Renal and Vascular Effects of Uric Acid Lowering in Normouricemic Patients with Uncomplicated Type 1 Diabetes

Diabetes Volume 66, July 2017 1939

Renal and Vascular Effects of Uric Acid Lowering in Normouricemic Patients With Uncomplicated Type 1 Diabetes

Yuliya Lytvyn,1,2 Ronnie Har,1 Amy Locke,1 Vesta Lai,1 Derek Fong,1 Andrew Advani,3 Bruce A. Perkins,4 and David Z.I. Cherney1

Diabetes 2017;66:1939–1949 | https://doi.org/10.2337/db17-0168

Higher plasma uric acid (PUA) levels are associated with at the efferent arteriole. Ongoing outcome trials will de- lower glomerular ﬁltration rate (GFR) and higher blood termine cardiorenal outcomes of PUA lowering in patients pressure (BP) in patients with type 1 diabetes (T1D). Our with T1D. aim was to determine the impact of PUA lowering on renal and vascular function in patients with uncomplicated T1D.

T1D patients (n = 49) were studied under euglycemic and Plasma uric acid (PUA) levels are associated with the PATHOPHYSIOLOGY hyperglycemic conditions at baseline and after PUA low- pathogenesis of diabetic complications, including cardiovas- ering with febuxostat (FBX) for 8 weeks. Healthy control cular disease and kidney injury (1). Interestingly, extracellu- subjects were studied under normoglycemic conditions lar PUA levels are lower in young adults and adolescents (n = 24). PUA, GFR (inulin), effective renal plasma flow with type 1 diabetes (T1D) compared with healthy control (para-aminohippurate), BP, and hemodynamic responses subjects (HCs) (2–4), likely due to a stimulatory effect of to an infusion of angiotensin II (assessment of intrarenal urinary glucose on the proximal tubular GLUT9 transporter, renin-angiotensin-aldosterone system [RAAS]) were mea- which induces uricosuria (2). Thus, PUA-mediated target sured before and after FBX treatment. Arterial stiffness, organinjurymaybemediatedbytheintracellularuric fl ow-mediated dilation (FMD), nitroglycerin-mediated dila- acid effects, uricosuria-related tubular cell exposure (5), tion (GMD), urinary nitric oxide (NO), and inflammatory sequestration of PUA along the vascular endothelium, or markers were measured before and after FBX treatment. PUA-mediated inflammation and activation of the renin- Gomez equations were used to estimate arteriolar affer- angiotensin (ANG)-aldosterone system (RAAS) (1). ent resistance, efferent resistance (R ), and glomerular E Consequently, even within the normal range, PUA is hydrostatic pressure (PGLO). FBX had a modest systolic BP–lowering effect in T1D patients (112 6 10 to 109 6 associated with impaired renal function (6,7), early glo- fi 9 mmHg, P = 0.049) without impacting arterial stiffness, merular ltration rate (GFR) loss (8), and an increased risk FMD, GMD, or NO. FBX enhanced the filtration fraction of the development of proteinuria in patients with T1D (9). response to hyperglycemia in T1D patients through larger Even in young adults and adolescents with T1D without increases in RE, PGLO, and interleukin-18 but without complications, higher PUA levels are associated with lower impacting the RAAS. FBX lowered systolic BP and mod- GFR (2–4), which may be driven by a PUA-mediated ulated the renal RE responses to hyperglycemia but with- increase in afferent renal arteriole resistance potentially out impacting the RAAS or NO levels, suggesting that PUA promoting ischemia to the renal microcirculation (4). Ad- may augment other hemodynamic or inflammatory mech- ditionally, accumulating evidence suggests that PUA levels anisms that control the renal response to hyperglycemia are independently associated with increased intimal medial

