Reduced Renal Methylarginine Metabolism Protects Against Progressive Kidney Damage

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Reduced Renal Methylarginine Metabolism Protects against Progressive Kidney Damage

† James A.P. Tomlinson,* Ben Caplin, Olga Boruc,* Claire Bruce-Cobbold,* Pedro Cutillas,* † ‡ Dirk Dormann,* Peter Faull,* Rebecca C. Grossman, Sanjay Khadayate,* Valeria R. Mas, † | Dorothea D. Nitsch,§ Zhen Wang,* Jill T. Norman, Christopher S. Wilcox, † David C. Wheeler, and James Leiper*

*Medical Research Council Clinical Sciences Centre, Imperial College, London, United Kingdom; †Centre for Nephrology, UCL Medical School Royal Free, London, United Kingdom; ‡Translational Genomics Transplant Laboratory, Transplant Division, Department of Surgery, University of Virginia, Charlottesville, Virginia; §Department of Non-communicable Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, United Kingdom; and |Hypertension, Kidney and Vascular Research Center, Georgetown University, Washington, DC

ABSTRACT Nitric oxide (NO) production is diminished in many patients with cardiovascular and renal disease. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthesis, and elevated plasma levels of ADMA are associated with poor outcomes. Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is a methylarginine- metabolizing enzyme that reduces ADMA levels. We reported previously that a DDAH1 gene variant associated with increased renal DDAH1 mRNA transcription and lower plasma ADMA levels, but counterintuitively, a steeper rate of renal function decline. Here, we test the hypothesis that reduced renal-specific ADMA metabolism protects against progressive renal damage. Renal DDAH1 is expressed predominately within the proximal PT2/2 tubule. A novel proximal tubule–specific Ddah1 knockout (Ddah1 ) mouse demonstrated tubular cell accu- PT2/2 mulation of ADMA and lower NO concentrations, but unaltered plasma ADMA concentrations. Ddah1 mice were protected from reduced kidney tissue mass, collagen deposition, and profibrotic cytokine expression in two independent renal injury models: folate nephropathy and unilateral ureteric obstruction. Furthermore, a study of two independent kidney transplant cohorts revealed higher levels of human renal allograft methylarginine- metabolizing enzyme gene expression associated with steeper function decline. We also report an association among DDAH1 expression, NO activity, and uromodulin expression supported by data from both animal and human studies, raising the possibility that kidney DDAH1 expression exacerbates renal injury through uromodulin- related mechanisms. Together, these data demonstrate that reduced renal tubular ADMA metabolism protects against progressive kidney function decline. Thus, circulating ADMA may be an imprecise marker of renal methylarginine metabolism, and therapeutic ADMA reduction may even be deleterious to kidney function.

J Am Soc Nephrol 26: 3045–3059, 2015. doi: 10.1681/ASN.2014030280

CKD poses an increasing global disease burden and dimethylaminohydrolase isoforms 1 and 2 (DDAH 1 contributes to cardiovascular disease (CVD), which is and 2) and alanine-glyoxylate aminotransferase-2 the leading cause of death worldwide.1,2 CKD is associated with reduced bioavailability of nitric oxide Received March 25, 2014. Accepted February 16, 2015.

(NO), which is required to maintain normal vascular Published online ahead of print. Publication date available at 3,4 and kidney function. Asymmetric dimethylarginine www.jasn.org. (ADMA) is released into the cytoplasm during nor- Correspondence: Dr. James Tomlinson, Medical Research mal protein turnover and competes with L-arginine Council Clinical Sciences Centre, Imperial College, Hammer- binding at the active site of nitric oxide synthase smith Hospital Campus, DuCane Road, London, W12 0NN, UK. (NOS) to block NO synthesis. The methylarginine- Email: [email protected] metabolizing enzymes (MAMEs)—dimethylarginine Copyright © 2015 by the American Society of Nephrology

