Targeting Proximal Tubule Mitochondrial Dysfunction Attenuates the Renal Disease of Methylmalonic Acidemia

Targeting proximal tubule mitochondrial dysfunction attenuates the renal disease of methylmalonic acidemia

Irini Manolia,1, Justin R. Sysola,1, Lingli Lib, Pascal Houillierb,c, Caterina Garoned,e, Cindy Wanga, Patricia M. Zerfasf, Kristina Cusmano-Ozogg, Sarah Youngh, Niraj S. Trivedii, Jun Chengj, Jennifer L. Sloana, Randy J. Chandlera, Mones Abu-Asabk, Maria Tsokosk, Abdel G. Elkahlounl, Seymour Rosenm,n, Gregory M. Ennsg, Gerard T. Berryo, Victoria Hoffmannf, Salvatore DiMaurod, Jurgen Schnermannb, and Charles P. Vendittia,2

aGenetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health (NIH), Bethesda, MD 20892; bKidney Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20814; cInstitut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche_S 872, Department of Physiology, Georges Pompidou European Hospital, Assistance Publique–Hôpitaux de Paris, Paris Descartes University, 75015 Paris, France; dColumbia University Medical Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032; eHuman Genetics Joint PhD Program, University of Bologna and Turin, 10126 Turin, Italy; fOfﬁce of Research Services, Division of Veterinary Resources, NIH, Bethesda, MD 20892; gDivision of Medical Genetics, Stanford University, Stanford, CA 94305; hBiochemical Genetics Laboratory, Division of Medical Genetics, Duke University Medical Center, Durham, NC 27713; iGenome Technology Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892; jEmbryonic Stem Cell and Transgenic Mouse Core Facility, National Human Genome Research Institute, NIH, Bethesda, MD 20892; kUltrastructural Pathology Section, Center for Cancer Research, NIH, Bethesda, MD 20892; lCancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892; mDepartment of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; and nDepartment of Pathology and oManton Center for Orphan Disease Research, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115

Edited by Arthur L. Beaudet, Baylor College of Medicine, Houston, TX, and approved July 3, 2013 (received for review February 12, 2013) Isolated methylmalonic acidemia (MMA), caused by deficiency of and symptomatic care, although liver, kidney, and combined the mitochondrial enzyme methylmalonyl-CoA mutase (MUT), is liver–kidney transplantation have been used in the severely af- often complicated by end stage renal disease that is resistant to fected patients (3). Kidney disease in MMA can manifest with conventional therapies, including liver transplantation. To estab- proximal and/or distal tubular dysfunction and is associated with lish a viable model of MMA renal disease, Mut was expressed in progressive tubulointerstitial disease (1, 4), eventually leading to MEDICAL SCIENCES − − the liver of Mut / mice as a stable transgene under the control of ESRD in >50% of those with severe forms of MMA by the age − − Mut an albumin (INS-Alb-Mut) promoter. Mut / ;TgINS-Alb- mice, al- of 8 y (5). Although renal disease can be present as early as 18 mo though completely rescued from neonatal lethality that was dis- (6), the decreased muscle mass seen in the patients (1, 7) renders − − played by Mut / mice, manifested a decreased glomerular filtration routine laboratory markers, such as creatinine, poorly predictive rate (GFR), chronic tubulointerstitial nephritis and ultrastructural of kidney involvement early in disease evolution. changes in the proximal tubule mitochondria associated with ab- Patients with MMA who have been treated by orthotopic liver errant tubular function, as demonstrated by single-nephron GFR transplantation display metabolic stability yet develop extrahe- −/− INS-Alb-Mut studies. Microarray analysis of Mut ;Tg kidneys identi- patic disease, including ESRD, and, therefore, provide a clinical fied numerous biomarkers, including lipocalin-2, which was then paradigm for the development of animal models to study the used to monitor the response of the GFR to antioxidant therapy in renal pathophysiology of MMA (8). In the present work, we have − − the mouse model. Renal biopsies and biomarker analysis from a large used transgenesis to create Mut / mice that express Mut in and diverse patient cohort (ClinicalTrials.gov identifier: NCT00078078) hepatocytes under the control of the mouse albumin promoter −/− − − precisely replicated the findings in the animals, establishing Mut ; (Mut / ;TgINS-Alb-Mut). These mice are protected from the neo- INS-Alb-Mut − − Tg mice as a unique model of MMA renal disease. Our natal lethality that characterizes the Mut / mice (9, 10) but studies suggest proximal tubular mitochondrial dysfunction is a manifest CTIN and a decreased glomerular filtration rate (GFR) key pathogenic mechanism of MMA-associated kidney disease, iden- associated with megamitochondria formation (11) and decreased tify lipocalin-2 as a biomarker of increased oxidative stress in the cytochrome c oxidase (COX) activity in the proximal tubules. renal tubule, and demonstrate that antioxidants can attenuate the The murine studies prompted the search for similar pathology in renal disease of MMA. kidney biopsies from MMA patients and suggested a therapeutic approach directed at alleviating mitochondrial dysfunction. Ge- cobalamin | chronic renal failure | megamitochondria | organic acidemia nomic and biochemical characterization of disease progression in − − the Mut / ;TgINS-Alb-Mut mice identified >50 biomarkers associ- enal tubular dysfunction with progression into chronic tubu- ated with MMA renal disease, including lipocalin-2 (Lcn2), that Rlointerstitial nephritis (CTIN) and end stage renal disease (ESRD) is a cardinal manifestation of methylmalonic acidemia (MMA), a common and severe organic acidemia characterized Author contributions: I.M., J.S., and C.P.V. designed research; I.M., J.R.S., L.L., P.H., C.G., by metabolic instability, multisystemic complications, and high C.W., P.M.Z., S.Y., N.S.T., J.L.S., M.A.