1Department of Medicine, Division of Nephrology, Toronto General Hospital, Uni- Received 7 February 2017 and accepted 5 April 2017. versity of Toronto, Toronto, Ontario, Canada Clinical trial reg. no. NCT02344602, clinicaltrials.gov. 2Department of Pharmacology and Toxicology, University of Toronto, Toronto, B.A.P. and D.Z.I.C. are co–senior authors. Ontario, Canada 3Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge © 2017 by the American Diabetes Association. Readers may use this article as Institute of St. Michael’s Hospital, Toronto, Ontario, Canada long as the work is properly cited, the use is educational and not for profit, and the 4Department of Medicine, Division of Endocrinology and Metabolism, Mount Sinai work is not altered. More information is available at http://www.diabetesjournals Hospital, University of Toronto, Toronto, Ontario, Canada .org/content/license. Corresponding author: David Z.I. Cherney, [email protected]. 1940 Cardiorenal Effects of Uric Acid Lowering in T1D Diabetes Volume 66, July 2017 thickness, endothelial dysfunction, and vascular stiffness (10), promoting the development of hypertension, cardiovascular disease, and chronic kidney disease (6,11). Although not observed in adolescents with T1D, the association between higher PUA levels within the normal range and higher blood pressure (BP) has been reported in young adults with uncomplicated T1D (2). Such relationships between PUA and early renal and cardiovascular risk factors in young patients with T1D suggest that lowering PUA may be an important strategy to reduce renal and cardiovascular complications related to diabetes. From the limited data available in patients with diabetes, lowering of PUA levels may improve endothelial function (12), lower BP (13–15), slow GFR decline, reduce proteinuria, and suppress renal inflammation (1,16–18). Given the lack of effective therapy that protects against initiation and progression of diabetic complications, it is of the utmost importance to evaluate the renal and vascular effects of pharmacological PUA lowering in patients with T1D. Ac- cordingly, the goals of our physiological study were to determine whether PUA lowering with the xanthine oxidase Figure 1—Flow diagram for study participants. inhibitor febuxostat (FBX) modifies 1) the effect of hyperglycemia and infused ANG II on renal hemodynamic function, 2) systemic BP, and 3) arterial stiffness and endothelial function during clamped euglycemia and hyperglycemia in normoalbuminuria on a 24-h urine collection; 3) BMI of an even earlier cohort of young T1D adults without any 18–35 kg/m2; 4) normal renal and liver function; 5)normal complications. electrocardiogram; 6)clinicBP,140/90 mmHg; 7)T1D Using a study design with both euglycemic and hyper- duration .5 years; 8) normal PUA levels ,450 mmol/L glycemic clamp conditions, we wanted to assess the effects based on Clinical Reference Laboratory Guidelines at the of uricosuria-related tubular exposure that is augmented by time of study initialization; and 9) no history of renal or hyperglycemia and the consequent glycosuria. As we have cardiovascular complications and no intake of concomitant previously shown, glycosuria stimulated by hyperglycemia medications that would alter BP or cardiovascular outcomes leads to uricosuria (2). It was therefore important to com- or interfere with purine metabolism. The study was ap- pare PUA-lowering effects during euglycemic conditions proved by the University Health Network Research Ethics with those during hyperglycemic conditions. In addition, Board (Toronto, ON, Canada), and all subjects gave written unlike other PUA-lowering agents, FBX lowers PUA levels informed consent. sequestered along the vascular endothelium (1), allowing us to potentially target PUA-mediated injury mechanisms that Experimental Design are due to either paracellular or even intracellular uric acid Patients with T1D were studied at baseline (day 1, levels. Finally, because of interactions among PUA, the euglycemic; day 2, hyperglycemic) and after 8 weeks of RAAS, and inflammation, we measured plasma aldoste- FBX therapy 80-mg daily (day 3, euglycemic; day 4, rone and renin levels as well as urinary inflammatory hyperglycemic). Medication compliance was assessed by marker levels and hemodynamic responses to an exoge- pill counting and was .90% in all participants. During nous ANG II infusion to better elucidate PUA lowering as clamped euglycemic study days, the blood glucose level was a modulator of these traditional renal and cardiovascular maintained between 4 and 6 mmol/L, and during clamped injury pathways. hyperglycemic study days the blood glucose level was maintained between 9 and 11 mmol/L. Studies were RESEARCH DESIGN AND METHODS performed after 7 days on a controlled diet consisting Subject Inclusion Criteria and Study Preparation of $150 mmol/day sodium and #1.5 g/kg/day protein. Twenty-four HCs and 49 patients with T1D completed this The sodium-replete diet was used to avoid circulating vol- open-label, proof-of-principle, 8-week FBX treatment study ume contraction, RAAS activation, and between-subject het- (Fig. 1) (ClinicalTrials.gov identifier NCT02344602). T1D erogeneity and in an attempt to keep study conditions study participants included 42 patients with T1D with nor- similar to typical North American dietary patterns. Prestudy mofiltration (T1D-N; GFR ,135 mL/min/1.73 m2)and7 protein intake was modest to avoid the hyperfiltration ef- patients with T1D with hyperfiltration (T1D-H; GFR $135 fect of high-protein diets (19). Compliance was ascertained mL/min/1.73 m2). Detailed inclusion criteria were as fol- by the measurement of 24-h urine sodium and urea excre- lows: 1) male and female participants 18–40 years old; 2) tion on the seventh day prior to the studies. All study diabetes.diabetesjournals.org Lytvyn and Associates 1941