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(AGXT2)5—metabolize ADMA and other methylarginines to in- active products. Thus, MAMEs represent alternative pathways of endogenous NO regulation.6 The kidney expresses all MAME and NOS isoforms in a cell- specific distribution,5,7,8 thereby providing the means for in- tricate, cell type–specific control of NO bioavailability. Renal NO regulates hemodynamics,9 afferent arteriolar vascular re- activity,10 glomerular filtration,11 tubular reabsorption, and solute excretion.8,12 Specifically, the proximal tubule (PT) is Figure 1. Immunohistochemical staining for DDAH1. Human allograft protocol biopsy tissue. (A) DDAH1 staining is seen in the the principal renal cell type expressing the enzyme with the epithelial cells of the proximal tubule (arrows) and not in the greatest activity for metabolism of ADMA, DDAH1;7,13 how- fl glomerulus (g) or control. (B) DDAH1 antibody preincubated with ever, how this in uences tubular reabsorption and response to DDAH1 peptide. Original magnification, 3200. injury through NO-ADMA regulation is unclear. Human studies demonstrate arteriovenous ADMA gradients across the kidney, which represent a large proportion of total-body codon-optimized Cre (iCre) expression in response to andro- ADMA clearance through enzymatic metabolism and urinary gen stimulation. excretion.14 Consequently, plasma ADMA levels rise with de- Two double transgenic mouse strains were created, both clining kidney function, as demonstrated by Vallance and col- possessing the KAP2iCre gene along with either ROSA26eYFP (a leagues’ early report of elevated plasma ADMA, lower NO, and reporter strain designed to confirm PT-specificgenemanipula- vascular dysfunction in patients with ESRD.15 tion [loxP sites flank a YFP STOP codon; Figure 2A]) or floxed Increased circulating ADMA is a powerful predictor of Ddah1 (loxP sites flank exon 1 of Ddah1,whichencodesthe progressive CVD and CKD, independent of eGFR and other initiating methionine residue and the first 100 amino acids of traditional risk factors.16–20 Because plasma ADMA increases theprotein;Figure2D).23 To achieve temporal control over PT- with declining kidney function, the direction of causality is specificKAP2iCreexpressionandgenedisruption,onlyfemale difficult to resolve by correlation alone. Although increased mice were treated with exogenous testosterone and used for study. plasma ADMA levels may correlate with reduced total-body YFP expression in female KAP2iCre/ROSA26eYFP reporter NO bioavailability, they do not necessarily reflect the balance mice treated with testosterone was demonstrated using Image- of NOS inhibition within the renal interstitium during pro- Stream analysis of isolated renal tubular cells (Figure 2B) and gressive CKD. Our published data suggest that lower plasma by renal tissue histologic testing (Figure 2C). Mice not pos- ADMA does not necessarily protect against renal function de- sessing the KAP2iCre transgene did not express YFP. cline and that increased DDAH1 activity limited to the kidney may in fact be harmful. Thus, we reported a noncoding PT-Specific Ddah1 Deletion DDAH1 variant (rs17384213) that was associated with higher The Ddah1 gene was deleted after testosterone exposure in 2 2 renal DDAH1 mRNA expression and lower plasma ADMA KAP2iCre/Ddah1-floxed mice (Ddah1PT / )butnotin fl fl concentrations. Counterintuitively, however, this correlated Ddah1-floxed (Ddah1 / ) controls (Figure 2E). Reduced with a faster rate of eGFR decline in two independent cohorts Ddah1 expression was clearly observed in the kidney, where of patients with CKD.21 Competitive inhibition of NOS by it exceeded 50% (P,0.05) (Figure 2F). Nonsignificant reduc- ADMA may play both protective and pathogenic roles,6 de- tions were detected in the liver and brain. Whole kidney lysate 2 2 termined by such factors as disease site, time course, and mag- DDAH1 protein expression in Ddah1PT / mice was reduced nitude of response. by approximately 80% (P,0.001) (Figure 2G) and enzymatic In light of these uncertainties, it is clearly important to DDAH1 activity was reduced by approximately 70% (P,0.05) define the role of PT-specific DDAH1 in CKD progression. We (Figure 2H). hypothesized that reduced renal DDAH1 activity protects against renal function decline, independent of circulating Downstream Effects of PT-Specific Ddah1 Deletion on NO and ADMA. Methylarginines and NO ADMA recovered from isolated renal tubules was almost 7-fold 2 higher (P,0.05) and nitrogen oxide species, nitrite NO2 and ni- 2 PT2/2 RESULTS trate NO3 (NOx) was 2.5-fold lower (P,0.05) in Ddah1 fl fl mice versus Ddah1 / controls (Figure 3, A and B). The effect of PT-Specific Gene Targeting PT-specific Ddah1 deletion was restricted to tubular isolates The PT is the principal renal cell type to express DDAH1 and was not evident in whole kidney tissue, plasma, or urine, (7,13 and Figure 1). Conditional PT-specific gene manipulation indicating tubule-specific ADMA-NO perturbation (Figure 3, was achieved using the KAP2iCre gene construct.22 Regulatory A–H). This was further demonstrated by an absence of baseline elements of the kidney androgen–regulated protein (KAP) pro- effects on renal expression of alternative MAME isoforms and moter and angiotensinogen genes confer highly PT-specific, NOS enzymes (Figure 3I), systolic BP (Figure 3J), renal function

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2 2 Figure 2. Proximal tubule-specific gene targeting. Generation of the ROSAYFP reporter mouse (A–C) and Ddah1PT / mouse strains (D–H). (A) Schematic of conditional YFP expression in the reporter mouse. (B) ImageStream flow cytometry reveals YFP expression in renal tubular cells isolated from the KAPiCre+ genotype but not KAPiCre2. R2 gating for sphericity, R4 gating for fluorescence. Bright-field and yellow fluorescent single-cell images are displayed (B) (KAPiCre+). (C) YFP fluorescence visible in unstained kidney sections from KAPiCre+ mice 2 2 and not KAPiCre2. Representative of three experiments. Bars=50 mm. (D) Schematic of conditional Ddah1 deletion in the Ddah1PT / mouse. (E) Kidney tissue end point PCR genotyping for KAPiCre (KC), floxed Ddah1, and deleted Ddah1 sequences reveals gene excision in 2 2 KAPiCre +(Ddah1PT / ) but not KAPiCre–(Ddah1fl/fl) genotypes (two per genotype shown for representation). Excision is detected within liver and brain, but quantitative RT-PCR analysis reveals significant Ddah1 mRNA reduction within kidney but not other organs (F). (G) Kidney tissue DDAH1 protein (4 for representation in Western blot) and (H) enzymatic activity are significantly reduced (70%–80%). Mann– 2 2 Whitney U test, n=6 (Ddah1fl/fl)andn=8 (Ddah1PT / ). *P,0.05; ***P,0.001. Error Bars, mean6SEM. TIC, total ion count. according to plasma creatinine (Figure 3K), and urinary vol- (Figure 4, A–F). However, through use of a proteomic approach, umes (Figure 3L). tubular Ddah1 disruption significantly affected several urinary peptides under baseline conditions. Of 1057 peptides screened 2 2 fl fl Effects of PT-Specific Ddah1 Deletion on Urine in Ddah1PT / and Ddah1 -/ mouseurine,relativeconcentra- Biochemistry tions of 82 peptides were significantly altered (P,0.05) (Figure 2 2 Urinary electrolyte, protein, and amino acid concentrations 5). Most significantly downregulated in Ddah1PT / mouse were not significantly affected by PT-specific Ddah1 deletion urine was uromodulin (UMOD) (8.5-fold lower in urine from

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Figure 3. Effects of PT-specific Ddah1 deletion on ADMA and NO are tubule-specific. (A and B) Tubular cells isolated from 2 2 Ddah1PT / mice contained significantly higher ADMA and lower NO concentrations. SDMA, a methylarginine that is not a substrate for DDAH1, was unaffected. (C–H) These changes were not evident in whole kidney tissue, plasma, or urine. (I) Tubular cell expression 2 2 of other MAMEs and NOS isoforms (neuronal, inducible, and endothelial) was unaffected in Ddah1PT / mice. (J–L) Similarly, systolic BP, plasma creatinine, and urinary volumes were not altered. Mann–Whitney U test, n=6 each group; *P,0.05. (Note: The mea- surement of urinary NOx is not necessarily an accurate determination of renal NOS activity, particularly in the context of a nitrate- containing diet). ns, not significant. Error bars, mean6SEM.

