Hydroxyproline Metabolism and Oxalate Synthesis in Primary Hyperoxaluria

BASIC RESEARCH www.jasn.org

Sonia Fargue,1 Dawn S. Milliner,2 John Knight,1 Julie B. Olson,2 W. Todd Lowther,3 and Ross P. Holmes1

1Department of Urology, University of Alabama at Birmingham, Birmingham, Alabama; 2Mayo Clinic Hyperoxaluria Center, Division of Nephrology and Hypertension, Rochester, Minnesota; and 3Center for Structural Biology, Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina

ABSTRACT

Background Endogenous oxalate synthesis contributes to calcium oxalate stone disease and is markedly increased in the inherited primary hyperoxaluria (PH) disorders. The incomplete knowledge regarding oxalate synthesis complicates discovery of new treatments. Hydroxyproline (Hyp) metabolism results in the formation of oxalate and glycolate. However, the relative contribution of Hyp metabolism to endogenous oxalate and glycolate synthesis is not known.

Methods To define this contribution, we performed primed, continuous, intravenous infusions of the 15 13 stable isotope [ N, C5]-Hyp in nine healthy subjects and 19 individuals with PH and quantified the levels 13 13 of urinary C2-oxalate and C2-glycolate formed using ion chromatography coupled to mass detection. Results The total urinary oxalate-to-creatinine ratio during the infusion was 73.1, 70.8, 47.0, and 10.6 mg oxalate/gcreatinineinsubjectswithPH1,PH2,andPH3 and controls, respectively. Hyp metabolism accounted for 12.8, 32.9, and 14.8 mg oxalate/g creatinine in subjects with PH1, PH2, and PH3, respectively, compared with 1.6 mg oxalate/g creatinine in controls. The contribution of Hyp to urinary oxalate was 15% in controls and 18%, 47%, and 33% in subjects with PH1, PH2, and PH3, respectively. The contribution of Hyp to urinary glycolate was 57% in controls, 30% in subjects with PH1, and ,13% in subjects with PH2 or PH3. Conclusions Hyp metabolism differs among PH types and is a major source of oxalate synthesis in individuals with PH2 and PH3. In patients with PH1, who have the highest urinary excretion of oxalate, the major sources of oxalate remain to be identified.

J Am Soc Nephrol 29: ccc–ccc, 2018. doi: https://doi.org/10.1681/ASN.2017040390

Endogenous oxalate synthesis makes an important aminotransferase (AGT1), prevents the conversion of contribution to calcium oxalate stone disease and is glyoxylate to glycine.2,3 In PH2, the deficiency in the main cause for the pathologies that develop in glyoxylate reductase/hydroxypyruvate reductase the rare, recessive, inherited primary hyperoxaluria (PH) disorders.1 Three genetic forms of PH have been identified, associated with mutations in AGXT Received April 7, 2017. Accepted March 6, 2018. for PH1, GRHPR for PH2, and HOGA1 for PH3 Published online ahead of print. Publication date available at (Figure 1, Supplemental Figure 1). PH1 and PH2 www.jasn.org. fi both result from de ciencies in enzymes that me- Correspondence: Dr. Ross P. Holmes, Department of Urology, tabolize glyoxylate to other compounds. In PH1, University of Alabama at Birmingham, 720 20th Street, South, the deficiency in the peroxisomal, pyridoxine- Kaul 816, Birmingham, AL 35294. Email: [email protected] dependent, liver-specific enzyme, alanine:glyoxylate Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: ccc–ccc, 2018 ISSN : 1046-6673/2906-ccc 1 BASIC RESEARCH www.jasn.org

(GRHPR; also known as GR) prevents the conversion of glyoxylate Significance Statement to glycolate in mitochondria and cytosol.4–6 PH3 results from mutations in the mitochondrial enzyme 4-hydroxy-2-oxoglutarate al- Theprimaryhyperoxalurias (PH)areagroupofrare,inheriteddisorders dolase (HOGA).7,8 HOGA catalyzes the last step of hydroxyproline characterized by kidney stone formation and severe kidney damage (Hyp) catabolism converting 4-hydroxy-2-oxoglutarate (HOG) to resulting from excessive endogenous oxalate synthesis. Limited treatment options are available for PH. Hydroxyproline metabolism, glyoxylate and pyruvate. It has been shown that HOG inhibits GR known to contribute to oxalate synthesis, is a potential target for ox- activity.9 Thus, PH3 has also been proposed to be a disorder alate reduction therapy. Using a stable isotope of hydroxyproline, the of glyoxylate metabolism. Outside of supportive measures, there contribution to urinary oxalate excretion was measured in healthy and are few therapeutic options for individuals with PH apart from PH subjects. Our results show that hydroxyproline accounts for up to the approximately 30% of patients with PH1 who respond half of the endogenous oxalate synthesis in PH2 and PH3, more than twice that of healthy subjects. In PH1, surprisingly, the bulk of the fully or partially to therapeutic doses of the cofactor for AGT, oxalatesynthesizedinPH1wasnotderivedfromhydroxyprolineandits pyridoxine.10 Liver/kidney transplantation is often the only treat- source remains to be determined. ment option for those with severe disease. Thus, development of new treatments is an important focus of current PH research. 12 Our understanding of how oxalate is synthesized in PH is in- most severe clinical outcomes. Dietary studies in mice, rats, pigs, complete. Glyoxylate is thought to be the major precursor of and humans have indicated that Hyp ingestion results in increased 13–17 endogenous oxalate synthesis in PH.1 Sources of glyoxylate syn- endogenous oxalate synthesis and urinary oxalate excretion. thesis include Hyp, glycine, glyoxal, and glycolate1,11 (Figure 1). Although it cannot be synthesized de novo, Hyp is a normal blood However, the relative contributions of these compounds to glyox- and tissue constituent with collagen turnover as its major source. ylate and oxalate synthesis are unknown in healthy individuals Hyp is produced by a post-translational hydroxylation of proline and notably in the different PH types. In addition to the increased residues principally in collagen.18 Our recent studies in normal 15 13 oxalate, there is often an increased urinary excretion of glycolate in mice infused with [ N, C5]-Hyp indicated that, under the ex- PH1, which is the most frequent of all PH types and carries the perimental conditions used, Hyp contributed approximately 20% of the oxalate excreted in urine.19 These results have recently been confirmed in HYPDH null mice (Prodh2 gene) that cannot metabolize Hyp and excreted approximately 20% less oxalate than wild-type mice on a low-oxalate diet (J. Knight, W.T. Lowther, andR.P.Holmes,unpublishedresults). The objective of this study was to determine the contribution of Hyp metabolism to urinary oxalate and glycolate in healthy subjects and individuals with PH, using continu- 15 13 ous, intravenous infusions of [ N, C5]-Hyp and measurements of 13C-labeled metabolites of Hyp in plasma and urine. This information will be of value in the identification of novel therapeutic targets for treatment of patients with PH.

