Selvaraj, Manikandan

~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.

Background: Cardiac fibrosis is a central pathological process that leads to worse outcomes in various cardiomyopathies. Recent evidence shows that CDCP1 (CUB domain-containing protein 1) modulates myocardial recovery by attenuating human cardiac fibroblast (HCF) proliferation. This study investigates the hypothesis that selective targeting of the CDCP1 cleavage domain can mitigate fibroblast proliferation, thereby offering a novel therapeutic approach to reducing cardiac fibrosis. Methods: A high-throughput in silico screening was performed using the Enamine compound library, focusing on structural regions that include the cleavage and adjacent sites of CDCP1 . A total of 147 candidate compounds were prioritized based on docking scores and binding stability profiles. Selected compounds were subsequently evaluated in vitro using HCF cultures seeded at 3,000 cells/well in 96-well plates. Each compound was administered at 10 μM concentration, and cell proliferation and viability were assessed by time-lapse imaging in the Incucyte® system at 24 and 96 hours. All experiments were done in triplicate with appropriate DMSO vehicle controls. Results: Compound C3 significantly reduced HCF proliferation (Fig B) to 27.3±2.4% relative to DMSO controls (99.9±0.5%, P <0.001) at 96 hours. Detailed structural modeling indicated that C3 binds to the CDCP1 cleavage site (Fig C) with a favorable Glide XP docking score of -7.66 kcal/mol. The interaction is stabilized by hydrogen bonds with Lys365 and Cys374 (Fig D), and supplementary hydrophobic interactions between aromatic rings and aliphatic groups, which contribute to a strong inhibitory effect. Conclusion: Targeting the CDCP1 cleavage domain with compound C3 effectively diminishes HCF proliferation in vitro. Therefore, these results substantiate the therapeutic potential of CDCP1 inhibition in cardiac fibrosis. Ongoing in vivo studies will explore the translational relevance of these findings.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)–derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration–approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.