2 2 fl fl Ddah1PT / mice compared with Ddah1 -/ controls) (P,0.001 casts, and interstitial inflammatory cell infiltrates (Figure 6; see or q=0.00083) (Figure 5). In addition, urinary Col1a1 was down- Supplemental Material for a full characterization of folate ne- 2 2 regulated 6.5-fold in Ddah1PT / mice (P,0.001; q=0.0017) at phropathy in wild-type mice). At 12 weeks after folate adminis- 2 2 baseline. tration, significant differences emerged between Ddah1PT / fl fl and Ddah1 -/ control mice. Renal collagen deposition was sig- 2 2 Effect of PT-Specific Ddah1 Deletion in Folate nificantly lower in Ddah1PT / mice treated with folate than in fl fl Nephropathy Ddah1 -/ controls (P,0.05) (Figures 7 and 8). In addition, re- A single intraperitoneal injection of folate induced acute tubular duction in renal mass according to kidney-to-body weight ratios 2 2 fl fl injury in all mice, manifested by gross tubular dilatation, luminal was significantly attenuated in Ddah1PT / versus Ddah1 -/

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Figure 4. Effects of PT-specific Ddah1 deletion on urinary electrolytes, protein, and amino acids at baseline. No consistent changes in 2 2 urine biochemistry were identified in Ddah1PT / mice including concentrations of sodium (A), phosphate (B), glucose (C), protein (D; according to urine protein:creatinine ratio [UPCR]) or creatinine (F). (E) Amino acids L-citrulline and L-arginine were product and substrate for NO synthesis, respectively; L-glutamine was selected because of its abundance in urine and has been shown to be elevated in patients with tubular dysfunction (Fanconi syndrome).51 All values corrected for creatinine concentration. Mann–Whitney U test, n=8 fl fl 2 2 (Ddah1 / )andn=20 (Ddah1PT / ). ns, not significant; KC, KAPiCre. Error bars, mean6SEM.

Figure 5. Heat map of urinary proteomic analysis after PT-specific Ddah1 deletion at baseline. Eighty-two of 1057 peptides screened were significantly altered by Ddah1 deletion. The 10 most significantly down- and upregulated peptides are represented here. (The q values represent values of significance adjusted for multiple comparisons; n=4.)

2 2 mice (P,0.01) (Figure 7B). Kidney tissue proﬁbrotic cytokine Furthermore, at 12 weeks, Ddah1PT / mice were protected 2 2 expression was 3- to 5-fold lower in Ddah1PT / mice (Col1a2: from other manifestations of advanced renal impairment, such P,0.05; TGF-b:P,0.01; endothelin-1: P,0.05) (Figure 7C). as hypertension (systolic BP; P,0.01) (Figure 7D) and raised

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2 2 Figure 6. Histologic appearances of folate nephropathy in Ddah1PT / mice and controls. At 2 days and 2 weeks after a single intraperitoneal folate injection, significant disease was evident, including tubular dilatation (td), epithelial cell flattening, luminal casts (lc), and more significantly at 2 weeks, tubulointerstitial inflammatory cell infiltrates (inc). At 12 weeks after folate injury, tubulointerstitial 2 2 fibrosis/extracellular matrix (ecm) deposition was evident. There were no gross histologic differences between Ddah1fl/fl and Ddah1PT / mice; however, analysis of collagen deposition and profibrotic cytokine gene expression revealed protection from PT-specificDdah1de- 2 2 letion (Figure 7). Images representative of n=9 (Ddah1fl/fl)andn=12 (Ddah1PT / ). Bars=50 mm. Original magnification, 3200. plasma creatinine (P,0.001) (Figure 7E). There was a trend to- and eGFR decline in two independent human renal transplant co- 2 2 ward lower levels of proteinuria in Ddah1PT / mice, although horts (Table 1). In both the London and the Virginia cohort, there this did not reach statistical significance (P=0.21) (Figure 7E). was a strong positive correlation between corrected DDAH1 expression and corrected AGXT2 expression (Supplemental Material). Effect of PT-Specific Ddah1 Deletion in Unilateral Therefore, for further analysis of eGFR decline we used a composite Ureteric Obstructive Nephropathy methylarginine gene expression calculated from the geometric To confirm that the protection against fibrosis observed with mean of the DDAH1 and AGXT2 expression. In both cohorts, PT-specific Ddah1 deletion was not a phenomenon unique to higher MAME expression was associated with a steeper decline in 2 2 folate toxicity, Ddah1PT / mice were exposed to a second eGFR during the 1 year after allograft biopsy (Table 2). Although renal injury model: unilateral ureteric obstruction (UUO). there was an association between MAME expression and baseline (See Supplemental Material for a detailed characterization of eGFR in the Virginia cohort, this did not reach statistical significance UUO nephropathy in wild-type mice). in the London participants. The multilevel approach allows estima- At 2 weeks after UUO, severe tubulointerstitial disease was tion of the association between gene expression and eGFR decline evident in kidney sections from obstructed kidneys, with gross independent of the baseline eGFR, as illustrated in Figure 10. tubular dilatation, epithelial cell flattening, parenchymal thin- ning, and collagen deposition (Supplemental Material). Com- Association among Kidney DDAH1 Expression, NO fl fl 2 2 pared with Ddah1 / controls, kidney tissue from Ddah1PT / Activity, and UMOD mice exhibited reduced loss of renal mass (P,0.05) and collagen Given the findings of lower urinary UMOD peptide in 2 2 deposition (P,0.05) (Figure 9, A–C). Furthermore, kidney tis- proteomic analysis of Ddah1PT / mouse urine, we examined sue expression of profibrotic genes Col1a2andTGF-b was sig- the association between DDAH1 gene expression and urinary 2 2 nificantly lower in Ddah1PT / mice (P,0.05) (Figure 9D). UMOD protein concentration in the London patient cohort. Although there was only a trend toward a positive correlation Allograft MAME Expression and eGFR Decline in Humans in the cohort as a whole, DDAH1 gene expression and urinary To confirm the preceding laboratory findings, we measured UMOD were significantly associated when the analysis was kidney tissue MAME (DDAH1 and AGXT2) transcript levels restricted to recipients of live-donor allografts (to minimize

