Citations

~80% of kidney stones (KS) are composed of calcium oxalate and small increases in urinary oxalate significantly enhance the stone risk. We previously discovered Oxalobacter formigenes (Of)-derived secreted factors which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion (JASN, 2017). The small Sel1-like protein-derived peptides (P8 and P9) were identified as the major Of-derived factors (AJP Cell Physiol, 2023), acting via the oxalate transporters SLC26A6 (A6) and SLC26A2 (A2). P8/9-induced stimulation of oxalate transport in C2 cells is likely PKA-dependent based on studies using the PKA inhibitor H89. Enhancing gut oxalate secretion with subsequent fecal excretion represents a novel therapeutic pathway for getting rid of body oxalate. Building on these findings, the study objective is to test the hypothesis that the observed regulation is indeed PKA-mediated, through mechanisms potentially involving other signaling pathways and/or altering A6 total and/or surface protein expression. To provide further evidence that it is PKA-dependent, we successfully identified the binding mode of P8/9 bound to PKA using molecular docking (ZDOCK). The regulatory subunit of PKA binds two cyclic AMPs and P8/P9 are predicted to interact at the cyclic AMP binding site according to our PKA-peptide modeling studies. To directly confirm the role of PKA, we used siRNA to knockdown its α catalytic subunit (PKA-α) in C2 cells. PKA-α mRNA expression is reduced by >90% as assessed by qPCR. Importantly, siRNA knockdown of PKA-α completely abolished P8/P9-induced stimulation of 14C-oxalate uptake by C2 cells in pilot studies, confirming the role of PKA in P8/9-mediated stimulatory regulation of intestinal oxalate transport. To identify the signaling pathways potentially upstream and/or downstream of PKA we used relatively selective pharmacological inhibitors. Interestingly, the stimulatory effects of P9 on 14C-oxalate uptake by C2 cells are significantly blocked by the ERK1/2 inhibitor U0126 (~90%), the PI3K inhibitor LY294002 (~84%), and the p38 inhibitor SB 202190 (~73%), while the general tyrosine kinase inhibitor genistein has no effect. Inhibitors alone did not affect baseline transport. These results support the involvement of the MAPK ERK1/2, PI3K, and p38 in the signaling pathways regulating intestinal oxalate transport by P8/9. We used immunoblotting and surface biotinylation studies to evaluate whether P8/9 stimulates oxalate transport in C2 cells by increasing its total and/or surface protein expression. P8/9 peptides did not affect A6 total and surface protein expression levels in preliminary studies. We could not assess A2 total and surface protein expression due to lack of good working antibodies. In view of our previous findings that the observed regulation is due to P9-induced increase in Vmax and reduction in Km, and since P8/9 did not affect A6 surface protein expression, it is likely possible that the observed stimulation is due to mechanisms including P8/9-induced enhanced A6 transport activity resulting from an increase in the intrinsic activity of the preexisting A6 membrane transporters. We conclude that P8/9 stimulates intestinal oxalate transport in C2 cells via signaling pathways including PKA, ERK1/2, PI3K, and p38 and through mechanisms including P8/9-induced enhanced A6 transport activity. Having the P8/9-PKA bound conformation determined, we plan to optimize P8/P9 into small molecule like drugs with better stability and clinical translational potential in terms of stimulating intestinal oxalate secretion, leading to reduced plasma and urinary oxalate levels. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Objective: This study aims to develop a contrast-free, high-sensitivity ultrasound method, denoted as Radon transform-based flow measurement (R-Flow), for in vivo mapping of microvascular flow vectors and for establishing R-Flow-derived vector-field metrics to noninvasively quantify microcirculatory patterns in liver cirrhosis. Impact Statement: R-Flow enables robust, contrast-free imaging of microvascular dynamics and demonstrates translational feasibility in the human liver. Its direction-aware indices offer pilot in vivo quantification of flow redistribution and remodeling, providing unique insights into hepatic flow dynamics. Introduction: Microvascular dysfunction is a hallmark of many diseases, yet noninvasive visualization and quantitative assessment of abnormal microcirculation remain limited. Methods: R-Flow leverages Radon transform to decode red blood cell dynamics from the spatiotemporal domain of ultrasound flow signals, reconstructing velocity vectors at microvascular scale. From these vector maps, unique direction-aware indices are further derived to characterize flow distribution and heterogeneity. Results: Validated across simulations, phantoms, and in vivo studies, R-Flow provides robust velocity estimation across a wide range (1 to 60 mm/s). Notably, it enables high-sensitivity microvascular flow vector mapping of human liver, showing strong agreement with references ( r > 0.9). In a rat model, these direction-aware indices revealed a shift from healthy multipath perfusion in control livers to directionally biased vascular pattern in cirrhotic livers, demonstrating significant correlation with pathological indicators. Conclusion: R-Flow enables noninvasive, contrast-free mapping of microvascular blood flow velocity and offers a promising approach for high-resolution assessment of microvascular flow characteristics.