-A., M.T., A.G.E., J.S., and C.P.V. performed research; K.C.-O., J.C., R.J.C., S.R., G.M.E., G.T.B., and C.P.V. contributed new reagents/analytic tools; mortality (1, 2). Isolated MMA is primarily caused by mutations I.M., J.R.S., L.L., P.H., C.G., C.W., K.C.-O., S.Y., N.S.T., J.L.S., M.A.-A., M.T., A.G.E., S.R., G.M.E., in the vitamin B12-dependent, mitochondrial matrix-localized G.T.B., V.H., S.D., J.S., and C.P.V. analyzed data; and I.M., J.S., and C.P.V. wrote the paper. methylmalonyl-CoA mutase (MUT), an enzyme that mediates The authors declare no conflict of interest. the entry of carbon skeletons derived from branched-chain This article is a PNAS Direct Submission. amino acid, odd-chained fatty acid, and cholesterol oxidation Freely available online through the PNAS open access option. into the Krebs cycle (3). Although the MUT enzyme is expressed Data deposition: The data reported in this paper have been deposited in the Gene Ex- ubiquitously, the clinical features observed, such as pancreatitis, pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE41044). metabolic “stroke” of the globus pallidus, optic nerve atrophy, 1I.M. and J.R.S. contributed equally to this work. immune dysfunction, and especially renal disease, indicate a tis- 2To whom correspondence should be addressed. E-mail: [email protected]. fi sue-speci c vulnerability in this metabolic disorder. There are no This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. treatments available for MMA other than dietary management 1073/pnas.1302764110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1302764110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 was subsequently validated in a large cohort of MMA patients. A and multiple matrix granules/deposits in the renal tubular cytosol of − − therapeutic regimen, directed at reducing oxidant injury with Mut / ;TgINS-Alb-Mut kidneys (Fig. S2A). CoQ10 and vitamin E (VitE), ameliorated the loss of GFR in the − − Mut / ;TgINS-Alb-Mut mice and was predicted by plasma Lcn2 Acceleration of MMA Renal Disease by Ingestion of a High-Protein Diet. concentrations. Our studies establish LCN2 as a biomarker of To recapitulate the renal disease observed in some patients post- oxidative stress and renal mitochondrial dysfunction and define liver transplantation with liberalized protein intake (8), the mice an approach for the treatment and monitoring of kidney disease were fed a high-protein (HP) (casein) chow. A dietary challenge = −/− INS-Alb-Mut in patients with MMA. study for 6 mo (n 8) was performed in female Mut ;Tg mice, because only rarely did male littermates survive beyond 2 mo − − Results of age on the same diet. On the HP chow, the Mut / ;TgINS-Alb-Mut − − Hepatic Expression of Mut Provides Phenotypic Attenuation in Mut / mice experienced a rapid weight loss of 14 ± 4% within the first Mice. A construct was engineered to express the Mut gene under month and failed to regain their weight (Fig. S3A). Plasma − − the control of the chimeric murine minimal albumin promoter methylmalonic acid concentration in the Mut / ;TgINS-Alb-Mut and α-fetoprotein enhancer (Fig. S1 A and B). Three trans- mice was 863 ± 288 μmol/L at baseline, 1,500 ± 620 μmol/L after mitting C57BL/6 TgINS-Alb-Mut founder lines were established and 2 mo, and 1,938 ± 418 μmol/L after 6 mo on a HP diet (P = 0.013 + − −/− INS-Alb-Mut crossed to C57BL/6 Mut / mice (9, 12). To accelerate the de- for Mut ;Tg mice compared with baseline values and velopment of extrahepatic manifestations, the lowest expressing P < 0.0001 compared with heterozygotes) (Fig. S3B). Studies in − − line was selected for further study. Mut / ;TgINS-Alb-Mut mice plasma vs. brain tissue extracts, obtained 2 mo after HP diet in were uniformly rescued from neonatal lethality (Fig. 1A) and a separate group of mice, showed significantly higher methyl- appeared comparable to their heterozygote littermates (Fig. 1B). malonic acid concentrations in the brain tissue compared with = = Immunoreactive Mut protein was detected only in the liver of the mice fed regular chow (RD) (Fig. S3 C and D; n 6 per group; P − − Mut / ;TgINS-Alb-Mut mice and was expressed at levels compara- 0.007), similar to the trend in plasma. These concentrations closely ble to mRNA expression (Fig. 1C and Fig. S1C). [1-13C]Pro- approximate methylmalonic acid levels observed in our MMA pionate oxidative capacity (13) was measured to evaluate the in patient cohort at baseline and after the onset of kidney failure in vivo metabolic capacity provided by the INS-Alb-Mut transgene. the older subgroup, respectively (Table S1). − − The Mut / ;TgINS-Alb-Mut mice metabolized 54.7 ± 9.2% of the ad- Severe Proximal Tubular Mitochondrial Ultrastructural Changes in ministered [1-13C]propionate isotopomer in 25 min, compared +/− −/− MMA Mice Replicate the Renal Pathology Seen in MMA Patients. with 76.5 ± 4.5% in the Mut and 10 ± 