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Figure 7. Effects of PT-specific Ddah1 deletion at 12 weeks after folate administration. (A) Renal collagen deposition was significantly 2 2 fl fl reduced in Ddah1PT / mice versus Ddah1 / controls (4.5 versus 7.2%; P,0.05). Two-way ANOVA with Bonferroni post-test, *P,0.05. 2 2 fl fl (B) Ddah1PT / mice had significantly attenuated kidney-to-body weight ratio reduction compared with Ddah1 / controls (P,0.001; values are deviations from means of vehicle-treated controls). (C) Quantitative RT-PCR analysis revealed significantly lower renal tissue 2 2 profibrotic cytokine expression in Ddah1PT / mice. Col1a2 was decreased .3-fold (P,0.05), TGFb was decreased .5-fold (P,0.01), 2 2 and endothelin-1 (ET-1) was decreased almost 3-fold (P,0.05). (D and E) Systolic BP and serum creatinine were lower in Ddah1PT / mice; **P,0.01 and ***P,0.001, respectively). (F) Proteinuria according to urine protein-to-creatinine ratio (UPCR) was reduced in 2 2 fl fl Ddah1PT / mice, but this change was not statistically significant. Mann–Whitney U test unless otherwise stated, n=9 (Ddah1 / )and 2 2 n=12 (Ddah1PT / ). *P,0.05; **P,0.01; ***P,0.001. ns, not significant. Error bars, mean6SEM. the effect of ischemia and other confounding variables asso- two human cohorts. We conclude that local imbalances of the ciated with deceased donor organs) (P=0.039) (Figure 11A). NO-ADMA-MAMEaxis maydetermine kidney function decline To establish whether the positive correlation between renal more reliably than circulating ADMA. PT cell–specific Ddah1 DDAH1 activity and UMOD protein expression observed in the deletion was dissociated from baseline plasma ADMA and sys- 2 2 Ddah1PT / mouse and human cohort was NO dependent, we temic BP. The observed protection against kidney collagen de- performed a further in vivo study in wild-type mice receiving a position and functional decline was therefore independent of nonselective NOS inhibitor (L-NG-nitroarginine methyl ester systemic confounders and distinguishes a critical difference be- [L-NAME]). Changes in renal UMOD protein expression were tween local (protective) and systemic (harmful) effects of assessed. After 2 weeks of L-NAME treatment (1 mg/ml dis- ADMA in renal function decline. solved in drinking water; concentrations previously established Data from two human renal transplant cohorts support the to systemically inhibit NOS in vivo24–26), kidney tissue NOx animal studies. Modeling renal function over time in the post- (nitrites and nitrates) were reduced by 25% (P,0.01). Further- transplant period using laboratory values is not straightfor- more, UMOD protein expression was significantly reduced after ward. Our estimates show substantial unexplained variation L-NAME treatment: 40% in kidney tissue (P,0.01) and 30% in that was not significantly improved with addition of other urine (P,0.01) (Figure 11, B–E). clinical variables. Despite this, we detected an association between eGFR decline and increased MAME expression in both renal transplant cohorts. The relationship between the DISCUSSION DDAH1 and AGXT2 enzymesisunclear,buttheirrelative expression was strongly correlated. A summary composite This study focused on MAME activity limited to kidney tissue by (geometric mean) of both the enzymes was used to demon- exposinganoveltransgenicmousestraintotwodifferentformsof strate that raised kidney tissue MAME expression correlated renal injury and examining renal biopsy and outcome data from with poorer outcomes in terms of eGFR.

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mechanisms. Furthermore, DDAH1 overexpression occurs indiscriminately in cell types that do not normally express DDAH1, but play significant roles in inflammation and fibrosis (e.g., macrophages). Here, PT-specific Ddah1 gene deletion raises ADMA and reduces NO availability only within tubules and provides protection against fibrosis and functional decline in two different models of nephropathy. 2 2 Proteomic urinalysis data in Ddah1PT / mice suggests that reduced tubular NO activity may confer protection against progressive fibrosis in folate and UUO nephropathy through reduced expression of collagen and UMOD proteins. A stimulatory role of NO activity upon collagen deposition and wound-healing in skin and tendon tissue has been shown in in vitro and in vivo studies using exogenous NO donors,31 iNOS over- expression32 and even dietary L-arginine supplementation.33 Published literature confirms NO as a pleiotropic molecule in the kidney as well as other systems34 with both protective and deleterious effects exerted by NO upon collagen deposition and fibrosis in renal disease.35–39 What is clear is that Figure 8. Reduced renal collagen deposition in PT-specific Ddah1 deleted mice at 12 maladaptive, pro-fibrotic responses to in- weeks after folate administration. Kidney sections were stained with picrosirius red jury are, at least in part, driven by cross- fi 3 and viewed through circularly polarized light. Images (original magni cation, 25) talk between high NO activity and fibrotic were processed using ImageJ software. Images were stitched to produce a complete mediators such as TGFb and matrix metal- kidney section image, total section area was defined by texture thresholding, and collagen (yellow) was quantified as a percentage of total kidney section area. n=3 loproteases through SMAD signaling and fl fl 2 2 37 fi replicates (i–iii) shown for representation of n=9 (Ddah1 / )andn=12 (Ddah1PT / ). other pathways. Relevant to our own nd- ings, pharmacologic DDAH1 inhibition (causing reduced NO activity) has been Our findings suggest a reanalysis of previously published demonstrated to reduce collagen deposition in a TGF-b and human observational data showing that elevated plasma ADMA SMAD-dependent manner in a bleomycin model of pulmo- was associated with progression of renal disease.19,20,27 Interest- nary fibrosis.40 ingly, the largest study of patients with CKD to date found no An association between urinary UMOD and renal DDAH1 2 2 association between serum methylarginine levels and renal pro- expression was identified in both the Ddah1PT / mouse gression once baseline GFR was considered.19 Previously, using a and human cohort data. An 8.5-fold reduction of UMOD in 2 2 Mendelian randomization approach (with less susceptibility to Ddah1PT / suggests that functional changes in MAMEs and reverse causation and residual confounding), we reported a gene NO synthesis within the proximal tubule are communicated variant that conferred higher kidney tissue DDAH1 mRNA ex- downstream along the nephron to the thick ascending limb, pression and was associated with more rapid renal decline.21 where UMOD is exclusively expressed.41 A link between NO Previously published rodent studies of kidney fibrosis have activity and UMOD expression was confirmed in wild-type reported that DDAH1 and NO are protective while ADMA is mice exposed to a nonselective NOS inhibitor (L-NAME), pathogenic; however, significant study design issues limit their which resulted in reduced UMOD protein expression in kidney interpretation. ADMA or L-NAME infusion into mice exac- tissue and urine. Of note, NO exerts regulatory control over two erbated renal fibrosis, but both raised SBP by approximately transcription factors—nuclear factor of activated T cells42 and 60 mmHg, suggesting hypertensive injury28;globalDDAH1 Oct-143—both of which have consensus-binding sites in the pro- overexpression, however, protected against fibrosis in angio- moter region of UMOD.44 These observations raise the possibil- tensin and surgical nephron-reduction models of CKD.29,30 ity of direct regulation of UMOD expression by NO, a suggestion Genetic DDAH1 overexpression decreases circulating ADMA that certainly warrants further study. Genome-wide association and introduces unmeasured effects upon normal regulatory studies identify many UMOD polymorphisms that associate with