Figure 1. Metabolism of Hyp and glyoxylate in hepatocytes. Oxalate synthesis by METHODS lactate dehydrogenase (LDH) is stimulated in three types of patients with PH by defects in either AGT (PH1, “*”), glyoxylate reductase (GR; PH2, “**”), or HOGA (PH3, Subjects “***”) (see Supplemental Figure 1 for detailed changes to metabolism that occur for A total of nine healthy subjects (5 males each PH type). Hyp is converted enzymatically to HOG in three steps. The action of [M]/4 females [F]) were studied at Wake HOGA results in the formation of glyoxylate and pyruvate. Mutations in HOGA lead to Forest School of Medicine and the Univer- a buildup of HOG and its reduced form, DHG. Importantly, the glyoxylate and gly- sity of Alabama at Birmingham. Nineteen colate are interconverted by the action of GR and glycolate oxidase (GO). D-amino patients with PH (12M/7F) attended the oxidase (DAO) is also a potential source of glyoxylate. Whereas GR is both cytosolic and mitochondrial and expressed ubiquitously, AGT and GO are peroxisomal and liver Hyperoxaluria Center at the Mayo Clinic specific. The factors that influence movement of metabolites between different sub- (Rochester, MN) for treatment. The inclu- cellular compartments are not known, but previous work and this study support ex- sion criteria for patients were age 15–85 change of glyoxylate metabolites between mitochondrial and peroxisomal pathways, years; confirmed eGFR.50 ml/min per as well as a level of redundancy and/or plasticity in these two pathways.21 1.73 m2 using plasma creatinine20;a

2 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2018 www.jasn.org BASIC RESEARCH

Chemicals Reagent grade chemicals were obtained from either Sigma-Aldrich Chemicals (St Louis, MO) or Fisher Scientiﬁc (Pittsburgh, 15 13 PA). [ N, C5]-Hyp (the nitrogen and all ﬁve carbons were isotopically labeled) was obtained from Almac following a custom synthesis (Craigavon, United King- 19,21 dom), and D3-leucine from Cam- bridge Isotopes (Andover, MA).

Sample Preparation Aliquots of urine samples from the infusion Figure 2. Infusion scheme for study subjects. After 3 days on a controlled diet and the fi collection of two 24-hour urine samples, fasted subjects were admitted to the infusion were acidi ed to a concentration of 50 mM 15 13 HCl (final pH,2) before freezing to pre- units. The infusion of [ N, C5]-Hyp and D3-leucine was initiated with a bolus and maintained at a constant rate for 6 hours. Collection of plasma (every 30 minutes) and vent possible oxalate crystallization that urine (every hour) was started 1 hour before the start of the infusion (defined as T0). could occur with cold storage or oxalogenesis associated with alkanization. Nonacidified urine was frozen for the measurement of genetically confirmed diagnosis of PH1, PH2, or PH3; and no other urinary parameters. All plasma and urine samples from history of transplantation for the PH group. Six of the PH1 patients with PH were shipped frozen and stored at 280°C. For subjects had at least one missense mutation and were treated plasma analyses by ion chromatography coupled with mass spec- with pyridoxine, which was maintained during the study. trometry (IC/MS), samples were filtered through Spin-X UF cen- Four of these patients with PH1 carried a single allele with a trifugal filters (Corning, NY) with a 10-kD nominal molecular known B6-responsive mutation (p.Gly170Arg or p. mass limit, and the ultrafiltrate was acidified by the addition of Phe152Ile). For control subjects, a normal serum and urine 2MHCltoafinal concentration of 60 mM (final pH,2). Cen- profile and good health assessed by their medical history was trifugal filters were washed with 0.01 M HCl before sample filtra- required. To eliminate the confounding effect of dietary oxa- tion to remove any contaminating trace organic acids trapped in late, the intake of dietary oxalate and calcium was controlled the filter device. for the infusion. For 3 days preceding the infusions, but not for baseline assessment, the study subjects consumed a low- Analytic Methods oxalate diet (50–100 mg of oxalate) containing 16% protein, Urinary creatinine was measured in nonacidified urine on an 30% fat, and 54% carbohydrate; 200 mg of ascorbate; and EasyRA chemical analyzer. Plasma Hyp concentration was 1000–1200 mg of calcium, in order to normalize their metab- measured by high-pressure liquid chromatography coupled olism. Foods high in Hyp were avoided. The study was ap- with UV detection using AccQ-Fluor reagents (Waters, Mil- proved by the Institutional Review Boards of the respective ford, MA) according to the manufacturer’s instructions. The 15 13 participating institutions. mole percent enrichment of [ N, C5]-Hyp and D3-Leucine in plasma was measured by gas chromatography–mass spec- 15 13 [ N, C5]-Hyp Intravenous Infusion troscopy by Metabolic Solutions (Nashua, NH). Ion chroma- The infusions were performed in the postabsorptive state. tography using an AS15, 23150 mm, anion exchange column After a 10-hour overnight fast, and waking at 6:00 AM,sub- coupled with negative-mode electrospray mass spectrometry jects emptied their bladders and drank 750 ml of water. (IC/MS) (Thermo Fisher ScientificInc.,Waltham,MA)was Subjects were admitted to the Clinical Research Unit at used to measure glycolate and mole percent enrichment with 13 7:00 AM. Infusions were initiated at 8:00 AM, with a priming C2-glycolate in acidified urines and plasma, as previously 15 13 22 dose of [ N, C5]-Hyp (0.75 mmol/kg) and D3-leucine described. Total oxalate and mole percent enrichment with 13 (2 mmol/kg) administered over a 5-minute period in 0.9% C2-oxalate were determined in acidified urine by an IC/MS saline. This was followed immediately by a constant infusion method recently developed in our laboratory. An IonPac 5 mm 15 13 of 0.75 and 2 mmol/h per kg for [ N, C5]-Hyp and D3-leucine, AS22, 23150 mm, anion exchange column with guard was respectively, for 6 hours. Separate catheters were used for the operated at 0.20 ml/min and room temperature using 20 mM infusion (antecubital vein) and blood collection (superficial ammonium carbonate as the mobile phase. The use of ammo- hand vein of the opposite arm). A 1-hour preinfusion urine nium carbonate for IC renders the detection of conductivity sample was collected. Blood and urine samples were collected impossible, but its volatility makes it compatible with the mass every half-hour and every hour, respectively, during the infusion. detector under these conditions. The mass detector (MSQ; Subjects drank 250 ml water per hour during the infusion (in- Thermo Fisher Scientific Inc., Waltham, MA) was operated fusion scheme in Figure 2). in ESI negative mode, needle voltage 0.5V, cone voltage 30V,