2% in the Mut mice − − Mut / ;TgINS-Alb-Mut mice that ingested a HP diet for 6 mo de- (P < 0.0001) (Fig. 1D). Despite the grossly normal phenotype, veloped severe multifocal tubulointerstitial nephritis (Fig. S3 Ea the animals displayed elevated plasma MMA levels (825.7 ± −/− INS-Alb-Mut + − vs. Eb), causing the kidney cortex in the Mut ;Tg mice to μ Mut / INS-Alb-Mut 331.4 mol/L) compared with their ;Tg littermates have a granular appearance (Fig. S3Eb, black arrows), whereas (5.8 ± 1.4 μmol/L; n = 7and5;P < 0.001). The histology of various fi fi tubules in the convoluted proximal segment had ne micro- organs showed no signi cant changes, whereas electron micro- vesicular cytoplasmic changes (Fig. S3Eb, Inset). Ultrastructural − − scopic examination showed increased number of mitochondria examination of the kidneys in the Mut / ;TgINS-Alb-Mut mice revealed a large number of mitochondria with electron-dense matrix and abnormal cristae in the proximal tubules (Fig. 2 Aa − − vs. Ab). Of note, the glomeruli in the Mut / ;TgINS-Alb-Mut mice appeared normal, and the podocyte foot processes were not effaced (Fig. 2 Ac vs. Ad). In contrast to the striking renal pathology − − observed in Mut / ;TgINS-Alb-Mut mice, there were no light or electron microscopic abnormalities in the liver, despite the prolonged exposure to HP and the massive (greater than 1,000× increased) plasma methylmalonic acid concentrations (Fig. S2B). To determine whether kidneys have a different susceptibility to methylmalonic acid than liver, we administered 20 mg/100 g body weight of methylmalonic acid via daily i.p. injections for 2 mo to wild-type mice. Nephrotoxicity was not observed (Fig. S4 A and B). In parallel to the murine studies, we collected kidney biopsies or explants from three MMA patients enrolled in a natural history study (ClinicalTrials.gov identifier: NCT00078078), who were undergoing solid-organ transplantation. The microscopic findings in the MMA patient kidneys closely resembled the − − changes seen in the Mut / ;TgINS-Alb-Mut mice (Fig. S3 E vs. F), whereas electron microscopic examination revealed similarly Fig. 1. Phenotypic characterization of the Mut−/−;TgINS-Alb-Mut mice. (A) enlarged mitochondria in the proximal tubule, with increased Mut−/−;TgINS-Alb-Mut mice (n = 23) exhibit normal survival compared with the electron-dense matrix and shortened, abnormal cristae (Fig. 2B). immediate neonatal lethality of the knockout strain (n = 29; P < 0.0001) and comparable to their heterozygote littermates with or without the transgene Abnormalities of Glomerular Filtration and Tubular Reabsorption Are Early −/− − − Mut (n =32 and 24, respectively; P = NS for both). (B) At 6 mo of age, Mut ; Manifestations of the Renal Disease in Mut / ;TgINS-Alb- Mice. INS-Alb-Mut − − Tg mice appear normal and are 14 ± 0.56% smaller than their lit- After ingesting a HP diet for 2 mo, the Mut / ;TgINS-Alb-Mut mice termates (n = 4 per group; P < 0.001). (C) Mut (78 kDa) was detected in fi −/− INS-Alb-Mut manifested signi cant weight loss, increased mortality, and mas- whole-tissue lysates from Mut ;Tg liver but not in kidney or sively elevated plasma methylmalonate concentrations. We, there- muscle. (D)[1-13C]Propionate oxidation showed a significant improvement of the in vivo oxidative capacity by the hepatic transgene-mediated Mut fore, selected this time point to measure the GFR using FITC- + − − − − − expression. (n = 11, 9 and 6 for Mut / , Mut / ;TgINS-Alb-Mut, and Mut / ,re- labeled inulin clearance and to assess proximal tubular function. −/− −/− INS-Alb-Mut spectively; values are means ± SEM, ****P < 0.0001 between Mut ;TgINS-Alb-Mut Mut ;Tg animals showed a significant reduction in − − vs. Mut / mice.) GFR (42.1 ± 3.35%; n = 11; P < 0.0001) compared both to the

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302764110 Manoli et al. Downloaded by guest on September 28, 2021 tubule, that was independent of the GFR (Fig. 3D). Blood and urine collected during the micropuncture SNGFR studies also showed a lower plasma bicarbonate and higher chloride concen- − − trationintheMut / ;TgINS-Alb-Mut mice compared with controls, consistent with chronic hyperchloremic metabolic acidosis. Despite the metabolic acidosis, urinary ammonium excretion was not increased (Table S2), an expected observation in the setting of renal tubular acidosis (14). Whole-body GFR studies on RD were not signiﬁcantly decreased up until 9 mo to 1 y of age (Fig. S4 C and D), suggesting that liver-restricted expression of Mut may offer protection from renal failure unless the pathway is challenged by an increased propiogenic load. MEDICAL SCIENCES

Fig. 2. Dietary challenge with HP in Mut−/−;TgINS-Alb-Mut mice and patient − − correlations. Mut / ;TgINS-Alb-Mut mice experienced signiﬁcant growth failure and plasma MMA concentrations of 1,938 ± 418 μmol/L after 6 mo on HP diet (Fig. S3). (A) Transmission EM showed enlarged mitochondria with whorl-like cristae (Aa vs. Ab; white arrows, mitochondria; white asterisks, brush border of the proximal tubule). Glomeruli appeared normal (Ac and Ad; white asterisks, podocyte foot processes). (B) Transmission EM of patient kidneys showed enlarged mitochondria with disorganized cristae. There were also large remnant vacuoles that contained amorphous membranous inclusions (white arrows, mitochondria; white asterisks, brush border of the proximal tubule; black arrow, vacuoles). (Scale bars: 1 μm.)