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Figure 9. Effects of PT-specific Ddah1 at 2 weeks after UUO. (A and B) Renal collagen deposition was significantly lower than in fl fl Ddah1 / controls (determined by nonbiased software analysis of picrosirius red– stained sections; 6.6% versus 10.4%; P,0.05). (C) 2 2 Renal parenchyma was less contracted in Ddah1PT / mice (assessed by percentage reduction of whole kidney section area [compared fl fl with unobstructed contralateral kidney in same mouse]; 35% versus 47% in Ddah1 / controls; P,0.05). (D) Quantitative RT-PCR 2 2 analysis revealed significantly lower renal tissue profibrotic cytokine expression in Ddah1PT / mice. Col1a2 was decreased .4-fold fl fl fl fl (0.07 versus 0.29 AU in Ddah1 / controls; P,0.05); TGFb was decreased 2-fold (0.19 versus 0.37 AU in Ddah1 / controls; P,0.05); fl fl and endothelin-1 was decreased approximately 40% (0.22 versus 0.35 AU; P=NS). Mann–Whitney U test, n=13 (Ddah1 / )andn=20 2 2 (Ddah1PT / ). *P,0.05. ns, not significant. Error bars, mean6SEM. raised UMOD expression, hypertension, CKD, and kidney func- with lower UMOD expression downstream and, consistent tion.45 A recent report confirmed that common UMOD gene with published data, may in part explain the protective effect variants increase UMOD expression and that (salt-sensitive) hy- observed against kidney disease progression. pertension and CKD progression may be induced through Thisstudyhasseverallimitations.Aminordegreeofextrarenal 2 2 NKCC2 cotransporter stimulation.46 In our study, reduced (liver and brain) Ddah1 gene deletion in the Ddah1PT / mouse Ddah1 metabolism of methylarginines (in the PT) correlates was suggested using sensitive PCR-based assays, although this did

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Table 1. Demographic characteristics of two human renal transplant cohorts CONCISE METHODS Characteristic London Cohort Virginia Cohort Women, n (%) 19 (51.35) 14 (38.9) Animal Studies Median age (yr) 47 (40–58) 55 (44.5–61) Animalexperimentsandhusbandrywereperformed Ethnicity, n (%) under United Kingdom Home Office License after South Asian 4 (10.81) 0 (0) approval by the local ethics committee. The KAPiCre Black 13 (35.14) 7 (19.4) transgenic mouse on a C57B6 background was Other 3 (8.11) 1 (2.8) purchased from The Jackson Laboratory (strain White 17 (45.95) 28 (77.8) name: B6.Cg-Tg(Kap-cre)29066/2Sig/J). The Live donor, n (%) 9 (24.32) 1 (2.8) ROSAYFP mouse (mixed background) was a kind – – Median donor age (yr) 53 (48 61) 47 (23 53) gift from Professor Graham Williams (Imperial Col- Delayed graft function, n (%) 21 (56.75) 14 (38.9) lege, London, UK). Ddah1 floxed mice on a mixed Rejection before protocol biopsy, n (%) 0 (0) 1 (2.8) background were previously developed and sup- Mean eGFR at biopsy6SD (ml/min per 1.73 m2) 52.9617.7 56.2619 fl Median UMOD (mg/mL) 8.1 (2.7–15.2) plied by our group. LoxP sites ank exon 1 of Median values are expressed with interquartile ranges in parentheses. Ddah1 (exon 1 contains the initiating methionine

