identify the chemical and biological constituents of patient-derived calcium stone fragments and their distribution within the stone. We will perform a high-resolution characterization of the stone’s chemical and microbial composition by combining advanced characterization and imaging techniques. In addition, we will conduct an extensive mesoscopic and microscopic characterization of whole stone fragments to understand the 3D distribution of different biological and chemical constituents as well as crystal phases.

We are confident that the meticulously crafted experiments, coupled with high-resolution techniques, will yield intriguing and insightful information on the connection between biofilms and calcium stones. I am sincerely grateful to the urology community for their support, which will enable me to continue my work and grow as a young scientist on my path to becoming an independent investigator. I am thankful to my advisor, Dr Joseph Liao, for his support and the opportunity to leverage my background in chemistry in both translational medical device development and basic science research, both related to benign urological diseases. Additionally, I would like to extend my gratitude to the Department of Urology at Stanford University and external collaborators for their support.