− − heterozygotes on HP and to the Mut / ;TgINS-Alb-Mut mice maintained on RD (Fig. 3A). We observed no differences between male and female GFRs (Fig. S4E); however, an increase in serum creatinine was evident in male (P = 0.003) but not in fe- −/− INS-Alb-Mut Fig. 3. Renal function at the whole-kidney and single-nephron level and male Mut ;Tg mice on HP. The overall survival in a −/− INS-Alb-Mut −/− INS-Alb-Mut COX/SDH staining in the kidney sections of Mut ;Tg mice. (A) − − larger group of age- and sex-matched Mut ;Tg mice Whole-animal GFR in the Mut / ;TgINS-Alb-Mut mice fed a HP diet (n = 11; 3 +/− fed a HP diet was 52% (P < 0.0001 compared with their Mut male and 8 female) was 42.1 ± 3.35% of the average GFR in heterozygote littermates with or without the transgene; Fig. S5A), with males mice on RD (n = 5; P < 0.0001). No change was observed in mutants on RD being more severely affected (P = 0.005 between sexes, Fig. (n = 5) or heterozygotes fed a HP diet (n = 9). (B) SNGFR (nanoliters per minute) −/− in male Mut−/−;TgINS-Alb-Mut mice on RD was lower than in Mut+/−;TgINS-Alb-Mut S5B). Histology and ultrastructure of the kidneys in both Mut ; + + INS-Alb-Mut littermates and Mut / control animals (n = 20 vs. 27 SNGFR determi- Tg male and female mice did not reveal any significant − − + − nations on 6 Mut / ;TgINS-Alb-Mut and 6 control, 2 Mut / ;TgINS-Alb-Mut litter- + + differences compared with heterozygotes at this time point mates, and 4 Mut / mice, respectively; P < 0.0001). (C and D) The proximal (Fig. S5E). tubule fluid reabsorption (C) and fractional reabsorption (D) obtained from − − Measurements of single-nephron (SN) GFR and tubular theSNGFRstudiesweresignificantly impaired in the male Mut / ;TgINS-Alb-Mut function were performed on anesthetized male mice fed RD mice (P < 0.0001and P = 0.026, respectively). No differences were ob- − − − − because male Mut / ;TgINS-Alb-Mut mice fed the HP diet did not served in arterial pressure (93 ± 2.2 mm Hg in Mut / ;TgINS-Alb-Mut mice vs. + − + + fi 95 ± 2.6 mm Hg in Mut / and Mut / mice; n = 6 per group) or kidney survive the procedure. SNGFR was signi cantly lower in the − − + − −/− INS-Alb-Mut weight (385 ± 8.5 mg in Mut / ;TgINS-Alb-Mut vs. 395 ± 24 mg in the Mut / Mut ;Tg mice (Fig. 3B), and this reduction was accom- + + and Mut / mice; n = 6 per group). (E) Immunohistochemistry for COX and panied by a parallel diminution of the proximal tubule fluid reab- fi SDH on kidney sections of mutant mice fed a HP diet revealed patchy/focal sorption rate (Fig. 3C). However, there was also a signi cantly decrease in COX staining (20×) in the presence of increased staining for the −/− INS-Alb-Mut lower fractional fluid reabsorption in the Mut ;Tg nuclear-encoded SDH (10×) in sections of the proximal tubules. Data are mice, indicating reduced reabsorptive capacity of the proximal means ± SEM. **P < 0.01; ****P < 0.0001.

Manoli et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 Bioenergetic Profiling of the Proximal Tubule. The abnormal ultrastructure and function of the proximal tubules prompted an in- vestigation of respiratory-chain enzyme expression and function. Using in situ immunohistochemistry, we compared COX (complex IV) and succinate dehydrogenase (SDH) (complex II) + − − − staining patterns in kidneys from Mut / compared with Mut / ; − − TgINS-Alb-Mut mice fed a HP diet for 2 mo. Mut / ;TgINS-Alb-Mut kidneys demonstrated a mixture of COX-positive and COX-negative tubules with uniformly SDH-hyperreactive staining compared with the heterozygotes (Fig. 3E). The enzymatic activities of the respiratory-chain complexes in both liver and kidney protein extracts showed no significant differences (Table S3). Furthermore, we measured levels of CoQ, isoforms 9 and 10, to examine whether a primary or secondary synthetic defect might be associated with the mitochondrial abnormalities observed. − − −/− Mut / ;TgINS-Alb-Mut mice fed a HP diet for 2 mo showed signif- Fig. 4. Lipocalin-2 correlates with kidney disease progression in Mut ; TgINS-Alb-Mut mice. (A) There was no difference in plasma Lcn2 (nanograms icantly lower CoQ10 levels than their heterozygote littermates (P < per milliliter) between heterozygote [n = 24 (12 male and 12 female); solid 0.001) (Table S4); however, CoQ levels were comparable, − − 9 square] and Mut / ;TgINS-Alb-Mut [n = 23 (11 male and 12 female) solid circle] suggesting that the biosynthetic capacity for CoQ was pre- mice on RD or heterozygotes fed a HP diet [n = 26 (17 male, 9 female); open − − served in the mutant kidneys. square]. However, Lcn2 was elevated in Mut / ;TgINS-Alb-Mut mice [n = 12 (5 male and 7 female); open circle] after ingesting a HP diet (P = 0.0007). (B) −/− INS-Alb-Mut Expression Profiling of MMA Renal Disease in Mut ;Tg Plasma Lcn2 (nanograms per milliliter) showed a negative correlation with Kidneys. We compared gene-expression profiles of whole-kidney measured GFR in mice on RD or HP [n = 19; 11 Mut−/−;TgINS-Alb-Mut (open + + + − − − mRNA samples between four female Mut / , Mut / ,andMut / ; triangles) and 8 heterozygote (solid triangles) mice; r = −0.533; P = 0.016; TgINS-Alb-Mut mice after they ingested a HP diet for 2 mo. Fifty-five R2 = 