Table 2. Estimated SDs for eGFR values Estimated SDs (95% CI ) Variable London Cohort Virginia Cohort GFR change over time (ml/min per 1.73 m2 per d) 0.032 (0.014 to 0.049) 20.01 (20.025 to 0.005) Increase in GFR at biopsy (ml/min per 1.73 m2 per unit MAME) 16.2 (27.3 to 43.4) 32.7 (13.4 to 52.0) Change in GFR over time (ml/min per 1.73 m2 per unit MAME per d) 20.065 (20.107 to 20.023) 20.050 (20.098 to 20.001) (1) Between patients at baseline (intercept): London, 16.2 ml/min per 1.73 m2 (95% confidence interval [95% CI], 12.8 to 20.6); Virginia, 14.9 ml/min per 1.73 m2 (95% CI, 11.4 to 19.5); (2) between patients over time (slope): London, 0.02 ml/min per 1.73 m2 per day (95% CI, 0.01 to 0.03); Virginia, 0.03 ml/min per 1.73 m2 per day (95% CI, 0.02 to 0.05); (3) within patients: London, 7.1 ml/min per 1.73 m2 (95% CI, 6.6 to 7.5); Virginia, 7.3 ml/min per 1.73 m2 (95% CI, 6.8 to 7.9). Correlation between baseline eGFR (intercept) and slope: London: 0.06 (95% CI, 20.49 to 0.57); Virginia: 0.17 (95% CI, 20.25 to 0.54). A decline of 0.05–0.065 ml/min per 1.73 m2 per day per unit MAME may appear trivial, but a 50% higher renal MAME expression would be reflected in an additional annual rate of renal function decline of 9– 12.0 ml/min per 1.73 m2 (MAME expression units across these cohorts were normalized to values between 0 and 1). not reach statistical significance in Ddah1 mRNAtranscription in codon and encodes the first 100 amino acids of DDAH1 [approximately 35% these tissues. This may represent a constitutive leak of Cre ex- of the total protein]). To achieve temporal activation of KAPiCre, female pression autonomous of androgen stimulation, or Cre activation mice were treated with a subcutaneous testosterone pellet (Innovative Re- in response to even very low levels of circulating testosterone in search of America, Sarasota, FL) for a minimum of 10 days. Animals were women. It is unlikely that such changes in neuronal or hepatic placed in metabolic cages for a maximum of 24 hours and urine collected was Ddah1 activity, in the absence of altered BP or systemic ADMA, centrifuged at 8000 g for 10 minutes, and the supernatant stored at 280°C. could influence renal fibrosis. Off-target Cre expression and ac- Invasive BP recording was performed under terminal (1%) tivity are widely under-reported.47 Cre toxicity causes cardiomy- isoflurane anesthesia. A Mikro-Tip pressure transducer catheter opathy,48 brain development defects,49 and widespread apoptosis (Millar Instruments, Houston, TX) was placed into the carotid artery. in embryos.50 To our knowledge, no studies have described Cre Recordings were obtained using the Powerlab system with analysis on toxicity relating to fibrosis, and any effects to date suggest that Chart software (both from ADInstruments, Oxfordshire, UK). Cre disrupts normal cellular function or architecture rather than PT2/2 preserve it, as seen in the Ddah1 mouse. Other limitations, Folate Nephropathy such as relatively small patient sample numbers, are counterbal- Mice received a single intraperitoneal injection of folate (240 mg/g) dis- anced by findings that are consistent across two very different solved in 0.3M NaHCO3 vehicle (20 mg/ml) or vehicle control alone (both experimental models of renal injury and human observational from Sigma-Aldrich, Dorset, UK). Mice were euthanized by CO2 narcosis data examining gene expression and clinical outcomes in two and cervical dislocation at day 2, day 14, or day 84 (12 weeks). Blood independent cohorts. collected by direct cardiac puncture was stored on ice before centrifugation Inconclusion,wedemonstratethatareductioninrenalADMA at 8000 g to remove plasma. The right kidney was halved (sagittal plane) metabolism protects against renal function decline in the context for histologic assessment and quartered for future RNA and protein ex- of renal injury. Whether therapeutic reduction of circulating traction, while the left kidney was weighed. Samples were stored at 280°C. ADMAwould improve cardiovascular outcomes at the same time as accelerating renal fibrosis requires further investigation. Our UUO Nephropathy findings suggest that, at least in the kidney, nonselective ADMA Under isoflurane inhalation anesthesia, micewereshaved, draped, and reduction could be detrimental. positioned on a heat mat. In the supine position, a left flank incision

3054 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 3045–3059, 2015 www.jasn.org BASIC RESEARCH

water or the same water containing dissolved (L-NAME; 1 mg/ml, Enzo Life Sciences). There were eight mice in each group. Water was

changed every 3 days and mice were euthanized by CO2 narcosis and cervical dislocation. Urine was collected for 24 hours before eutha- nasia; kidney tissue was harvested, snap frozen in liquid nitrogen, and stored at 280°C before further analysis.

Renal Tubule Isolation Mice were euthanized by CO2 narcosis and cervical dislocation. The abdominal aorta was ligated superior to the renal arteries, and magnetic beads (Dynabeads M-450 Tosylactivated, Invitrogen, Paisley, UK) sus- pended in ice-cold HBSS were infused via an aortic catheter placed distally. Kidney perfusion was confirmed by rapid blanching. Kidneys Figure 10. Relationship between kidney tissue MAME expression were then dissected out, decapsulated, and minced with a scalpel before and eGFR decline in renal transplant recipients. Estimates of incubation in collagenase (1 mg/ml; Sigma-Aldrich) at 37°C for 30 (change in) eGFR over time in London cohort stratified by MAME minutes with agitation. The resulting homogenate was successively expression (above or below median) derived from the multilevel sieved through 100-micron and 50-micron pore sizes, and fragments model. Dotted line, MAME expression below median; solid line, caught by the latter were resuspended in cold HBSS. Magnetic separation MAME expression above median. of glomerular and tubular fragments was confirmed by microscopic examination (bead entrapment visible within glomerular capillary loops). Purified tubules were pelleted by centrifugation at 1000 g and exposed the left ureter and was ligated twice with 4–0 nylon sutures stored at 280°C for future use or subjected to further digestion (Accu- and transected. Following suture closure, animals received intramus- max; protease/ DNAse solution; Innovative Cell Technologies Inc., San cular diclofenac analgesia (Vetagesic) and recovered in heated cham- Diego, CA) before ImageStream analysis (Amnis Corp., Seattle, WA). bers. Mice were euthanized by cervical dislocation, followed by organ Cells were gated according to spherical shape and yellow fluorescence harvesting at three time points: 1, 2, and 3 weeks. before automated single-cell image capture.

