Pathological biomineralization, particularly the formation of calcium oxalate (CaOx) kidney stones, affects approximately 10% of the world's population and is linked to serious health issues like hypertension and chronic kidney disease. Current treatments primarily involve surgical removal, which has a high recurrence rate. Medical management focuses on altering urinary chemistry to decrease CaOx crystallization and crystal-cell interactions. Citrate, a urinary constituent, inhibits CaOx crystallization. Natural polyphenols have also shown promise as inhibitors. Many *in vitro* studies focus on individual inhibitors, but higher concentrations often lead to toxicity. This study investigates the synergistic effects of combining citrate with natural polyphenols to enhance inhibition efficacy at lower concentrations, providing a potentially safer and more effective treatment strategy. The combination of inhibitors offers the possibility of synergistic, additive, or antagonistic effects, with synergistic effects being particularly desirable for therapeutic application. Previous research has shown both synergistic and antagonistic effects depending on the combination of inhibitors, making the exploration of effective synergistic combinations critical.

Existing literature highlights the role of various molecules in inhibiting COM crystallization. Metal ions like Zn²⁺, citrate derivatives, polyphosphates, and urinary proteins with specific functional groups are potential inhibitor candidates. Citrate, in particular, has shown effectiveness in suppressing CaOx crystallization and crystal-cell interactions. The study by Rimer et al. demonstrated hydroxy-citrate's unique strain-induced dissolution mechanism for COM growth retardation. Furthermore, previous research by the authors established the effectiveness of natural polyphenols as COM crystallization inhibitors. The mechanism of inhibition involves step pinning and kink blocking, where molecules either impede step propagation or hinder solute incorporation into kinks. Citrate functions as both a step pinner and a kink blocker, a property mimicked by some of the studied polyphenols. Existing research on CaOx crystallization highlights the limitations of single inhibitors requiring high concentrations, prompting the investigation of binary inhibitor combinations to improve efficacy and reduce toxicity.

The study employed multiple methodologies to evaluate the synergistic effects of combining citrate with four natural polyphenols: gallic acid (GaA), ellagic acid (EA), pyrogallic acid (PGA), and protocatechuic acid (PrA). Bulk crystallization assays at physiological pH (6.4) were performed to assess the impact of inhibitor pairs on COM crystal morphology and size using scanning electron microscopy (SEM). The growth kinetics of COM crystals were quantitatively assessed using a calcium ion-selective electrode (ISE) to monitor free Ca²⁺ depletion over time. Time-elapsed *in situ* atomic force microscopy (AFM) provided microscopic insights into the molecular mechanisms of COM (100) surface growth in the presence of various inhibitors. Step velocities in different growth directions were measured from sequential AFM images to quantify the growth impedance imposed by the inhibitors. Isobolograms were used to quantitatively evaluate the cooperative effects of inhibitor pairs on step propagation. Theoretical models were developed to describe the synergistic cooperativity in suppressing step velocity, incorporating common crystal growth models and experimental measurements. The models account for the dual inhibitory actions (step pinning and kink blocking) of both individual and combined inhibitors. These models allowed for correlation of inhibitor concentration with step propagation velocity. *In vitro* cytotoxicity and adhesion assays were conducted using human proximal tubular (HK-2) cells to evaluate the effects of inhibitor combinations on crystal-cell interactions, assessing cell viability, LDH release, and crystal adhesion. Finally, an *in vivo* study was performed on a mouse model of CaOx-induced nephrocalcinosis to assess the efficacy of inhibitor combinations in reducing renal CaOx deposition and kidney injury. Blood urea nitrogen (BUN) and serum creatinine levels were measured to assess kidney function. Statistical analyses included one-way ANOVA and Student's t-tests for *in vitro* studies and bootstrap distribution analysis for *in vivo* studies.

The study's key findings demonstrate a strong synergistic cooperativity between citrate and natural polyphenols in inhibiting COM crystallization. SEM images showed significant changes in COM crystal shape and size when citrate was combined with the polyphenols, indicative of enhanced inhibition. ISE measurements revealed that the combination of citrate with polyphenols significantly increased the inhibition percentage compared to citrate alone. *In situ* AFM measurements showed that the inhibitor pairs significantly reduced the step velocity compared to citrate alone, highlighting the synergistic effect at the microscopic level. Isobolograms confirmed the synergistic effect of the inhibitor pairs. The theoretical model accurately predicted the synergistic effect observed in the experiments, providing a mechanistic explanation. *In vitro* studies showed that the inhibitor combinations synergistically reduced COM crystal cytotoxicity and adhesion to HK-2 cells, as indicated by improved cell viability, reduced LDH release, and decreased crystal adhesion. The *in vivo* mouse model study demonstrated that the combinations of citrate with PGA, EA, and GAA synergistically reduced renal CaOx deposition and kidney injury, improving kidney function as measured by BUN and serum creatinine levels. The CA/EA combination showed superior *in vivo* performance in reducing crystal deposition compared to other pairs. The combination index (CI) values, a quantitative measure of synergistic effects, showed significantly lower values for the citrate-polyphenol combinations compared to previously reported synergistic pairs like citrate-chondroitin sulfate, indicating superior synergistic performance of the novel combinations.

The findings address the research question by demonstrating the substantial synergistic benefit of combining citrate with natural polyphenols in preventing CaOx kidney stone formation. The significance of the results lies in the identification of a novel therapeutic strategy that leverages the synergistic action of multiple inhibitors targeting the same binding site. This approach has the potential to improve the efficacy and reduce the toxicity of existing treatments. The mechanism of synergy, involving the reduction of both growth kinetic constants and the crystallization driving force, is explained by a theoretical model and supports the observed experimental results. The results are relevant to the field as they offer a promising avenue for developing more effective and less toxic treatments for kidney stone disease. The in vivo validation of these synergistic effects in a mouse model strengthens the clinical implications of these findings.

This study successfully demonstrated the synergistic cooperativity of combining citrate with natural polyphenols (GaA, EA, PGA, or PrA) for inhibiting COM crystallization and reducing kidney stone formation. The combination significantly boosted inhibition efficacy compared to citrate alone, both *in vitro* and *in vivo*. A theoretical model supports the experimental findings, elucidating the synergistic mechanism. These results highlight a novel therapeutic strategy for preventing CaOx kidney stones. Future research could focus on optimizing the ratios of the inhibitor pairs, exploring the long-term effects of the combinations, and evaluating their efficacy in human clinical trials. Further investigation into the specific molecular interactions between the inhibitors and the COM crystal surface would also enhance our understanding of the synergistic mechanism.

The study used a mouse model, which might not fully recapitulate the complexity of human kidney stone formation. The study focused on a specific pH range (6.4) relevant to kidney stone formation, and the efficacy of the inhibitor combinations might vary at different pH levels. The long-term effects of the inhibitor combinations on kidney health were not extensively evaluated in the *in vivo* study. The theoretical model made certain assumptions and simplifications, which could affect the accuracy of the predictions.