0.244). Lcn2 and GFR data are expressed as percentage of the mean genes fulfilled criteria for twofold differential expression. Sig- value for heterozygote mice on RD. Data are means ± SEM. ****P < 0.0001 – nificant enrichment was identified in 10 pathways, including (Kruskal Wallis test). those involved in immune response, lipid, branched-chain amino acid and ketone metabolism, and cell survival (Tables S5 and S6). − − MMA (n = 46), enrolled in a dedicated natural history study. We Genes showing highly discordant expression between the Mut / ; + + + − documented significant correlations between LCN2 and serum TgINS-Alb-Mut mice and both Mut / or Mut / ;TgINS-Alb-Mut lit- A termates included lipocalin-2 (Lcn2, or neutrophil-gelatinase- markers of renal function including creatinine (Fig. S6 ), cys- − associated lipocalin) (P = 6.74 × 10 5) (15), as well as other tran- tatin-C (Fig. S6B), and estimated GFR (Fig. S6C), as well as scripts expressed in the setting of kidney damage such as Fabp1 markers of oxidant stress, including plasma oxidized LDL (Fig. (P = 0.0024) (16) and Havcr1 [kidney injury molecule (Kim)-1; S6D) and urinary F2iPs (Fig. S6E). No correlation was observed P = 0.0012] (17). Targets of PPARα were up-regulated in the between LCN2 and dietary protein intake (grams per kilogram − − Mut / ;TgINS-Alb-Mut kidneys, including Cyp4a12a and Cyp4a12b, body weight per day) of complete or deficient protein and serum two subtypes of a cytochrome P450 system that are involved in total protein or albumin concentrations (grams per deciliter) the metabolism of arachidonic acid in the proximal tubule and (Fig. S6 F and G). result in the generation of 20-hydroxyeicosatetraenoic acid (20- HETE), a powerful vasoconstrictor (18). A sexually dimorphic Treatment with Antioxidants Ameliorates the Renal Dysfunction – Induced by the HP Diet. To assess the role of oxidative stress in expression was observed for Cyp4a isoforms (Fig. S5 F I). − − the deterioration of renal function in the Mut / ;TgINS-Alb-Mut Lipocalin-2 As a Biomarker of Renal Dysfunction and Oxidative Stress mice, we included 0.5% wt/wt ubiquinone, a highly bioavailable Mut−/− INS-Alb-Mut fi in ;Tg Mice and MMA Patients. To con rm the in- form of CoQ10, and 0.2% wt/wt VitE in the HP mouse chow. duction of Lcn2 observed in the microarray studies, Lcn2 was This regimen has been used previously with success to treat an measured in both mice and patients. Plasma concentrations of isolated case of MMA with optic nerve atrophy (19), whereas the −/− INS-Alb-Mut Lcn2 in the Mut ;Tg mice after 2 mo of ingesting dose and formulation of CoQ10 was identical to that shown to a HP chow were elevated (260.7 ± 59.7% of the mean Lcn2 in significantly decrease oxidative stress and increase survival in ± −/− INS- heterozygotes on RD) compared with their littermates (100 a mouse model of Huntington disease (20). While Mut ;Tg = 6.05%; P 0.0004) (Fig. 4A). Moreover, an increased expression Alb-Mut mice fed a HP diet lost >25% of their weight after 3 d, of Lcn2 correlated with the severity of chronic kidney disease, as those that consumed the same diet plus antioxidants showed demonstrated by the inverse correlation of Lcn2 plasma levels = − = = a trend toward increased survival and decreased weight loss (Fig. with GFR (r 0.533; n 19; P 0.016) (Fig. 4B). Lcn2 cor- S5 C and D; P = NS and 0.013, respectively). Moreover, the related with the decreased GFR even in female animals [r = CoQ /VitE-treated mice (n = 6) showed a significantly im- −0.52; P = not significant (NS)], despite the normal creatinine 10 proved GFR (215.08 ± 57.01 μL/min or 58.6 ± 4.94% hetero- levels and a much milder phenotype, suggesting that Lcn2 is an − − ± μ ± early biomarker of the renal dysfunction in Mut / ;TgINS-Alb-Mut zygote GFR from 151.22 37.6 L/min or 42.1 3.35% on HP mice. To investigate the relation between the Lcn2 induction and diet, which represents a 42.2% improvement in absolute num- the abnormal tubular mitochondrial ultrastructure and focally bers, or 16.5% improvement in percent heterozygote GFR) vs. − − = = decreased COX activity evident in the Mut / ;TgINS-Alb-Mut mice on HP diet (n 11; P 0.0128; Fig. 5A). The improvement mouse kidneys, we evaluated for increased radical oxygen species in the measured GFR was accompanied by a total abrogation of (ROS) production as a byproduct of disturbed mitochondrial increased plasma Lcn2 levels and a reduction in Lcn2 mRNA −/− function. Urinary F2-isoprostanes (F2iPs) are a well-character- expression in the kidney of the CoQ10/VitE-treated Mut ; INS-Alb-Mut ized marker of ROS-induced lipid peroxidation and were Tg mice (Fig. 5 B and C), as well as by the normali- found to positively correlate with urine Lcn2 concentrations in zation of the increased plasma creatinine concentrations seen − − Mut / ;TgINS-Alb-Mut mice (r = 0.712; n =14; P = 0.0063). We next in males (Fig. 5D). The decrease in Lcn2 was paralleled by a sought to validate these biomarkers in a cohort of patients with similar decrease in urinary F2iPs (Fig. 5E). On the other hand,

