Pre-Specified

oxypurinol

Background: High urinary oxalate levels (UOx) in patients with enteric hyperoxaluria (EH) can lead to recurrent kidney stones, nephrocalcinosis and chronic kidney disease.SYNB8802 is an engineered E. coli Nissle 1917 that contains an oxalate degradation pathway which converts oxalate to formate within the gastrointestinal (GI) tract, thereby reducing urinary oxalate.SYNB8802 is an oral, non-colonizing live biotherapeutic developed for the treatment of EH.SYNB8802 is explored in a Phase 1a/b study in healthy volunteers (HV) and Roux-en-Y (RYGB) patients with hyperoxaluria.
Methods: In Part A of the study [NCT04629170] hyperoxaluria was induced in adult HV by a high oxalate (400-600mg), low calcium (400mg) diet over 4 days.Subjects were then randomized (6 active:3 placebo) to receive 5 days of SYNB8802 or placebo TID.24hr UOx levels were measured daily.Primary outcome was safety and tolerability.Part B is a double-blind, placebo-controlled crossover study of SYNB8802 in subjects with enteric hyperoxaluria and a history of RYGB.Up to 20 subjects will be randomized in a crossover design with a 2-week washout period to receive SYNB8802 (at 3e11 live cells/dose) or placebo, dosed up to TID with meals.Urine samples for determination of 24hr UOx levels will be collected over 3 days at baseline and on the last 3 days of each dosing period.Subjects will maintain their normal diet throughout the study.The primary endpoint is change from baseline in 24hr UOx amount excreted with SYNB8802 treatment versus placebo.Secondary endpoints include change from baseline in UOx:creatinine ratio with SYNB8802 treatment versus placebo.
Results: In Part A, a well-tolerated dose of 3e11 live cells was identified in HV.At this dose, the percent change from baseline UOx levels was -28.6% (90% CI: -42.4 to -11.6) compared to placebo in diet-induced hyperoxaluria.This dose is being studied in Roux-en-Y patients with hyperoxaluria in Part B. The results from the RYGB population will be reported.
Conclusions: These results provide proof of mechanism for UOx lowering by SYNB8802 through GI consumption of oxalate in diet-induced hyperoxaluria.Part B seeks proof-of-concept in patients with enteric hyperoxaluria.

PO2027 Poster
Clinical Pharmacology, Pharmacokinetics, and Drug Toxicity in Kidney Diseases Background: Although calcineurin inhibitors (CNIs) such as tacrolimus and cyclosporin have dramatically improved the quality of patient care, long-term therapy causes irreversible damage to the kidneys in the form of renal fibrosis.These morphologic changes ultimately lead to a decline in renal function and can progress to end-stage renal failure.These detrimental outcomes present a critical need to identify the driving mechanisms by which CNIs cause renal damage.It is well established that TGFb is a major contributor to CNI-induced renal fibrosis.However, the underlying mechanisms remain unknown.The objectives of this study are to 1) investigate whether TGFb secretion is required to stimulate TGFb receptor signaling in a model of CNIinduced renal fibrosis and 2) investigate whether calcineurin plays a critical role in regulating TGFb receptor activity.
Methods: To examine the role of calcineurin inhibition in altered TGFb receptor signaling, wild type mice were treated with either vehicle (100% ethanol) or 10 mg/kg tacrolimus for 7 days.To confirm in vivo findings, wild-type mouse renal cortical fibroblasts were treated with either vehicle (100% ethanol) or 1nM tacrolimus for 24 hours in the presence and absence of anti-TGFb neutralizing antibodies.TGFb receptor expression and activation, TGFb receptor downstream signaling mediators, profibrotic markers and calcineurin activity were analyzed.
Results: Findings demonstrate that tacrolimus-induced loss of calcineurin activity is accompanied with enhanced TGFb receptor activation and signaling.Notably, increasing concentrations of anti-TGFb neutralizing antibodies failed to abolish aberrant TGFb signaling and increased expression of profibrotic markers.
Conclusions: Together, these results demonstrate that 1) CNIs promote ligandindependent TGFb signaling and 2) calcineurin plays a functional role in regulating TGFb receptor activity.
Funding: NIDDK Support, Other U.S. Government Support