participants were instructed to avoid caffeine-containing The DPF was calculated as follows: products and to have the same light breakfast on the morn- ¼ = ing of each study visit. HCs were studied during normogly- DPF GFR KFG cemic conditions only. The glomerular oncotic pressure from the plasma protein Assessment of Renal Hemodynamic Function mean concentration (CM)withinthecapillaries: Subjects presented to the Renal Physiology Laboratory on ¼ = 3 ð1=1 2 Þ days 1 and 2 for the baseline euglycemic and hyperglycemic CM TP FF ln FF studies. After the respective clamped glycemic level was achieved for 5 h, blood samples were collected for mea- pG ¼ 5 x ðCM 2 2Þ suring levels of inulin and p-aminohippuric acid (PAH) and for baseline circulating levels of RAAS mediators PGLO: (plasma renin concentration and aldosterone). Oscillometric ¼ þ þ brachial artery BP measurements were obtained in dupli- PGLO DPF PBow pG cates in a reclining position at 30-min intervals throughout the study (Critikon, Tampa, FL). Subjects remained supine at RA and RE were estimated using the principles of Ohm’s all times. Baseline renal hemodynamic function (GFR and law, where 1,328 is the conversion factor to dyne $ s $ 2 effective renal plasma ﬂow [ERPF]) was measured using in- cm 5 (22): ulin and PAH clearance according to the plasma disappear- ¼½ð - Þ= 3 1;328 ance technique (20,21). The mean of the ﬁnal 2 clearance RA MAP PGLO RBF periods represented baseline GFR and ERPF, expressed per 2 1.73 m . RE ¼ ½GFR=ðKFG3ðRBF 2 GFRÞ The following parameters were calculated as follows:

GFR Assessment of ANG II Infusion Response ð Þ¼ Filtration fraction FF ERPF On euglycemic day 1 only, after baseline clearance periods were complete, ANG II (Clinalfa; Bachem AG, Bubendorf, ERPF Switzerland) was administered at incremental doses of 1 ð Þ¼ Renal blood flow RBF 1 2 Hematocrit and 3 ng/kg/min, each over 30 min, followed by a 30-min recovery phase (26). Oscillometric brachial artery BP mea- MAP surements were obtained in duplicate in a reclining position ð Þ¼ Renal vascular resistance RVR RBF every 5 min during each ANG II infusion (Critikon). Blood was collected during each ANG II infusion period and after a 30-min recovery for hematocrit, inulin, and PAH to assess where MAP is mean arterial pressure. Indirect intraglomer- renal hemodynamic parameters. ular hemodynamic parameters were estimated using equations of Gomez (22) based on data from animal studies. These equations were successfully used in a similar manner Urinary Nitric Oxide and Inflammatory Marker to evaluate patients with conditions such as hypertension, Assessments endocrine disorders, and T1D (4,23,24). The following Assessments of nitrite and nitrate levels, metabolites of assumptions were imposed by the Gomez equations: 1) nitric oxide (NO), were performed on midstream urine intrarenal vascular resistances are divided into afferent, samples collected on each of the 4 study days after postglomerular, and efferent; 2) hydrostatic pressures respective euglycemic and hyperglycemic clamps were within the renal tubules, venules, and Bowman space and stabilized for 3 h. Nitrite assessments were performed interstitium (PBOW) are in an equilibrium of 10 mmHg; 3) based on the ELISA kit instructions provided by the the glomerulus is in filtration disequilibrium; and 4)the manufacturer (R&D Systems, Minneapolis, MN). Similarly, gross filtration coefficient (Kfg) is 0.1733 mL/s/mmHg (glo- urine specimens were used to measure levels of cytokines/ merular hydrostatic pressure [PGLO] = 47.5 mmHg) for HCs chemokines using an established Cytokine/Chemokine and Kfg = 0.1012 mL/s/mmHg (PGLO = 56.4 mmHg) for Panel Luminex Assay (Eve Technologies, Calgary, Alberta, fl patients with T1D to re ect the different PGLO values in Canada). The following urinary cytokine/chemokines were diabetic and control conditions observed in previous micro- measured: epidermal growth factor, fibroblast growth puncture studies in Munich-Wistar rats (25). MAP (in factor-2, Eotaxin 1, transforming growth factor-a,gran- mmHg), ERPF (in mL/s), GFR (in mL/s), and total protein ulocyte colony-stimulating factor, Flt-3 ligand, granulocyte- (in g/dL) measurements were used to calculate efferent re- monocyte colony-stimulating factor, Fractalkine, interferon 25 sistance (RE;indyne$ s $ cm ), afferent resistance (RA;in (IFN)-a2, IFN-g,growth-relatedoncogene-a, interleukin 25 dyne $ s $ cm ), PGLO (in mmHg), change in filtration pres- (IL)-10, MCP-3, IL-12p40, macrophage-derived chemokine, sure across glomerular capillaries (DPF;inmmHg),andglo- IL-12p70, platelet-derived growth factor-AA, IL-13, platelet- merular oncotic pressure (in mmHg). derived growth factor-BB, IL-15, soluble CD40L, IL-17A, 1942 Cardiorenal Effects of Uric Acid Lowering in T1D Diabetes Volume 66, July 2017