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1302764110 Manoli et al. Downloaded by guest on September 28, 2021 − − mitochondrial abnormalities seen in Mut / mice (11) in the face of >1,000× elevated plasma methylmalonic acid concentrations suggests that the cellular phenotype could be rescued by tissue- specific expression of Mut (Fig. S2B). Moreover, a significant functional improvement in the GFR was observed in the CoQ10/ − − VitE treated Mut / ;TgINS-Alb-Mut mice despite the continuous exposure of the kidneys to a HP diet and elevated circulating metabolites (Fig. 5 A and F), whereas daily i.p. administered methylmalonic acid for 2 mo did not have significant nephrotoxic effects in wild-type mice (Fig. S4A). Together, these findings suggest that cell and/or mitochondrial autonomous effects, rather than elevations in methylmalonic acid per se, are re- sponsible for the hepatic mitochondrial changes and the GFR decrease associated with proximal tubular mitochondrial dysfunction. This challenges previous theories about methylmalonate as a toxic agent (21, 22) and further supports ob- servations from gene therapy studies showing that the long-term − − correction of Mut / mice is mediated by a small number of stably transduced cells (13, 23). The concept that a kidney with normal Mut activity may be protected from MMA nephrotoxicity is reinforced by a recent study that proposed providing renal allografts as a form of “cellular” therapy for MMA, despite the implicit knowledge that the allograft will be exposed to very high metabolite concentrations in the recipient patient (24). Further support of a cell-autonomous effect was demonstrated by the local production of methylmalonic acid in the brain tissue on HP diet (Fig. S3D), as well as by the focal decrease in COX in − − a subset of the Mut / ;TgINS-Alb-Mut tubules (Fig. 3E), with no obvious impairment in respiratory-chain complex activities ana- MEDICAL SCIENCES lyzed in whole kidney extracts, similar to what is observed in the skeletal muscle of patients with mitochondrial myopathies (25). Given that HP seems necessary for the progression of chronic renal disease in our model (Fig. S4 C and D), we suggest that both mitochondrial dysfunction and toxic metabolite accumula- tion play a role in disease pathophysiology; hence, conditional cell-autonomy more accurately reflects the current findings. Gene-expression profiling on whole kidneys collected 2 mo after initiating a HP diet revealed significant enrichment in several inflammatory, signaling, and metabolic pathways. A number of gene-targets of the retinoid X receptor/peroxisome proliferator-activated receptor α (PPARα), including Cyp4a12a and -b, Fabp1, and Hmgcs2, were up-regulated, consistent with the pronounced number of mitochondria in the tubules of patients and mice. Biomarkers of acute and chronic kidney injury, Havcr1 (KIM-1) and Lcn2 (neutrophil gelatinase-associated − − lipocalin), were up-regulated in the Mut / ;TgINS-Alb-Mut kidneys. Fig. 5. Dietary supplementation with CoQ10 (0.5% wt/wt) and VitE (0.2% Lcn2, originally identified as a 25-kDa protein associated with wt/wt) ameliorated the loss of GFR and was predicted by plasma Lcn2 levels −/− INS-Alb-Mut −/− INS-Alb-Mut neutrophil gelatinase (26), is largely produced by the tubular in Mut ;Tg mice. (A) Mut ;Tg mice on the HP plus anti- epithelium of the distal nephron after cellular damage and is oxidants diet [n = 6 (2 male, 4 female)] showed a significantly improved GFR − − compared with Mut / ;TgINS-Alb-Mut mice fed solely HP diet [n = 11 (3 male, 8 associated with acute kidney injury (15), as well as chronic kidney female); P = 0.0104]. (B and C) Antioxidants reduced Lcn2 in the plasma (B) disease progression (27). The murine and patient studies pre- and in the kidney tissue (C)ofMut−/−;TgINS-Alb-Mut mice (P = NS compared with sented here demonstrate a strong correlation of Lcn2 with treated heterozygote mice). (D–F) Plasma creatinine normalized in the trea- markers of oxidative stress. Moreover, Lcn2 mRNA expression ted male mice (D), and urine F2iPs improved after antioxidant therapy (P = NS in the tissue and its resulting plasma concentrations were re- compared with treated heterozygote mice) (E), despite the persistence of duced after the administration of antioxidants, supporting a link elevated serum MMA levels (P < 0.0001 between mutant and heterozygote between oxidative stress and Lcn2 in the renal disease of MMA mice on either diet) (F). Data are presented as means ± SEM. *P < 0.05; **P < < < – and potentially other forms of tubulointerstitial nephritis asso- 0.01; ***P 0.001; ****P 0.0001 (Kruskal Wallis test). ciated with mitochondrial dysfunction. The proximal tubular dysfunction was further validated by − − −/− INS-Alb-Mut plasma MMA concentrations were identical between the Mut / ; micropuncture studies of single Mut ;Tg nephrons TgINS-Alb-Mut mice receiving either diet (Fig. 5F). (Fig. 3 B–D) that demonstrated aberrant ammonium excretion in the presence of hyperchloremic metabolic acidosis (Table S2). Discussion The diminished GFR can thus be viewed as protective, because it Similar to MMA patients who have received orthotopic liver reduces the absorptive load upon the functionally impaired − − transplantation, Mut / ;TgINS-Alb-Mut animals demonstrated signifi- proximal tubule cells. Although it is unclear which exact mech- cant elevations of plasma methylmalonic acid, CTIN, and di- anism(s) underlies the drastic reduction of GFR, single-nephron minished GFR following HP-diet administration. Our observation measurements documented a functional impairment well before that transgenesis corrected the severe hepatic ultrastructural any overt structural or ultrastructural changes in the tubules or