J Am Soc Nephrol 29: ccc–ccc, 2018 Hydroxyproline and Oxalate Synthesis 3 BASIC RESEARCH www.jasn.org

À Á and an MS source temperature of 400°C. The column eluent was Hyp Contributionð%Þ¼ EUox or EUGlc EHyp 3100 (2) mixed with 50% acetonitrile at 0.4 ml/min using a zero-dead volume mixing tee before entry into the mass spectrometer. Se- where EUox and EUGlc are the mole percent enrichment of urine with lected-ion monitoring (SIM) at the following mass/charge ratios, 13 13 “ 12 13 C2-oxalate and C2-glycolate, respectively (referred to as en- C2-oxalate (SIM89) and C2-oxalate (SIM91), was used to richment” in the text). determine total oxalate and the mole percent enrichment of 13 13 The amount of urinary oxalate and glycolate derived from C2-oxalate in the samples. Percent mole C2-oxalate enrichment 13 Hyp metabolism is estimated as the absolute amount synthe- was determined using standards with known amounts of C2- sized, deﬁned as the percent contribution of Hyp to urinary – oxalate in the range 0% 50% mole percent enrichment. Total metabolite (calculated by Equation 2), multiplied by the size of oxalate levels in samples were determined by standard isotope 13 the relevant pool (the urinary excretion of that metabolite), dilution analysis using C2-oxalate. Urine isoptomers of HOG expressed as ratio to creatinine excretion. (SIM 161, 166), dihydroxyglutarate (DHG) (SIM 163, 168), and glycerate (SIM 105) were determined by IC/MS, using an AS11-HC 4 mm anion exchange hydroxide column operated at 0.38 ml/min Statistical Analyses and 30°C. The limits of detection for total and 13Cmolepercent Statistical analyses were conducted in GraphPad Prism v. 7. enrichment (above natural abundance) for each analyte were the Comparison of data between groups was analyzed with one-way 13 following: total glycolate (0.02 mM), C2-glycolate (0.8% mole ANOVA followed by Tukey’s test for multiple comparisons if

Hyp derived metabolite ðmg=g creatinineÞ¼mean hourly urine excretion during infusion (3) in mg=g creatinine 3 ð% Hyp contribution at end of infusion T360 minutes=100Þ

13 enrichment), total oxalate (0.1 mM), C2-oxalate (0.2% mole en- P,0.05. A paired t test was used to compare repeated measures richment), total glycerate (0.02 mM), and total HOG (2 mM). between different time points, such as preinfusion (T0) and post- BecausepurestandardsforDHGarenotavailable,2-hydroxyglu- infusion (T360). Equilibration over time was analyzed by re- tarate was used as a standard to estimate DHG concentration. peated-measures one-way ANOVA analysis followed by Tukey’s post hoc test. In addition to parametric tests, nonparametric anal- Calculations yses (Kruskall–Wallis and Wilcoxon tests) were also performed Calculations are on the basis of 13C-labeled samples obtained and confirmed the conclusion drawn. Data are expressed as 15 13 after equilibration of the isotopic tracer ([ N, C5]-Hyp mean6SEM, unless otherwise indicated. The criterion for statis- steady state). Detailed methods and the rationale for tracer tical significance was P,0.05; all tests were two-sided. use are described in the Supplemental Material. The whole-body turnover rate or flux of Hyp (QHyp)was calculated as described in Equation 1.23–25 RESULTS ÂÀ Á Ã ¼ 2 Q i Ei EHyp 1 (1) Demographic and clinical characteristics of the control and PH groups are shown in Table 1; individual subject data and PH 15 13 where “i” is the [ N, C5]-Hyp infusion rate in micromoles per genotype information are presented in Supplemental Tables 1 kilogram per hour and Ei/EHyp is the ratio of isotopic enrichment of and 2. No significant differences between the subject groups 15 13 the [ N, C5]-Hyp infusate (Ei, 94%) and the mole percent en- were observed for body mass index and GFR. On the basis of 15 13 richment of the total Hyp plasma pool with [ N, C5]-Hyp (EHyp) preinfusion (T0) urine collections on an oxalate-controlled (referred to as “enrichment” in the text). diet (Table 1), urinary oxalate excretion was higher in PH 15 13 Because the [ N, C5]-Hyp tracer is also the precursor to groups compared with normal subjects. PH1 subjects showed 13 13 the labeled products C2-oxalate and C2-glycolate, the pro- significant individual variation that appeared related to their duction of oxalate and glycolate from Hyp metabolism was pyridoxine responsiveness. Urinary glycolate excretion was quantified using the following method: significantly different between the groups; highest in the The overall contribution of Hyp catabolism to urinary oxalate PH1 and lowest in the PH3 group, with some PH1 subjects 10 (UOx)andglycolate(UGlc) excretion is determined by the mole per- in the normal range as expected. Urinary glycerate was high- 13 13 cent enrichment of urine with C2-oxalate and C2-glycolate cor- est in patients with PH2, whereas HOG and DHG were highest 15 13 rected for the fraction of labeled [ N, C5]-Hyp that is circulating in in patients with PH3, as expected given their specificPHmo- the plasma (Equation 2).19,23 lecular defects (Figure 1, Supplemental Figure 1). Mean