IL-1RA, IL-1a,IL-9,IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, to RAAS modulation is ;19 mL/min/1.73 m2 (26,29). To IL-8, IFN-inducible protein-10, MCP-1, macrophage inflam- detect a 10 mL/min/1.73 m2 between-group difference in matory protein-1a,macrophageinflammatory protein-1b, the GFR response to PUA lowering, for a two-sided test regulated on activation normal T-cell expressed and se- with P = 0.01 (to correct for multiple comparisons) and creted, tumor necrosis factor-a, tumor necrosis factor-b, with Za = 2.58, the sample size equals 24 in each group vascular endothelial growth factor A, and IL-18. All markers (T1D-H and T1D-N) for a total of 48 patients with T1D in urine were corrected for urinary creatinine concentration. (29). Because only seven patients with T1D exhibited hyperfiltration in our cohort, the analysis based on hyperfiltra- Vascular Assessments tion status was only exploratory. Vascular assessments were performed on each of the 4 study The difference between renal hemodynamic parameters days after ambient glycemia had been stabilized and before at euglycemic clamp and hyperglycemic clamp was used to renal hemodynamic function testing. In brief, arterial analyzethehyperglycemicresponsebeforeandafterFBX compliance was measured noninvasively using a SphygmoCor treatment. The difference between renal hemodynamic device (AtCor Medical Systems Inc., Sydney, New South parameters at baseline euglycemic clamp and 30 min after the Wales, Australia). Right carotid artery waveforms were 1 and 3 ng/kg/min ANG II infusions were used to analyze the recorded with a high-fidelity micromanometer (SPC-301; ANG II response before and after FBX treatment. Millar Instruments) and using the validated transfer func- One T1D-H patient was excluded from the analysis tion, and corresponding central aortic pressure waveform examining the effect of FBX on renal hemodynamic func- data were generated. MAP and heart rate (HR) were deter- tion during the euglycemic clamp only due to issues with mined using the integral software. The augmentation index the blood sample collected for the inulin and PAH mea- (AIX), an estimate of arterial stiffness, was calculated as surements. For similar reasons, one patient with T1D-N was the difference between the second systolic peak and the excluded from the analysis of ANG II infusion responses inflection point, which was expressed as a percentage of before and after FBX treatment. One patient with T1D-N the central pulse pressure corrected to a HR of 75 beats per was excluded from the analysis of radial AIX during hyper- minute (bpm). The aortic pulse wave velocity (PWV) was glycemia because of an inability to obtain measurements measured using the same device. before and after FBX treatment. Endothelial function of the brachial artery was deter- Data are presented as the mean 6 SD. Within-group mined by the diameter change in response to an increased differences and responses to FBX treatment were analyzed blood flow generated by reactive hyperemia (flow-mediated using paired t tests. To assess for between-group differ- dilation [FMD]). Reactive hyperemia was stimulated by a ences, ANOVA with a post hoc Tukey test was used. Linear 5-min inflation of a pneumatic cuff placed distal to the regression analysis was used to determine correlations be- antecubital fossa followed by deflation. Endothelial- tween renal hemodynamic responses and PUA levels. Sta- independent function of the brachial artery was mea- tistical significance was defined as P , 0.05. All statistical sured by the diameter change in response to a sublingual analyses were performed using SAS version 9.1.3 and nitroglycerin spray (400 mg; nitroglycerin-mediated dila- GraphPad Prism software (version 5.0). tion [GMD]). A high-resolution B-mode ultrasound device (Vivid i [7- to 15-MHz linear-array transducer]; GE/ RESULTS Vingmed, Waukesha, WI) was used to capture longitudinal Baseline Characteristics electrocardiogram-gated end-diastolic images of the brachial The study population comprised 49 patients with T1D fl artery before and after cuff in ation. The diameter was (42 patients with T1D-N, 7 patients with T1D-H) and determined using an automated edge-detection algorithm 28 HC participants (Table 1). All patients had a mean T1D fl that has been described previously (27), and blood ow was duration of 14.3 6 7.2 years. Overall, baseline characteris- measured from the velocity-time integral of the Doppler tics were similar between the T1D and HC groups, whereas signal. FMD was defined as the maximal percentage change 24-h protein intake tended to be lower and hemoglobin A1c in vessel diameter after reactive hyperemia and was also level was higher in the T1D group. reported as FMD/flow, which was defined as the maximal percentage change in vessel diameter divided by the per- Effect of FBX on PUA Levels centage change in flow to create a stimulus-adjusted re- As expected, patients with T1D at baseline under euglyce- sponse measure (28). GMD was defined as the maximal mic conditions had lower PUA levels compared with HCs percentage change in vessel diameter within 5 min after (240 6 62 vs. 303 6 71 mmol/L, respectively; P = 0.0002), the sublingual nitroglycerin spray was administered. and PUA levels were further lowered in patients with T1D during hyperglycemic conditions (240 6 62 vs. 221 6 61 Statistical Analyses mmol/L; P , 0.0001). FBX treatment significantly de- The primary end point of this study was the change from creased PUA levels by ;50% in each group, as follows: baseline in GFR after 8 weeks of FBX treatment under HC group, 303 6 71 to 131 6 55 mmol/L (P , 0.0001); stable euglycemic and hyperglycemic conditions. Our pre- T1D group during euglycemic conditions, 240 6 62 to vious data have shown that the SD of the DGFR in response 124 6 53 mmol/L (P , 0.0001); and T1D group during diabetes.diabetesjournals.org Lytvyn and Associates 1943