Manoli et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 the podocytes and glomerular architecture were detected. (Fig. 5). Both compounds are orally administered, display an Tubuloglomerular feedback, the response of GFR to an in- excellent safety profile, even at high doses, and are given in creased delivery of NaCl to the macula densa region, might play combination to increase antioxidant capacity (20, 31). We found − − a role, although in our approach, all fluid is withdrawn at the end that treated Mut / ;TgINS-Alb-Mut mice experienced a significant of the proximal tubule, thus normalizing distal delivery to zero in amelioration in the loss of GFR and a normalization of plasma both wild-type and mutant mice. Therefore, it is likely that the Lcn2 levels compared with untreated animals fed a HP diet, − − marked reduction of GFR in Mut / ;TgINS-Alb-Mut mice reflects despite exhibiting the same magnitude of elevation of serum a functional process initiated by the proximal tubule mitochon- metabolites. Furthermore, a decrease in the GFR was accom- drial dysfunction, possibly mediated by altered vascular tone at panied by an increase in plasma Lcn2, even when the creatinine the afferent or efferent arteriole. That increased ROS and in- concentration was normal, establishing plasma Lcn2 as a sensi- duction of genes involved in the generation of metabolites of tive biomarker to monitor therapies aimed at the renal disease of − − arachidonic acid, such as 20-HETE, were identified in Mut / ; MMA. Our findings demonstrate that readily available anti- TgINS-Alb-Mut kidneys supports the concept that small-molecule oxidants can significantly affect the rate of decline of renal mediators may play an important role in the kidney disease of − − function in a mouse model that replicates the kidney disease of MMA (18). Mut / ;TgINS-Alb-Mut mice, therefore, represent MMA, a relentless disease with limited therapeutic options other a unique model to explore mechanisms of tubuloglomerular feed- than solid-organ transplantation. back in the context of mitochondrial dysfunction in the tubules. Materials and Methods The normal levels of renal CoQ9 indicate that impaired CoQ biosynthesis is not a primary determinant of the kidney disease of Animals were housed in an Association for Assessment and Accreditation of MMA (Table S4) and is consistent with the distinct renal pa- Laboratory Animal Care-accredited specific pathogen-free facility and were − − thology exhibited by the Mut / ;TgINS-Alb-Mut animals compared approved by the Institutional Animal Care and Use Committee of the Na- with a model of defective CoQ biosynthesis, the Pdss2kd/kd mice tional Human Genome Research Institute (NHGRI), National Institutes of − − (28). However, the decrease in renal CoQ in the Mut / ;TgINS- Health (NIH). The human studies were approved by the NHGRI institutional 10 review board as part of a NIH protocol (ClinicalTrials.gov identifier: Alb-Mut mouse kidney extracts paralleled a previous report of fi NCT00078078) and were performed in compliance with the Helsinki Decla- reduced CoQ10 concentrations in MMA patient broblasts (29) ration. Experimental details are described in SI Materials and Methods. and further suggested that markers of oxidant stress be explored in both animals and patients. Indeed, dietary stress caused urine ACKNOWLEDGMENTS. We thank all patients and their families; Shelley −/− INS-Alb-Mut F2iPs to increase in the Mut ;Tg mice compared with Hoogstraten-Miller for veterinary support; Irene Ginty, Cherry Yang, Tina their diet-matched littermates. Furthermore, oxidative markers, Aroyo, Theresa Ferrine, and Lemlem Alemu for mouse maintenance; and Isa Bernardini and Roxanne Fischer for processing patient samples. This work including urine F2iPs and plasma oxidized LDL, were elevated in was supported by the Intramural Research Program of the National Human MMA patients with advanced kidney disease (Fig. S6). These Genome Research Institute (I.M., J.R.S., C.W., N.S.T., J.C., J.L.S., R.J.C., A.G.E., findings align with previous reports (30) and suggest a possible and C.P.V.), the National Institute of Diabetes and Digestive and Kidney set of biomarkers to follow disease severity and/or progression. Diseases (L.L., P.H., and J.S.), the National Cancer Institute (M.A.-A. and M.T.), and the Office of the Director (P.M.Z. and V.H.) of the National Institutes of Because the renal disease of MMA correlated with markers of Health. J.R.S. and C.W. were also supported by the Angels for Alyssa (MMA oxidant injury in mice and patients, we devised a treatment Research Fund). P.H. was supported by the Philippe Foundation, the Oak Ridge protocol with two well-established antioxidants, CoQ10 and VitE Institute for Science and Education, and Paris Descartes University, France.