4 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2018 www.jasn.org BASIC RESEARCH

15 13 Table 1. Baseline characteristics and urinary and plasma values before and after [ N, C5]-Hyp infusion at 0.75 mmol/h per kg Parameters Controls PH1 PH2 PH3 P Value N 97 4 8 Age, yr 29.362.0 25.762.1 [PH2] 51.5611.1 3766.6 0.03 Sex (M/F) 6/3 4/3 0/4 8/0 BMI, kg/m2 25.261.2 24.661.1 26.263.3 28.762.6 0.45 GFR, ml/min per 1.73 m2 10566.5 112.164.0 129617.7 97.569.7 0.84 B6 treatment 0 6 of 7 0 2 of 8 24-h urine profile Urine oxalate, mg/g creat 18.962.2 [PH2] 63.2623.1 88.9619.1 52.466.6 0.02 Urine glycolate, mg/g creat 36.169.7 92.4630.8 [PH3] 33.065.1 16.365.5 0.03 Urine glycerate, mg/g creat 3.360.8 7.063.1 617.86189.7 [C, PH1,3] 9.365.8 ,0.001 Urine HOG, mg/g creat ,3 ,3 ,3 28.269.1 [C, PH1] 0.02 Urine DHG, mg/g creat 0.860.1 0.960.1 1.760.2 16.463.4 [C, PH1,2] ,0.001 Before infusion Plasma oxalate, mM2.860.3 [PH1] 6.661.6 4.360.4 4.760.7 0.02 Plasma glycolate, mM4.560.3 26.9610.2 [C, PH3] 7.461.1 a 3.560.3 0.01 Plasma Hyp, mM 10.960.4 [PH1,2,3] 6.160.5 a 8.460.8 7.560.7 a ,0.001 Urine oxalate, mg/g creat 13.261.5 72.3624.6 69.5612.2 43.466.1 0.07 Urine glycolate, mg/g creat 23.965.7 63.4623.1 [PH3] 33.066.8 9.462.0 a 0.04 End of infusion Plasma oxalate, mM3.460.5 5.761.5 3.660.2 4.560.7 0.18 Plasma glycolate, mM3.960.3 25.569.6 [C, PH3] 4.460.5 3.060.3 0.01 Plasma Hyp, mM 10.160.5 8.360.6 9.061.1 9.560.4 0.14 Urine oxalate, mg/g creat 10.461.4 [PH1,3] 65.2620.4 57.068.8 49.064.0 0.004 Urine glycolate, mg/g creat 30.467.3 54.1613.6 [PH3] 16.762.8 13.462.6 0.01 Urine glycerate, mg/g creat 4.661.3 2.160.4 248684.8 [C, PH1,3] 1.660.2 ,0.001 Urine HOG, mg/g creat ,3 ,3 ,38.562.5 0.10 Urine DHG, mg/g creat 0.460.1 0.460.1 1.160.2 11.662.3 [C, PH1,2] ,0.001 13 Urine C2-oxalate, mg/g creat 0.360.1 [PH1,2,3] 3.361.1 10.661.5 [PH1,3] 4.460.6 ,0.001 13 Urine C2-glycolate, mg/g creat 2.460.5 5.261.0 [C, PH2,3] 0.360.1 0.760.3 ,0.001 13 Urine C2-HOG, mg/g creat ,0.8 ,0.8 ,0.8 2.361.0 0.04 13 Urine C2-DHG, mg/g creat ,0.2 ,0.2 ,0.2 0.460.1 [C, PH1,2] ,0.001 Average excretion during infusion Urine oxalate, mg/g creat 10.660.6 [PH1] 73.1627.9 70.8618.8 47.064.0 0.02 Urine glycolate, mg/g creat 23.364.0 [PH1] 59.6616.7 [PH3] 25.461.6 12.863.1 0.01 Each subject provided two 24-h urine collections of which the mean was used for the 24-h urine profile determinations. Urine values before the start of the infusion 15 13 (also called T0, “before infusion”) and after the infusion of [ N, C5]-Hyp infusion (also called T360, “end of infusion”) were determined in hourly collections, started the hour before that time. The “average excretion during infusion” was calculated as the mean of all seven hourly values during the infusion and was used as total urine excretions for the calculation of contributions. Urine excretions are expressed as a ratio to creatinine excretion. Results expressed asmean6SEM, P values shown for comparisons between groups. Statistical analyses were done to compare data between groups and to compare data before and after infusion (see Methods). Significant differences between subject groups according to Tukey’s post hoc test are specified as [different from subject group] directly to the right of the means columns analyzed, [C]: different from the control group. M/F, male/female; BMI, body mass index; creat, creatinine. aSignificant difference between T0 and T360 infusion time points P,0.05.

13 13 plasma Hyp levels, before infusion (T0), were significantly C2-oxalate and C2-glycolate in urine yields information on lower in individuals with PH compared with control subjects. how much oxalate and glycolate in urine is derived from the The kinetics of Hyp metabolism and the formation of two of metabolism of Hyp. The latter is defined as the contribution, its products, glycolate and oxalate, were studied using stable using calculations described in the Methods (see Supplemental isotope tracer methodology, similar to methods established for Figure 2, Supplemental Material). 19,23,24 15 13 the study of other amino acids. A primed, continuous Six-hour infusions of [ N, C5]-Hyp and D3-leucine, a 15 13 infusion of the stable isotope tracer, [ N, C5]-Hyp, was per- common infusion control, were performed in all subjects formed intravenously and serial samples were collected until (Figure 2). The enrichment of the total plasma Hyp pool 15 15 13 equilibration of the tracer in plasma. The amount of the [ N, with [ N, C5]-Hyp reached steady state in 5–6hours 13 C5]-Hyp tracer at steady state relative to the total Hyp (Supplemental Figure 3), and was higher in PH1, PH2, and concentration (defined as the isotopic enrichment) yields PH3 subjects (32%, 40%, and 28% versus 19% in controls, information on the daily turnover of Hyp. The enrichment of respectively; P,0.001). This may be related to the lower