— Further analysis revealed normalization of the GFR in Table 1 Baseline demographic characteristics of HCs and 6 6 patients with T1D the six patients with T1D-H (150 13 to 129 2 P Patients with 10 mL/min/1.73 m ; = 0.0113) (Fig. 2) but not in patients 2 Parameter HCs (n =24) T1D (n = 49) with T1D-N (111 6 14 to 111 6 16 mL/min/1.73 m ; P = Males 12 (50%) 25 (51%) 0.9227). Age (years) 25.5 6 4.5 26.3 6 5.4 Effect of FBX on Renal Function, BP, and Vascular Diabetes duration Parameters During Hyperglycemic Conditions (years) 14.3 6 7.2 In patients with T1D during hyperglycemic conditions, FBX 2 6 6 BMI (kg/m )23.63.4 25.1 3.4 treatment did not affect renal hemodynamic function, Cholesterol 4.2 6 0.8 4.6 6 0.8 intraglomerular hemodynamics, BP, vascular parameters, HDL cholesterol 1.5 6 0.5 1.5 6 0.4 or measures of endothelial-dependent and endothelial- LDL cholesterol 2.4 6 0.7 2.6 6 0.7 independent vascular function (Table 3) (Fig. 3). Triglycerides 1.0 6 0.7 1.1 6 0.7 PUA Correlations With Renal Hemodynamic Parameters Hemoglobin A1c, 31.7 6 2.4 62.3 6 14.8 mmol/mol (%) (5.0 6 0.2) (7.8 6 1.3)* As observed in our previous studies in separate cohorts (2–4), a higher PUA level was correlated with a lower GFR 24-h urine sodium r 2 P (mmol/day) 155 6 65 150 6 76 (during euglycemia, = 0.37, = 0.009; during hyperglycemia, r = 20.46, P = 0.0009) and lower ERPF (during 24-h protein intake r 2 P (g/kg/day)† 1.1 6 0.3 1.0 6 0.3* euglycemia, = 0.29, = 0.047; during hyperglycemia, r 2 P Estradiol (females only) 226 6 169 217 6 250 = 0.39, = 0.006 during hyperglycemia). After FBX treatment, a higher PUA level was still correlated with a Progesterone (females only) 3.3 6 4.2 3.1 6 7.5 lower GFR during euglycemia (r = 20.30, P = 0.038) but Values are reported as the mean 6 SD, unless otherwise indicated. not during hyperglycemia (r = 20.18, P = 0.22), and asso- n, number of participants. *P , 0.05 vs. HCs. †24-h protein fi intake was estimated as follows: ([urine urea 3 0.18] + 14)/weight ciations with ERPF were not signi cant (during euglycemia, (in kg). r = 20.087, P = 0.56; during hyperglycemia, r = 20.15, P = 0.29). No correlations were observed between PUA and GFR or ERPF in HCs before or after FBX treatment. hyperglycemic conditions, 221 6 61 to 108 6 42 mmol/L P , ( 0.0001) (Table 2). Effect of FBX on Renal Hemodynamic Responses to Clamped Hyperglycemia Effect of FBX on Renal Function, BP, and Vascular FBX treatment led to an augmented FF response to hyper- Parameters During Euglycemic Conditions glycemia (0.01 6 0.04 to 0.03 6 0.04, P = 0.0296), which In the HC group, FBX treatment did not influence renal was accompanied by an exaggerated increase in RE (195 6 hemodynamic function (ERPF, GFR, FF, RBF, and RVR), 25 384 to 400 6 525 dyne $ s $ cm , P =0.0271)andPGLO intraglomerular hemodynamics (PGLO,RA,RE,andRA/RE (0.5 6 3.6 to 2.7 6 3.7 mmHg, P = 0.0053) but not in RA, ratio), BP (systolic BP [SBP], diastolic BP [DBP], and HR), GFR, or ERPF (Fig. 3). No significant differences were ob- or vascular parameters (aortic AIX, carotid AIX, carotid fem- served in BP or vascular parameters responses to hypergly- oral and carotid radial PWVs, FMD, and GMD) (Table 3). FBX cemia before versus after FBX treatment. treatment increased the root mean square of successive differ- 6 6 P ences (RMSSD; 64.1 29.1 to 80.9 46.9 ms; = 0.0293) Effect of FBX on ANG II Infusion Responses and Plasma but not the SD of all normal R-R intervals (SDNN; 77.4 6 RAAS Markers 22.8 to 88.2 6 37.8 ms, P = 0.0769) in HC participants. FBX treatment did not alter plasma aldosterone or renin In the overall T1D group, FBX treatment led to a modest levels in HCs or patients with T1D under euglycemic and decrease in SBP (112 6 9to1096 9mmHg;P = 0.0491) ) hyperglycemic conditions (Table 2) and did not alter the Table 3) but not in DBP (67 6 6to666 7mmHg;P = renal hemodynamic response to 1 or 3 ng/kg/min ANG II 0.2823) or HR (67 6 11 to 66 6 10 bpm; P = 0.8605). infusions (Fig. 4), nor was there an impact on changes in Although there were no differences observed in renal intraglomerular hemodynamic parameters or BP re- hemodynamic function under euglycemic conditions in sponses to ANG II in either group before versus after 6 response to FBX treatment, PGLO decreased (54.4 4.1 to FBX treatment. 53.4 6 3.2 mmHg; P = 0.0497) without significant changes in RA,RE,andRA/RE ratio in patients with T1D (Table 3). Effect of FBX on Glucose Control, Laboratory FBX treatment did not alter vascular stiffness parame- Parameters, and Adverse Events ters (aortic AIX, carotid AIX, and carotid femoral and ca- FBX treatment decreased hemoglobin A1c levels (5.05 6 0.22 rotid radial PWVs), measures of endothelial-dependent and to 4.97 6 0.23%, P = 0.0167) in HC participants but not in endothelial-independent vascular function (FMD and GMD, patients with T1D during euglycemic or hyperglycemic con- respectively), or HR variability measures (RMSSD and SDNN, ditions. FBX treatment did not alter 24-h protein intake, Table 3). 24-h urine sodium excretion, BMI, estradiol or progesterone 1944 Cardiorenal Effects of Uric Acid Lowering in T1D Diabetes Volume 66, July 2017