1. Walter JH, et al. (1989) Chronic renal failure in methylmalonic acidaemia. Eur J Pediatr 17. Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre JV (2002) Kidney Injury 148(4):344–348. Molecule-1 (KIM-1): A novel biomarker for human renal proximal tubule injury. 2. Matsui SM, Mahoney MJ, Rosenberg LE (1983) The natural history of the inherited Kidney Int 62(1):237–244. methylmalonic acidemias. N Engl J Med 308(15):857–861. 18. Kroetz DL, Xu F (2005) Regulation and inhibition of arachidonic acid omega- 3. Manoli I, Venditti CP (2005) Methylmalonic acidemia. GeneReviews, eds Pagon RA, hydroxylases and 20-HETE formation. Annu Rev Pharmacol Toxicol 45:413–438. et al. (University of Washington, Seattle, WA). 19. Pinar-Sueiro S, Martínez-Fernández R, Lage-Medina S, Aldamiz-Echevarria L, Vecino E 4. D’Angio CT, Dillon MJ, Leonard JV (1991) Renal tubular dysfunction in methylmalonic (2010) Optic neuropathy in methylmalonic acidemia: The role of neuroprotection. J Inherit Metab Dis, 10.1007/s10545-010-9084-8. acidaemia. Eur J Pediatr 150(4):259–263. 20. Smith KM, et al. (2006) Dose ranging and efficacy study of high-dose coenzyme Q10 5. Hörster F, et al. (2007) Long-term outcome in methylmalonic acidurias is influenced by formulations in Huntington’s disease mice. Biochim Biophys Acta 1762(6):616–626. the underlying defect (mut0, mut-, cblA, cblB). Pediatr Res 62(2):225–230. 21. Wolff JA, et al. (1985) Proximal renal tubular acidosis in methylmalonic acidemia. 6. Oberholzer VG, Levin B, Burgess EA, Young WF (1967) Methylmalonic aciduria. An J Neurogenet 2(1):31–39. inborn error of metabolism leading to chronic metabolic acidosis. Arch Dis Child 22. Kashtan CE, et al. (1998) Chronic administration of methylmalonic acid (MMA) to rats – 42(225):492 504. causes proteinuria and renal tubular injury. Pediatr Res 43:309. 7. Hauser NS, Manoli I, Graf JC, Sloan J, Venditti CP (2011) Variable dietary management 23. Carrillo-Carrasco N, Chandler RJ, Chandrasekaran S, Venditti CP (2010) Liver-directed of methylmalonic acidemia: Metabolic and energetic correlations. Am J Clin Nutr recombinant adeno-associated viral gene delivery rescues a lethal mouse model of 93(1):47–56. methylmalonic acidemia and provides long-term phenotypic correction. Hum Gene 8. Nyhan WL, Gargus JJ, Boyle K, Selby R, Koch R (2002) Progressive neurologic disability Ther 21(9):1147–1154. in methylmalonic acidemia despite transplantation of the liver. Eur J Pediatr 161(7): 24. Brassier A, et al. (2013) Renal transplantation in 4 patients with methylmalonic aciduria: 377–379. A cell therapy for metabolic disease. Mol Genet Metab, 10.1016/j.ymgme.2013.05.001. 9. Chandler RJ, et al. (2007) Metabolic phenotype of methylmalonic acidemia in mice 25. DiMauro S, Tanji K, Bonilla E, Pallotti F, Schon EA (2002) Mitochondrial abnormalities and humans: The role of skeletal muscle. BMC Med Genet 8:64. in muscle and other aging cells: Classification, causes, and effects. Muscle Nerve 26(5): 10. Peters H, et al. (2003) A knock-out mouse model for methylmalonic aciduria resulting 597–607. in neonatal lethality. J Biol Chem 278(52):52909–52913. 26. Kjeldsen L, Johnsen AH, Sengeløv H, Borregaard N (1993) Isolation and primary 11. Chandler RJ, et al. (2009) Mitochondrial dysfunction in mut methylmalonic acidemia. structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem 268(14):10425–10432. FASEB J 23(4):1252–1261. 27. Viau A, et al. (2010) Lipocalin 2 is essential for chronic kidney disease progression in 12. Chandler RJ, Venditti CP (2008) Adenovirus-mediated gene delivery rescues a neonatal mice and humans. J Clin Invest 120(11):4065–4076. lethal murine model of mut(0) methylmalonic acidemia. Hum Gene Ther 19(1):53–60. 28. Peng M, et al. (2008) Primary coenzyme Q deficiency in Pdss2 mutant mice causes 13. Chandler RJ, Venditti CP (2010) Long-term rescue of a lethal murine model of meth- isolated renal disease. PLoS Genet 4(4):e1000061. ylmalonic acidemia using adeno-associated viral gene therapy. Mol Ther 18(1):11–16. 29. Haas D, et al. (2009) Coenzyme Q(10) is decreased in fibroblasts of patients with meth- 14. Brenes LG, Sanchez MI (1993) Impaired urinary ammonium excretion in patients with ylmalonic aciduria but not in mevalonic aciduria. J Inherit Metab Dis 32(4):570–575. – isolated proximal renal tubular acidosis. J Am Soc Nephrol 4(4):1073 1078. 30. Atkuri KR, et al. (2009) Inherited disorders affecting mitochondrial function are as- fi 15. Mishra J, et al. (2003) Identi cation of neutrophil gelatinase-associated lipocalin as sociated with glutathione deficiency and hypocitrullinemia. Proc Natl Acad Sci USA a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 14(10): 106(10):3941–3945. 2534–2543. 31. Thomas SR, et al. (2001) Dietary cosupplementation with vitamin E and coenzyme 16. Kamijo A, et al. (2004) Urinary excretion of fatty acid-binding protein reflects stress Q(10) inhibits atherosclerosis in apolipoprotein E gene knockout mice. Arterioscler overload on the proximal tubules. Am J Pathol 165(4):1243–1255. Thromb Vasc Biol 21(4):585–593.

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