J Am Soc Nephrol 29: ccc–ccc, 2018 Hydroxyproline and Oxalate Synthesis 5 BASIC RESEARCH www.jasn.org

mmol/h per kg infusion rate (Table 1). Alto- gether, these data support that the Hyp infusion protocol we applied did not alter the normal synthesis of the metabolites under study, as appropriate for a metabolic tracer. The levels of the urine metabolites of [15N, 13 13 13 C5]-Hyp, C2-oxalate and C2-glycolate, did not reach steady state within the 6- hour-long infusion (Supplemental Figure 4). These observations are consistent with a delay in the time taken for metabolites to equili- Figure 3. Urinary oxalate and glycolate derived from Hyp catabolism. These stacked brate within intracellular pools, as we have bar graphs indicate the total amount (as ratio to creatinine) of urinary oxalate (A) and previously observed with glycine infusions.23 glycolate (B) and the amount derived from Hyp catabolism (black) and non-Hyp The excretion of total oxalate, glycolate, sources (gray). The Hyp-derived values were calculated using the average hourly ex- 13 13 C2-oxalate, and C2-glycolate levels were cretions of total oxalate and glycolate during the Hyp infusion and the contribution of determined in hourly urinary collections Hyp catabolism to urinary oxalate and glycolate at infusion steady state, T360 (Table 1). These measurements and those 6 , ’ (Equation 3 in Methods). Results: mean SEM, *P 0.05 with Tukey s post hoc test for in the final collection (T360 minutes) per- Hyp-derived oxalate. mitted the calculation of the amount of urinary oxalate derived from the catabo- baseline plasma Hyp levels seen in those groups (Table 1). In lism of Hyp; 12.8, 32.9, and 14.8 mg oxalate/g creatinine in contrast, the enrichment of the D3-leucine was not different PH1, PH2, and PH3, respectively, compared with 1.6 mg ox- between the groups and reached steady state within 2 hours alate/g creatinine in controls (Figure 3A). The isotopic enrich- 13 (results not shown), similar to previous reports by us and oth- ment of urinary oxalate with C2-oxalate and plasma Hyp 23,24 15 13 ers. To determine whether the increase in plasma Hyp with with [ N, C5]-Hyp at the end of the infusion were used to 15 13 [ N, C5]-Hyp infusion (0.75 mmol/h per kg) altered metab- determine the percent contribution of Hyp catabolism to total olism, we infused a subset of four normal subjects with a lower urinary oxalate excretion (see Supplemental Material). The 15 13 rate of [ N, C5]-Hyp (0.15 mmol/h per kg). This resulted in proportional contribution of Hyp catabolism (Figure 4) to enriching the plasma Hyp pool by 3.7%, a value approximately total urinary oxalate excretion was similar in PH1 subjects five-fold lower than that achieved with the higher infusion rate. and controls (18% versus 15%, respectively), but was higher There was no significant difference in the Hyp turnover rate in PH2 (47%) and PH3 (33%) (P,0.05). The contribution in betweeninfusionratesof0.15and0.75mmol/h per kg PH3 subjects, however, showed variable results, with three of (P=0.15). However, the lower infusion dose compromised the eight PH3 subjects in the range of controls (Figure 4A). 13 13 our detection of C2-oxalate, because the levels obtained The amounts of glycolate and C2-glycolate derived from were close to the limit of quantitation. Therefore, the Hyp Hyp catabolism are shown in Figure 3B. Hyp contributed a turnover rate was determined in all subjects at the 0.75 mean 57% of total urinary glycolate excretion in normal sub- mmol/h per kg infusion rate. There were no statistical differ- jects (Figure 4B). This contribution was lower in all PH types 13 ences between pre- and postinfusion samples for urinary oxa- compared with controls. Low amounts of C2-glycolate were late, glycerate, HOG, and DHG, and plasma oxalate at the 0.75 detected in the urine of all patients with PH2, indicating that a pathway of glyoxylate to glycolate exists in the absence of GR. In addition to oxalate, 13 urinary C2-glycolate was detected in four of eight PH3 subjects, suggesting that glyoxylate can be synthesized from HOG (Figure 1), and that this process is variable 13 in patients with PH3. C5-HOG (2.3 mg/g 13 creatinine) and C5-DHG (0.4 mg/g creatinine) were detected in the last urine collection from six of eight and eight of eight patients with PH3, respectively (Table 1). Figure 4. Percent contribution of Hyp catabolism to total urinary oxalate and glyco- fi late excretion. The contribution of Hyp to urinary oxalate (A) and glycolate (B) was These latter data further con rm that Hyp calculated using the mole percent enrichment in the final urine collection and the mole metabolism in individuals with PH3 in- 15 13 percent enrichment of [ N, C5]-Hyp in the final plasma collected at 360 minutes of in- cludes conversion of HOG to DHG. fusion time, T360 (Equation 2 in Methods). Line: mean. Individual points: separate subjects. The mean Hyp turnover rate was 3.1 *P,0.05 with Tukey’s post hoc test. mmol/h per kg in healthy subjects

6 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2018 www.jasn.org BASIC RESEARCH