levels (females only), or any other clinically relevant bio- chemical and hematological parameters assessed (including serum sodium, potassium, calcium, magnesium, chloride,

value phosphate, and liver enzymes) in T1D or HC study partic- 0.0001 P , ipants. No adverse events were reported, aside from some mild nausea after FBX intake in several patients, which re- 42 5.8 0.4028 47 0.9976

0.18] + 14)/weight solved after taking the agent with food. 6 6 6 3 Effect of FBX on Urinary NO and Inﬂammatory Markers FBX treatment did not signiﬁcantly affect levels of urinary 61 108 54 60

15.6 5.1 nitrite and nitrates in either HC or T1D study participants 6 6 6 during euglycemic or hyperglycemic conditions. In patients with T1D, PUA lowering with FBX caused an exaggerated increased response to hyperglycemia in urinary IL-5 (P = 0.0029), IL-9 (P = 0.0067), and IL-18 (P = 0.0136) levels d hyperglycemic clamp conditions value Baseline FBX ﬂ 0.0001 221 (Fig. 5). No other changes in the measured urinary in am- P ,

= 49) matory markers were observed. n

1.3) 0.8831 DISCUSSION T1D ( 6 In patients with T1D, PUA levels are linked with early renal 53 83 0.1126 3.8 0.5487 260 0.6742 16.5 0.9376 6.8 39 0.1265 60 0.4 0.8264 6 6 function loss (3,7). Although the association between PUA 6 6 6 6 6