PO2028 Poster
Clinical Pharmacology, Pharmacokinetics, and Drug Toxicity in Kidney Diseases

Immunoreactivity of CD3+CD4+ in Stable Young, Middle Aged, and Elderly Kidney Transplant Recipients Receiving Maintenance Tacrolimus and Mycophenolic Acid Immunosuppression
Kathleen M. Tornatore, 1,2 Aijaz A. Gundroo, 3,1 Shirley S. Chang, 1,2 Mahfuz Yonis, 1,2 Kris Attwood, 4,1 Brian M. Murray.Background: Tacrolimus and mycophenolic acid are the standard of care in most stable kidney transplant recipients (KTR) at U.S. transplant centers.However, there are limited data that determine within subject immunophenotypic responses over the adult age range.This study examined ex vivo immunoreactivity of CD3+CD4+ lymphocytes in stable young, middle age and elderly KTR receiving tacrolimus and mycophenolic acid.
Methods: Fifteen stable KTR greater than 1 yr post-transplant completed a 12-hour study with serial collections at pre-dose (trough-0 hr), 4, 8 and 12 hours.The immune response potential was evaluated by Interleukin-2 (IL-2) and Interferon gamma (IFN-γ) production by CD3+CD4+ T cells after ex-vivo treatment with PMA/Ionomycin with Brefeldin-A.Data was represented as within individual, timed collection and the mean for all time points of ex-vivo stimulation by cell sub-populations stratified by young, middle age and elderly.Comparisons were made using Kruskal-Wallis test.
Results: Table summarizes the major findings.There were no group differences between tacrolimus and mycophenolic acid troughs with all tacrolimus troughs within the therapeutic range.Increased IFN-γ from CD3+CD4+ T cells was quantitated by ex vivo immunoreactivity in middle age recipients at the 4 and 8 hours during the 12-hr study period.No significant differences were noted for interleukin-2 quantitated from CD3+CD4+.
Conclusions: These data indicate increased IFN-γ from CD3+CD4+ T cells for ex vivo immunoreactivity over a 12-hr dosing interval in middle age KTR receiving longterm maintenance immunosuppression.Variable immunodynamics and the implications of intra-and interpatient variability in immunoreactivity across the range of adult KTR require further investigation of clinical and allograft outcomes.
Funding: Other NIH Support -National Institute of Aging
Results: Empagliflozin treatment increased urinary glucose excretion in parallel to empagliflozin plasma levels in a dose-dependent manner starting at doses of 1 mg/kg.5/6Nx rats on HSD treated with this low empagliflozin dose showed significantly reduced cardiac (-34.85%;p<0.05) and renal (-33.68%; p<0.05) fibrosis in comparison to 5/6Nx rats on HSD treated with placebo.These effects were comparable to the effects of a standard dose (5mg/kg/day) of telmisartan (cardiac fibrosis: -36.37%; p<0.01; renal fibrosis; -43.96%; p<0.01).RNA-sequencing followed by confirmatory qRT-PCR revealed that both telmisartan and empagliflozin exert their cardiac effects on genes involved in vascular cell stability and cardiac iron homeostasis, whereas in the kidneys expression of genes involved in endothelial function and oxidative stress were differentially expressed.Urinary adenosine excretion, a surrogate marker of the tubuloglomerular feedback (TGF) mechanism, was not affected.
Conclusions: The antifibrotic properties of low dose empagliflozin were comparable to a standard dose of telmisartan.The underlying pathways seem to be TGF independent.
Funding: Commercial Support -Boehringer Ingelheim Pharma GmbH & Co. KG