13 with C2-glycine infusion suggest that glycine metabolism contributes up to 5%. Die- tary oxalate accounts for 40% of total urinary oxalate excretion,23 with the remaining 60% derived from endogenous oxalate synthesis. Thus, in healthy subjects, Hyp and glycine combined account for approximately one third of endogenous oxalate synthesis. Other sources that contribute to oxalate synthesis could include glycolate not derived from Hyp metabolism (e.g.,glycolatederived from phosphoglycolate formed during DNA repair22), glyoxal,12 and ascorbic acid.26 In PH2 subjects Hyp was responsible for almost half of the endogenous oxalate produced, the highest contribution of Hyp among all groups (Figures 4 and 5). This result demonstrates that GR, deficient in PH2, plays an important role in limiting the metabolism of Hyp-derived glyoxylate to oxalate (Supplemental Figure 1). Mito- chondrial GR activity may be particularly fi Figure 5. Overview of the endogenous synthesis and excretion of oxalate and gly- important in this glyoxylate detoxi cation colate in healthy subjects and patients with PH. The estimated amount of urinary process, and its role is likely to be accentu- oxalate and glycolate excreted in 24-hour urine that is derived from Hyp metabolism is ated in the renal proximal tubule, which expressed as mg/g creatinine (see Figures 3 and 4 for details). The relative contri- lacks AGT but expresses enzymes of the bution of oxalate and glycolate derived from Hyp to the urinary oxalate and glycolate Hyp catabolism pathway. The low but 13 pools is expressed as percentage of total urinary excretion. detectable amounts of C2-glycolate observed in individuals with PH2 (Supple- (Supplemental Figure 5) compared with 1.5 and 1.1 mmol/h mental Figure 4, Table 1) suggest that another enzyme, such per kg, respectively, in PH1 and PH2 subjects. In PH3 the as lactate dehydrogenase, could catalyze the conversion of mean Hyp turnover rate was also lower (2 mmol/h per kg), glyoxylate to glycolate.1 The contribution of Hyp to oxalate but the distribution of values was broader and demonstrated production observed in PH2 subjects raises the possibility an age-related decrease (Supplemental Figure 6). that a therapy blocking HYPDH activity could be efficacious in reducing urinary oxalate in this group. HYPDH deficiency (MIM #237000) in humans does not cause clinical symp- DISCUSSION toms27,28 and HYPDH null mice lack symptoms while having an approximately 20% reduced urinary oxalate excretion In treating PH, reduction in endogenous oxalate production is (J. Knight, W.T. Lowther, and R.P. Holmes, unpublished re- at the heart of therapeutic strategies. The search for effective sults). These observations support targeting liver and kidney strategies is hampered by a lack of knowledge of pathways of HYPDH as an approach for oxalate reduction therapy in PH2. endogenous oxalate synthesis (Figure 1). Hyp metabolism re- In PH3 subjects, Hyp catabolism was also an important sults in oxalate formation, but its contribution to urinary ox- source of urinary oxalate. The contribution of Hyp to urinary alate excretion in humans was not previously known. In this oxalate was on average higher (33%, Figure 4) than control study we used constant infusions of a stable isotope of Hyp to subjects but quite variable and in some cases even similar to demonstrate that Hyp metabolism is an important source of that of healthy subjects, despite the HOGA deficiency (Figure urinary oxalate excretion in all forms of PH, with the greatest 4).9 This heterogeneity extended to urinary levels of HOG, proportional contribution in PH2 and PH3. In PH1, which is DHG, and Hyp contribution to urinary glycolate. This sug- the most severe form of PH, sources other than Hyp appear to gests that additional enzymes may be capable of metabolizing play a prominent role in endogenous oxalate and glycolate HOG, to either glyoxylate or DHG, with varying degrees be- synthesis and remain to be clarified. tween individuals. Several clinical reports mention a decline of symptoms with age in some patients with PH3. These obser- Hyp Metabolism vations, in conjunction with the decrease in Hyp turnover rate We observed that Hyp metabolism contributes 15% of the with age in PH3 subjects (Supplemental Figure 6), hint at the oxalate excreted in urine of normal individuals. Prior studies possibility of age-related changes or disease adaptations in

J Am Soc Nephrol 29: ccc–ccc, 2018 Hydroxyproline and Oxalate Synthesis 7 BASIC RESEARCH www.jasn.org