L-NAME Experiment Biochemical Methods Female littermates wild-type C57BL/6 mice aged approximately 10 The Agilent 6400 Series Triple Quadrupole liquid chromatography– weeks were randomly allocated to receive plain deionized drinking tandem mass spectrometry (LC-MS/MS) system was used for this

Figure 11. Relationship between DDAH1 gene expression, NO activity, and UMOD protein expression. (A) Renal biopsy tissue DDAH1 mRNA expression was positively correlated with urinary UMOD in live donor allografts (P=0.04). When extended to the whole cohort, the association was rendered statistically insignificant, which likely reflects the heterogeneity of organs from deceased donors. (B–D) Non- selective NOS inhibition with L-NAME dissolved in drinking water (1 mg/ml for 2 weeks) in wild-type C57BL/6 mice. (B) L-NAME–treated mice had significantly reduced kidney tissue NOx as determined by chemiluminescence (P,0.01) and significantly lower UMOD protein expression in urine as determined by ELISA (C) (P,0.01) and kidney tissue as determined by Western blot (D and E) (P,0.01) (n=3, representative of eight in each group). Mann–Whitney U test. **P,0.01. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Error bars, mean6SEM.

J Am Soc Nephrol 26: 3045–3059, 2015 Methylarginines in Kidney Disease 3055 BASIC RESEARCH www.jasn.org study. Biologic compounds optimized for detection included methyl- cutting temperature compound and snap frozen onto cork discs in arginines (Calbiochem, Nottingham, UK), amino acids, and creatinine methanol cooled on dry ice and stored at 280°C. Sections (5 mM) (the latter two from Sigma-Aldrich). Biologic samples, including were cut at 220°C (cryotome CM 1850, Leica Microsystems, Wetzlar, plasma, urine, and tissue lysates, were prepared by methanol protein Germany) onto poly-L-lysine–coated glass slides. Slides were studied precipitation in a 1:5 dilution; the samples were vortexed and the pellet using a Leica SP5 microscope. removed by centrifugation. Sample solutions were evaporated to dry in a heat-block and resuspended in mobile phase (0.1% formic acid). An Paraffin Sections internal standard was added (7-deuterated ADMA; Cambridge Isotope Harvested organs were fixed in 4% paraformaldehyde overnight and Laboratories, Tewksbury, MA) before precipitation to allow correction then stored in 70% ethanol. Tissues were embedded in paraffin blocks fi for extraction ef ciency and ion suppression at the point of detection by an automated processor; 5-mM sections were cut on a microtome (ions in large abundance can outcompete minority ions at the detector). and attached to poly-L-lysine–coated glass slides before staining with fi A hypercarb (Thermo Fisher Scienti c) chromatography column was periodic–acid Schiff or picrosirius red. used, and the mobile phase consisted of 0.1% formic acid, 1% acetonitrile (increasing to 50% between 5 and 10 minutes), over a total run time of 15 minutes. Data were collected using Agilent (Berkshire, UK) Mass- Collagen Content Kidney sections were assessed for collagen deposition using a Manual Hunter data acquisition software and analyzed with MassHunter qual- Zeiss Axiophot microscope (Zeiss, Thornwood, NY) through circularly itative analysis software before downloading of raw data into Excel polarized light. Tissue images were obtained with a 2.53 objective lens, (Microsoft Corp., Redmond, WA) for processing. The mass spectrum recorded on a digital camera (Canon EOS-40D digital SLR; Canon Inc,, variables were as follows: ADMA, mass-to-charge ratio (m/z):203.3– Tokyo, Japan) and viewed through Canon Utilities EOS software 46.0; collision energy (CE): 12; symmetrical dimethylarginine, m/z: (Canon Inc.). Images were analyzed using a Fiji software macro (written 203.3–70.2, CE: 24; arginine, m/z: 175.2–60.1,CE:8;citrulline, by Dr. Dirk Dorman, head of microscopy, MRC Clinical Sciences Cen- m/z: 176.2–86.2, CE: 16; glutamine, m/z: 147.0–56.3, CE: 0; d7- tre, Imperial College, London, UK) to quantify collagen in each whole ADMA, m/z: 210.0–46.0, CE: 24; creatinine m/z:114.0–44.0, CE: 12. kidney slice, thus obviating bias. Percentage collagen deposition per total Standard curves were generated for each substance of interest. section area (pixels) could then be reported.

Molecular Biologic Methods Routine biochemical variables in patient samples were measured by Statistical Analyses National Health Service hospital laboratories. NOx (NO2 and NO3) was Statistical analyses were performed using the Prism software package 6 measured by chemiluminescence with a Sievers Nitric Oxide Analyzer (GraphPad Inc.). All data are presented as mean SEM. For compar- – (GE Analytical Instruments, Manchester, UK) as per protocol. Protein isons between two groups for a single variable, a Mann Whitney U concentrations were determined by Bradford assay and Nanodrop. test (nonparametric data or where a normal distribution could not be TissuegenotypingwasperformedusingReadyMixTaqPCRReaction assumed) was used. Differences between multiple groups were as- Mix (Sigma-Aldrich). Tissue mRNA expression was determined by sessed by one-way ANOVA,followed by Bonferroni post-test for mul- iScript cDNA reverse transcription kit, followed by quantitative RT-PCR tiple comparisons. Two-way ANOVA was used where comparisons (iTaqFastSYBRGreenSupermixwithROX(BioRad,Hertfordshire,UK) were being made between groups subject to two experimental vari- using a 7900HT Fast System, Applied Biosystems (Life Technologies, ables, followed by Bonferroni post-test for multiple comparisons. A , fi Paisley, UK). All reactions were performed in duplicate, and several P value 0.05 was considered to represent a statistically signi cant “housekeeper” sequences were optimized for different types of biologic difference. sample, with Pol2a the most stably expressed gene. Primer sequences are listed in the complete methods. Proteomic Analysis (LC-MS/MS) Proteins were separated and identified using Western blotting and Urine samples were retrieved from 280°C and thawed in an Eppendorf SDS-PAGE according to standard protocols. A polyclonal goat DDAH1 Thermomixer Comfort mixer at 22°C, 900 g for 10 minutes, and then antibody was produced by our own group. UMOD antibody was centrifuged at 5°C, 13,000 g for 3 minutes. Two hundred microliters of purchased from R&D Systems (Minneapolis, MN). Glyceraldehyde sample was mixed 1:5 with 0.1% trifluoroacetic (TFA) acid and sub- 3-phosphate dehydrogenase antibody was purchased from Sigma- jected to two different solid phase extraction (SPE) steps; reverse-phase Aldrich, and the LI-COR Odyssey system (Cambridge, UK) was used SPE (Waters Oasis HLB cartridges, Waters Corp., Milford, MA) and for protein detection and quantification. Mouse urine UMOD protein cation exchange SPE (Waters Oasis MCX cartridges). SPE was per- was quantified by ELISA according to manufacturers’ protocol (Uscn formed using a manual vacuum manifold (24-port vacuum manifold; Life Science Inc., Hubei, China). Thermo Scientific) and LC-MS grade solvents (Fisher Scientific). Dried samples were solubilized in 40 ml of 0.1% TFA, sonicated for 10 min- Histologic Methods utes, and then centrifuged at 5°C, 13,000 g for 10 minutes. Ten micro- Frozen Sections liters of supernatant was transferred to a Bioquote Ltd. 0.2-ml lubricated Kidneys from the ROSAYFP reporter mouse were fixed overnight in hydrophobic PCR tube and placed into an autosampler vial ready for 4% paraformaldehyde, then 30% sucrose in PBS until full tissue LC-MS injection and analysis. A Dionex (Thermo Scientific) Ultimate submersion (about 4 hours). Specimens were embedded in optimal 3000 nano-liquid chromatography system was used to separate peptides

