Category: Basic Science: Stones
Introduction & Objective : Endogenous oxalate synthesis primarily occurs via lactate dehydrogenase (LDH) activity in the liver. Liver specific RNAi therapeutics are currently in clinical trials for a number of diseases. Previous work using RNAi against liver glycolate oxidase demonstrated reduction of urinary oxalate in the Agxt knock out mouse, a model for primary hyperoxaluria Type 1 (PH1). Our objective was to evaluate the effects of siRNA knock down of liver LDHA in wild type (Wt) and PH1 mouse model (Agxt knock out (KO)).
Methods : Wild-type (n=6) and Agxt KO (n=6) mice were placed on an ultra-low oxalate diet. The mice were subcutaneously administered 4 weekly doses (10mg/kg) of LDHA siRNA (Alnylam Pharmaceuticals). 24-hour urine samples were collected at baseline and following 4 weeks of LDHA siRNA treatment. Plasma, liver and muscle tissue were collected at completion of the study. Urinary, plasma, and tissue metabolites were measured using ion chromatography/mass spectrometry (oxalate, glycolate and lactate) and high pressure liquid chromatography (pyruvate and glyoxylate). LDH specific activity was determined in tissue lysates with either lactate or glyoxylate as the assay substrate and measuring the rate of reduction of NAD at 340nm at pH 9.0. Statistical analysis was performed utilizing Student t-test.
Results : There was a >98% decrease in liver specific LDH activity utilizing glyoxylate as a substrate, and >56% decrease in liver specific LDH activity with lactate as a substrate. There was no change in LDH activity in muscle and heart tissue lysates following LDHA siRNA treatment. Changes in urine, plasma and liver metabolites with LDHA siRNA treatment are shown in Table 1.
Conclusions : Knock down of hepatic LDHA using siRNA results in a profound decrease in urinary oxalate excretion in both Wt and Agxt KO mice suggesting this approach may be an effective therapy for primary hyperoxaluria syndromes as well as idiopathic hyperoxaluria. Liver, plasma, and urinary glycolate levels were reduced after treatment suggesting liver LDH activity plays an important role in whole body glycolate metabolism. The long-term consequences of a decrease in the liver lactate pyruvate ratio with LDHA siRNA warrants further investigation.
Kyle Wood– Assistant Professor, University of Alabama at Birmingham, Birmingham, Alabama
John Knight– Assistant Professor, University of Alabama at Birmingham, Birmingham, Alabama
Ross Holmes– Professor, University of Alabama at Birmingham, Birmingham, Alabama
Dean Assimos– Professor, University of Alabama at Birmingham, Birmingham, Alabama
University of Alabama at Birmingham
Kyle David Wood MD is an Assistant Professor at the University of Alabama at Birmingham. He has completed an Endourology Fellowship. His clinical practice is focused on the medical and surgical management of complex kidney stones. He was an AUA Research Scholar and recently recieved a K08 NIH mentored grant. His research interest is on endogenous oxalate synthesis and its relationship to kidney stone disease.
University of Alabama at Birmingham
Dr. Dean George Assimos is currently the Anton J. Bueschen Professor and Chairman of the Department of Urology at the University of Alabama at Birmingham School of Medicine. He obtained a BS degree in biology from Purdue University in 1974 and an MD degree from Loyola University of Chicago in 1977. He subsequently completed his urology residency at Northwestern University in 1983. He was a fellow in intra-renal surgery and kidney stone disease at Wake Forest University in 1984 after which he was an A.U.A. research scholar at the same institution from 1984-1986. He has dedicated his career to the care of patients afflicted with complex renal stone disease including medical and surgical management. He has conducted clinical, translational and basic science research focusing on factors which impact urinary oxalate excretion. He and his research colleagues have received steady funding from the NIH for these endeavors. They have demonstrated the important contribution of dietary oxalate to the urinary oxalate pool, defined the importance of interactions between dietary calcium and oxalate, and characterized mechanisms of gastrointestinal oxalate absorption and endogenous oxalate synthesis. He is the author of numerous papers and chapters pertaining to these subjects. He has served as a member of the A.U.A Nephrolithiasis Guidelines and was the Chair of this committee. Dr. Assimos is on the editorial board of several urologic journals. He is also committed to urologic education participating as a speaker and course director for the A.U.A. and other organizations. He has received several prestigious honors including the American Urological Care Foundation Distinguished Scholar Alumnus Award, the Ralph Clayman Mentor Award from the Endourological Society, and the AUA Hugh Hampton Young Award. Dr. Assimos is the past president of the Southeastern Section of the A.U.A.