Hyp metabolism.29 The pathophysiology of PH3 and the mech- individuals was 3.1 mmol/h per kg, which is equivalent to a turn- anisms leading to the increased oxalate synthesis have been the over of 660 mg Hyp/d or 4.7 g collagen/d for a 70-kg individual. subject of debate. We have previously shown that HOG inhibits This is almost twice the rates published by Kivirikko,35 which were recombinant GR activity and have hypothesized that this results in on the basis of the urinary Hyp excretions of a 12-year-old female increased endogenous oxalate synthesis in PH3 (Supplemental and a 31-year-old male with hydroxyprolinemia due to a presumed Figure 1).9 The trend toward lower total glycolate excretion in HYPDH deficiency. Reasons for the prior underestimation of Hyp PH3 subjects compared with healthy individuals and other PH turnover rate may include inadequate methodology for estimating types supports this mechanism. On the other hand, the formation Hyp at that time and possible Hyp degradation through other 13 13 of urinary C2-glycolate and C2-oxalate in PH3 subjects also metabolic pathways in these individuals. The reasons for the lower supports the hypothesis that an alternative aldolase activity con- Hyp turnover rates (Supplemental Figure 5) and lower total plasma verts HOG to glyoxylate.9 Strategies that block HYPDH activity Hyp seen in patients with PH in our study are unclear. One possible could also benefitpatientswithPH3,giventhatHOGA,theen- explanation would be that a lower turnover of collagen takes place zyme affected by mutations in PH3, catalyzes a reaction down- inresponsetotheincreasedoxalateproductionthatoccursinPH, stream in the Hyp catabolic pathway. However, the heterogeneity thus partially limiting glyoxylate production upstream of the enzy- of the production of HOG and its metabolites in patients with matic deficiencies. Importantly, despite lower Hyp turnover from PH3 highlights the need for a greater understanding of the met- bone and other soft tissues and lower blood levels of Hyp in PH, the abolic pathways.29,30 metabolism of Hyp accounts for quantitatively larger amounts of PH1 subjects exhibited a greater synthesis of oxalate, total and oxalate excreted in the urine in patients with all three forms of PH derived from Hyp, compared with controls (Figure 3A). Interest- (Figure 5). ingly, the proportional contribution of Hyp catabolism to urinary oxalate was similar to that of healthy subjects (Figures 4 and 5). Limitations and Future Perspective Other pathways of endogenous oxalate synthesis seem to play an The number of subjects enrolled in each group was small due to important role in this disorder. Our observations underscore that the rarityofthedisease; thecomplexityofinfusions; and the time, fi the metabolic de ciencies leading to PH1 are quite different from expense, and travel involved, particularly for patients. Genotypic those of PH2 and PH3 and appear to rely on peroxisomal rather and age differences in individuals with PH, unavoidable in such a fi than mitochondrial pathways. The AGT de ciency in PH1 estab- study, may have contributed to variations in their responses. The lishes an open cycle of glycolate-glyoxylate interconversion in the truecontribution ofHypto oxalate andglycolate productionmay liver (Supplemental Figure 1B); i.e., oxidation of glycolate to glyox- be underestimated in our study, because steady states of the [15N, ylate by peroxisomal GO and reduction of glyoxylate to glycolate by 13 13 13 C5]-Hyp metabolites, C2-oxalate and C2-glycolate, were cytosolic GR. This cycling most likely plays an important role in the not reached at the end of the infusion and the contribution of pathophysiology of PH1, due to the hydrogen peroxide produced dietary oxalate absorption to urinary oxalate may change during with each cycle. Recent studies by our group and others have shown the course of the fast. Alternative study designs could be on the basis fi that reducing GO activity with siRNA in the Agxt-de cient mouse of longer infusions but metabolism may be significantly altered fi 19,31,32 results in a signi cant decrease in urinary oxalate excretion, with an extended fast, and studies conducted in the fed state could further supporting that glycolate metabolism is a major source of create complexities limiting their interpretation. Future studies will oxalate in PH1, independent of Hyp catabolism. Subsequent work be needed to elucidate the remaining sources of endogenous oxa- with rats and monkeys33 further supported GO as a viable siRNA late synthesis, which are prominent in PH1, and the role that the knock down target for oxalate reduction therapy, and clinical trials glycolate to glyoxylate cycling plays in oxalate synthesis. in patients with PH1 using GO siRNA are being conducted. This therapeutic approach, however, will increase glycolate levels.33 Pre- liminary studies in our laboratory show that isolated human proximal tubules, which lack GO, can convert glycolate to oxalate (S. ACKNOWLEDGMENTS Fargue,J.Knight,andR.P.Holmes,unpublishedresults),which could result in increased renal oxalate synthesis in the event of We thank Zhixin Wang, Nicole Holderman, and Alex Dowell for their increased glycolate production with siRNA treatment. excellent technical assistance. We greatly appreciate the participation of the subjects with primary hyperoxaluria and the healthy subjects and the Hyp Turnover cooperation of the Clinical Research Units at Wake Forest School of The bulk of the Hyp that enters the extracellular fluid compartment Medicine, UAB (supported by National Institutes of Health [NIH] grant is derived from collagen turnover with bone being a major source, TR001417), and the Mayo Clinic (supported by NIH grant TR000135). exceeding that of skin, muscle, and other tissues.34 It is possible that This work was funded by NIH grants DK054468, DK083527, some of the Hyp-rich body pools exchange slowly with the extra- DK083908, and K01DK114332 and the Oxalosis and Hyperoxaluria cellular Hyp pool in addition to an unknown intersubject variabil- Foundation. ity in these processes.35 Thiscouldinpartexplaintheextended This work was presented as an abstract at the 2016 annual meeting 15 13 time required for infused [ N, C5]-Hyp to approach steady state. of the American Society of Nephrology Kidney Week, Chicago, IL, In our study, the mean total body turnover rate of Hyp in normal November 15–20, 2016. ClinicalTrials.gov identifier NCT02038543.

8 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc,2018 www.jasn.org BASIC RESEARCH