3056 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 3045–3059, 2015 www.jasn.org BASIC RESEARCH before mass spectrometric analysis. A volume of 4 ml was loaded onto a (Applied Biosystems, Life Technologies). Quantitative RT-PCR re- trap column (Acclaim Pepmap 100; Thermo Scientiﬁc) at 8 ml/min in sults from the genes of interest were normalized to the three most

98% water, 2% acetonitrile, 0.1% TFA. Peptides were then eluted online stable of six control genes: ACTb, b-2 microglobulin (b2M), hypo- to an analytical column (Acclaim Pepmap RSLC; Thermo Scientific) xanthine phosphoribosyltransferase 1 (HPRT), ribosomal protein and separated using a ramped gradient with conditions: initial 5 minutes 13A (RPL13A), succinate dehydrogenase complex subunit A flavo- with 4% solvent B (96% solvent A), then 90 minute gradient 4%–55% protein (SDHA), and TATA box binding protein (TBP). In the Vir- solvent B, then 10 minutes isocratic at 100% solvent B, then 5 minutes ginia cohort, a Taqman primer approach was used. Expression of isocratic at 4% solvent B (solvent A: 98% water, 2% acetonitrile, 0.1% DDAH1 and AGXT2 was performed and corrected to a single refer- formic acid; solvent B: 20% water, 80% acetonitrile, 0.1% formic acid). ence gene (b2M). In both cohorts, genes of interest were normalized Eluted peptides were analyzed using LTQ XL Orbitrap (Thermo Fisher to vary between zero and one. Ten samples were quantified at both Scientific) operating in positive polarity, top four collision-induced dis- sites, demonstrating high levels of intercenter agreement. sociation (CID) method. Ions for dissociation were determined from Uromodulin in the human cohort was measured using ELISA (MD initial 15,000 resolution MS scan (event 1) followed by CID on the top Bioproducts, Zurich, Switzerland) as per manufacturer’s instructions. four most abundant ions. CID conditions were as follows: default charge state, 2; 2.0 m/z isolation width; normalized collision energy, 35.0; Ac- Statistical Analyses tivation Q value, 0.25; activation time, 30 ms; lock mass value, A multilevel multivariable approach was used to model eGFR over 445.120030 m/z. time in each of the two cohorts. Follow-up was commenced at the time of protocol biopsy, and GFR measurements (according to Modifica- Proteomic Data Analysis tion of Diet in Renal Disease) out to 1 year after examination of biopsy Raw files were searched against UniprotKB/Swiss-Prot database specimens. The eGFR values were nested within patients using both a (version 10032012) restricted to mouse entries using Mascot version random intercept and random slope and the slope of eGFR quantified 2.3.01. Relative quantification of peptides was performed by extracted by the inclusion time variable. The effect of gene expression on eGFR ion chromatograms of parent ions using software developed in-house. decline was quantified by the addition of an expression3time inter- action. Residuals were examined to ensure there was no substantial Human Cohort Data Methods deviation from normality. Participants An appropriate research ethics committee, National Health Service management, and institutional review board approvals were in place ACKNOWLEDGMENTS for bothcohortstudies. Studieswereconductedin accordance withthe Declaration of Helsinki principles, and all participants provided Thiswork wasfundedby theMedicalResearch Council(J.L.), Imperial written informed consent. College London Biomedical Research Centre (J.T.), Chain-Florey The London Protocol Biopsy Study consisted of patients undergoing Clinical Research Fellowship, Medical Research Council (J.T.), and the protocol biopsy following first-kidney-alone transplant as per routine British Heart Foundation (J.L.). British Heart Foundation Pro- clinicalcare.ParticipantsrecruitedfromtheRoyalFreeHospitalNational gramme Grant ref. RG002/05. Work in C.S.W.’s laboratory is sup- Health Service Trust transplant clinic. The Virginia cohort underwent ported by grants from the NIH (DK-49870 and HL-68686). similar protocol biopsies except that two patients with kidney-after-liver Some of the data presented here have previously been presented in transplantwerealsoincluded.Follow-upbiochemistrywasperformedin abstract form for poster presentation (American Society of Nephrology local laboratories as per routine clinical care; eGFR results were obtained Kidney Week 2013). with a minimum interval of 1 week between data points in the London study and monthly in the Virginia study. DISCLOSURES Biopsy Tissue Quantitative RT-PCR None. Protocol biopsy samples were preserved in RNA Later (Qiagen) at the bedside, before being stored at 270°C. Tissue was lysed in a 1% b-mercaptoethanol–containing proprietary lysis buffer (RLT, Qiagen). REFERENCES RNAwas extracted using a column system (RNeasy kit, Qiagen) according to the manufacturer’s instructions. No carrier RNA was used. 1. 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