PO2031 Poster
Clinical Pharmacology, Pharmacokinetics, and Drug Toxicity in Kidney Diseases
Background: Inhibition of prolyl-4-hydroxylase (PHD) enzymes leads to the stabilization of hypoxia inducible factor (HIF) and the expression of HIF target genes.Because of effects on erythropoiesis, several PHD inhibitors are undergoing clinical evaluation for the treatment of anemia with chronic kidney disease.However, the impact on other biological functions is not well investigated.We demonstrate that AKBX27922, a novel small molecule PHD inhibitor, can shift cellular metabolism from mitochondrial oxidative phosphorylation to glycolysis, mimicking adaptation to hypoxia.
Methods: Inhibition of PHD enzymatic activity was determined using the timeresolved fluorescence resonance energy transfer assay.HIF1a stabilization in Hep3B cells was measured by meso scale discovery technology and protein expression of HIF target genes by enzyme linked immunosorbent assay.Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in HepG2 cells with the Seahorse technology.Pharmacodynamics of HIF stabilization were confirmed in vivo.
Results: In vitro, AKBX27922 potently and in a concentration-dependent manner, and without chelating iron, inhibited PHD1 and PHD2 enzyme activity, leading to HIF1a stabilization and expression of HIF target genes implicated in erythropoiesis, angiogenesis, glycolysis, and cell survival.Pretreatment of HepG2 cells with AKBX27922 dosedependently reduced both basal and maximal OCR without affecting cellular viability, while ECAR was significantly increased.Reactive oxygen species production in human primary renal epithelial cells was decreased.In vivo, AKBX27922 stabilized HIF in the liver and kidneys, as measured by luciferase activity in the oxygen-dependent degradation domain (ODD)-luciferase reporter mouse.In rats, AKBX27922 induced time-dependent stabilization of HIF1a in the kidney medulla and papilla, and increased expression of glycolysis related (ALDOC, CAR9, PDK1, PFKFB4, LDH) and other HIF-target genes (EPO, ADM, HMOX-1) in the kidneys and liver.
Conclusions: PHD inhibitor AKBX27922 mimics hypoxia, leading to HIF-driven metabolic adaptation.This novel small molecule will be useful as an in vitro and in vivo research tool for additional mechanistic studies that probe the pleotropic biology of HIF.
Background: The proposed project comprises of the development of a kidney proximal tubule (KPT) microphysiological system (MPS) from human cells as well as two experimental animal species that are typically used in kidney toxicity screening: rat and dog.These KPT-MPS can serve as an important new tool in chemical toxicity screening, allowing cross-referencing animal-based MPS data within vivoanimal data and with human-based MPS data and clinical outcomes.It also has the potential to result in a significant reduction of the use of live animals in studies.
Methods: The Nortis chip is made from silicone in a polycarbonate casing and is designed to use the "mandrel" method for generating channels within a 3D extracellular matrix using retractable small glass fibers.The channels serve as starting points for generating tubular tissue structures, such as vessels or kidney tubules.The chip is compatible with high-quality imaging, tissue sampling, and up-and down-stream fluid collection.Multiple publications have documented the suitability of the Nortis system to generate functional human KPTs and how well they resemble the function of in vivo tubules.All 3D MPS experiments are accompanied by 2D controls for comparison, using a traditional culture dish system.To assess viability of tissue, Live-Dead staining assays were run on canine tubules with Calcein-AM (live) and the nucleic acid stain ethidium homodimer I (dead), the results of which indicated sufficiently viable tubules.Confocal imaging and 3D rendering of these tubules demonstrates presence of key ion and drug transport proteins in their respective basolateral and lumenal domains.
Results: Preliminary studies have shown that rat and canine derived KPT-MPS in the Nortis platform produce structurally viable tissue structures that elicit injury markers in response to nephrotoxic insults using in vivo relevant toxic compounds in a differential manner.
Conclusions: Our preliminary data suggests that Nortis kidney chip allows for an ideal predicitive platfrom for comparative toxicity studies, allowing for fast and highly predictive preclinical simulations.

Tacrolimus Induces Ligand-Independent TGF-β Receptor Signaling to Promote Renal Fibrosis
Adaku C. Ume, Clintoria R. Williams.Kidney Pathophysiology Research Group Wright State University Boonshoft School of Medicine, Dayton, OH.