R.P.H., J.K., W.T.L., and D.S.M. designed the study; S.F., J.K., and J. D, Childs B, Kinzler KW, et al., New York, McGraw-Hill, 2001, pp B.O. carried out experiments and patient recruitment; S.F. and J.K. 1821–1838 analyzed the data; S.F., J.K., R.P.H., W.T.L., and D.S.M. drafted and 19. Li X, Knight J, Fargue S, Buchalski B, Guan Z, Inscho EW, et al.: Me- fi tabolism of (13)C5-hydroxyproline in mouse models of Primary Hy- revised the paper; all authors approved the nal version of the peroxaluria and its inhibition by RNAi therapeutics targeting liver manuscript. glycolate oxidase and hydroxyproline dehydrogenase. Biochim Bio- phys Acta 1862: 233–239, 2016 20. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D; Modifica- DISCLOSURES tion of Diet in Renal Disease Study Group: A more accurate method to estimate glomerular filtration rate from serum creatinine: A new pre- None. diction equation. Ann Intern Med 130: 461–470, 1999 21. Jiang J, Johnson LC, Knight J, Callahan MF, Riedel TJ, Holmes RP, et al.: Metabolism of [13C5]hydroxyproline in vitro and in vivo: Impli- REFERENCES cations for primary hyperoxaluria. Am J Physiol Gastrointest Liver Physiol 302: G637–G643, 2012 22. Knight J, Hinsdale M, Holmes R: Glycolate and 2-phosphoglycolate 1. Holmes RP, Assimos DG: Glyoxylate synthesis, and its modulation and content of tissues measured by ion chromatography coupled to mass influence on oxalate synthesis. JUrol160: 1617–1624, 1998 spectrometry. Anal Biochem 421: 121–124, 2012 2. Danpure CJ: Primary hyperoxaluria. In: The Metabolic and Molecular 23. Knight J, Assimos DG, Callahan MF, Holmes RP: Metabolism of primed, Bases of Inherited Disease, 8th Ed., edited by Scriver CR, Beaudet AL, constant infusions of [1,2-13C ] glycine and [1-13C ] phenylalanine to Sly WS, Vallee D, Childs B, Kinzler KW, et al., New York, McGraw-Hill, 2 1 urinary oxalate. Metabolism 60: 950–956, 2011 2001, pp 3323–3367 3. Danpure CJ, Jennings PR: Peroxisomal alanine:glyoxylate aminotransferase 24. Matthews DE, Motil KJ, Rohrbaugh DK, Burke JF, Young VR, Bier deficiency in primary hyperoxaluria type I. FEBS Lett 201: 20–24, 1986 DM: Measurement of leucine metabolism in man from a primed, – 4. Williams HE, Smith LH Jr .: L-glyceric aciduria. A new genetic variant of continuous infusion of L-[1-3C]leucine. Am J Physiol 238: E473 primary hyperoxaluria. N Engl J Med 278: 233–238, 1968 E479, 1980 5. Cramer SD, Ferree PM, Lin K, Milliner DS, Holmes RP: The gene en- 25. Thompson GN, Pacy PJ, Merritt H, Ford GC, Read MA, Cheng KN, coding hydroxypyruvate reductase (GRHPR) is mutated in patients with et al.: Rapid measurement of whole body and forearm protein primary hyperoxaluria type II. Hum Mol Genet 8: 2063–2069, 1999 turnover using a [2H5]phenylalanine model. Am J Physiol 256: – 6. Rumsby G, Cregeen DP: Identification and expression of a cDNA for E631 E639, 1989 human hydroxypyruvate/glyoxylate reductase. Biochim Biophys Acta 26. Knight J, Madduma-Liyanage K, Mobley JA, Assimos DG, Holmes RP: – 1446: 383–388, 1999 Ascorbic acid intake and oxalate synthesis. Urolithiasis 44: 289 297, 7. Belostotsky R, Seboun E, Idelson GH, Milliner DS, Becker-Cohen R, 2016 Rinat C, et al.: Mutations in DHDPSL are responsible for primary hy- 27. Pelkonen R, Kivirikko KI: Hydroxyprolinemia: An apparently harmless – peroxaluria type III. Am J Hum Genet 87: 392–399, 2010 familial metabolic disorder. NEnglJMed283: 451 456, 1970 8. Monico CG, Rossetti S, Belostotsky R, Cogal AG, Herges RM, Seide BM, 28.StaufnerC,HaackTB,FeyhP,GramerG,RagaDE,TerrileC,etal.: et al.: Primary hyperoxaluria type III gene HOGA1 (formerly DHDPSL) Genetic cause and prevalence of hydroxyprolinemia. J Inherit Metab as a possible risk factor for idiopathic calcium oxalate urolithiasis. Dis 39: 625–632, 2016 Clin J Am Soc Nephrol 6: 2289–2295, 2011 29. Beck BB, Baasner A, Buescher A, Habbig S, Reintjes N, Kemper MJ, 9. Riedel TJ, Knight J, Murray MS, Milliner DS, Holmes RP, Lowther WT: 4-Hy- et al.: Novel findings in patients with primary hyperoxaluria type III and droxy-2-oxoglutarate aldolase inactivity in primary hyperoxaluria type 3 and implications for advanced molecular testing strategies. Eur J Hum glyoxylate reductase inhibition. Biochim Biophys Acta 1822: 1544–1552, 2012 Genet 21: 162–172, 2013 10. Cochat P, Hulton SA, Acquaviva C, Danpure CJ, Daudon M, De Marchi 30. MacDonald JR, Huang AD, Loomes KM: Cellular degradation of 4- M, et al.; OxalEurope: Primary hyperoxaluria Type 1: Indications for hydroxy-2-oxoglutarate aldolaseleadstoabsolutedeficiency in pri- screening and guidance for diagnosis and treatment. Nephrol Dial mary hyperoxaluria type 3. FEBS Lett 590: 1467–1476, 2016 Transplant 27: 1729–1736, 2012 31. Dutta C, Avitahl-Curtis N, Pursell N, Larsson Cohen M, Holmes B, 11. Lange JN, Wood KD, Knight J, Assimos DG, Holmes RP: Glyoxal formation Diwanji R, et al.: Inhibition of glycolate oxidase with dicer-substrate and its role in endogenous oxalate synthesis. Adv Urol 2012: 819202, 2012 siRNA reduces calcium oxalate deposition in a mouse model of primary 12. Cochat P, Rumsby G: Primary hyperoxaluria. N Engl J Med 369: 649– hyperoxaluria type 1. Mol Ther 24: 770–778, 2016 658, 2013 32. Martin-Higueras C, Luis-Lima S, Salido E: Glycolate oxidase is a safe and 13. Khan SR, Glenton PA: Experimental induction of calcium oxalate efficient target for substrate reduction therapy in a mouse model of nephrolithiasis in mice. JUrol184: 1189–1196, 2010 primary hyperoxaluria type I. Mol Ther 24: 719–725, 2016 14. Khan SR, Glenton PA, Byer KJ: Modeling of hyperoxaluric calcium ox- 33. Liebow A, Li X, Racie T, Hettinger J, Bettencourt BR, Najafian N, et al.: alate nephrolithiasis: Experimental induction of hyperoxaluria by hy- An investigational RNAi therapeutic targeting glycolate oxidase re- droxy-L-proline. Kidney Int 70: 914–923, 2006 duces oxalate production in models of primary hyperoxaluria. JAmSoc 15. Knight J, Holmes RP, Cramer SD, Takayama T, Salido E: Hydroxyproline Nephrol 28: 494–503, 2017 metabolism in mouse models of primary hyperoxaluria. Am J Physiol 34. Laitinen O: The metabolism of collagen and its hormonal control in the Renal Physiol 302: F688–F693, 2012 rat with special emphasis on the interactions of collagen and calcium in 16. Knight J, Jiang J, Assimos DG, Holmes RP: Hydroxyproline ingestion and the bones. Acta Endocrinol (Copenh) 56[Suppl 120]: 1–86, 1967 urinary oxalate and glycolate excretion. Kidney Int 70: 1929–1934, 2006 35. Kivirikko KI: Urinary excretion of hydroxyproline in health and disease. 17. Patel SR, Penniston KL, Iwicki L, Saeed I, Crenshaw TD, Nakada SY: Int Rev Connect Tissue Res 5: 93–163, 1970 Dietary induction of long-term hyperoxaluria in the porcine model. J Endourol 26: 433–438, 2012 18. Phang JM, Hu CA, Valle D: Disorders of proline and hydroxyproline metabolism. In: The Metabolic and Molecular Bases of In- This article contains supplemental material online at http://jasn.asnjournals. herited Disease, edited by Scriver CR, Beaudet AL, Sly WS, Vallee org/lookup/suppl/doi:10.1681/ASN.2017040390/-/DCSupplemental.

J Am Soc Nephrol 29: ccc–ccc, 2018 Hydroxyproline and Oxalate Synthesis 9