Introduction: Kidney stone disease affects between 7 and 13% of the North American population and continues to burden global healthcare systems. The majority of kidney stones are calcium-based, with approximately 80% composed of calcium oxalate (CaOx). Despite advances in current preventative strategies, first-time symptomatic stone formers have an approximate 39% recurrence rate after 15 years. Thus, it is clear that more effective strategies are needed to prevent disease recurrence. Osteopontin (OPN) is a urinary protein that can inhibit the formation of CaOx monohydrate (COM) crystals in vitro. Numerous studies have confirmed that human and other mammalian OPN isoforms are potent inhibitors of COM crystallization. Despite this, the efficacy of these peptides as a supplement to prevent kidney stones has never been assessed in a living organism. We therefore sought to determine if a 16-residue synthetic OPN phosphopeptide (hereafter denoted as P3) could reduce CaOx stone burden and adhesion to mammalian kidney cells using in vivo and in vitro models, respectively. Methods: To evaluate the effect of P3 on adhesion of COM crystals in vitro, synthetic crystals were allowed to adhere to Madin-Darby canine kidney (MDCK) cells in the presence of P3. An established Drosophila melanogaster model of CaOx-based urolithiasis was used to test the applicability of P3 to reduce stone burden in vivo. CaOx crystal burden and morphology were assessed using birefringence and confocal microscopy, respectively. Results: P3 directly reduced the adherence of synthetic COM crystals to MDCK cells. Furthermore, oral supplementation of P3 significantly reduced CaOx burden in D. melanogaster. Confocal microscopy of crystals in D. melanogaster Malpighian tubules revealed that P3 was able to interact with COM crystals and alter morphology similar to previous in vitro studies. Conclusions: Altogether, this study demonstrates that a synthetic OPN phosphopeptide can directly mitigate CaOx-based urolithiasis formation in vivo by modulating crystal morphology. While in vitro evidence from simplistic models supports the inhibitory role of OPN phosphopeptides on CaOx crystal formation, this is the first study to directly illustrate that they can mitigate crystal formation in a complex living system. These findings suggest that OPN supplementation is a promising therapeutic approach and may be clinically useful in the management of recurrent CaOx urolithiasis in humans. SOURCE OF Funding: N/A