13.9 (7.9 levels and renal risk is compelling, studies measuring the

6 effects of PUA-lowering therapies on renal outcomes fo-

24-h protein intake: estimated by the formula ([urine urea cused on older, hypertensive patients with type 2 diabetes †

Euglycemia Hyperglycemia and established chronic kidney disease. Our study examined the effect of PUA lowering on renal hemodynamic and vas-

1.3)* 62.4 cular function in young, normotensive patients with T1D 6 s with T1D studied under euglycemic an

0.05 vs. HC. who had normal renal function and normal baseline PUA 62 124 76 132 7.5 2.1 249 245 22.9 10.1 56 67 0.3* 1.0 , 6 6 levels. Our overall aim was to determine whether there is a 6 6 6 6 6 P physiological rationale for PUA lowering in the presence of 14.8 (7.8

6 uncomplicated T1D. We used FBX rather than allopurinol as a physiological probe because of the greater potency and better safety proﬁle of this agent compared with older agents. Furthermore, because of its mechanism of action, FBX does not stimulate the production of reactive oxygen value Baseline FBX 0.0001 240 P

, species, which may limit the vascular protective effects of

, number of participants. * allopurinol (30). n Although our ﬁrst major observation suggests that PUA 55 8.5 0.3343 10.3 74 0.7968 150 5.9 0.5248 3.1 220 0.1997 217 260 0.8852 76 0.3 0.2726 1.0 0.23 0.0167 62.3 6 6 6 lowering does not affect renal function measured by GFR 6 6 6 6 6 = 24)

n and ERPF in patients with T1D or HCs, we observed a ﬁ

HC ( signi cant reduction in GFR in each of the six patients with hyperﬁltration at baseline. Because of the lack of a placebo 71 131 65 152 4.2 4.3 169 285 10.4 12.1 164 283 0.3 1.1 0.22 4.97 group in this physiological analysis, it is unclear whether 6 6 6 6 6 6 6 6 such GFR normalization occurred due to a regression to the Baseline FBX 1.1 mean or to a physiological effect (31). Given the role of hyperﬁltration in predicting the subsequent development SD, unless otherwise indicated.

6 of microalbuminuria and nephropathy in patients with † T1D (32), placebo-controlled studies with larger T1D-H cohorts are needed to determine whether PUA lowering can normalize hyperﬁltration, thereby promoting renal protection. , mmol/mol (%) 5.05

1c Similar to our previous reports (2,4), in our current T1D cohort, higher baseline PUA levels within the normal range Diet parameters and plasma marker response to FBX treatment in HCs and patient mol/L) 303

m correlated with lower GFR and ERPF during euglycemia and — hyperglycemia. After FBX treatment, the correlation be- Progesterone (females only) 3.3 Estradiol (females only) 226 PUA ( Renin (ng/L) 14.4 Aldosterone (ng/dL) 291 24-h protein intake (g/kg/day) 24-h urine sodium (mmol/day) 155 Hemoglobin A tween PUA and renal hemodynamic function remained sig- Values are reported as the mean Plasma analysis Diet parameters Parameter Table 2 (in kg). niﬁcant only for ERPF during euglycemic conditions—likely 1945

Table 3—Renal, intraglomerular, and systemic hemodynamic function and vascular parameter response to FBX treatment in HCs and patients with T1D studied under euglycemic and hyperglycemic clamp conditions T1D (n = 49) HC (n =24) Euglycemia Hyperglycemia Lytvyn and Associates Parameter Baseline FBX P value Baseline FBX P value Baseline FBX P value Renal hemodynamic function ERPF (mL/min/1.73 m2) 654 6 111 639 6 91 0.3818 647 6 131 657 6 113 0.5329 676 6 133 665 6 124 0.4235 GFR (mL/min/1.73 m2)1176 17 119 6 15 0.2836 115 6 19 113 6 16 0.1893 130 6 21 133 6 19 0.2142 FF 0.18 6 0.04 0.19 6 0.03 0.3124 0.18 6 0.04 0.17 6 0.03 0.1019 0.20 6 0.04 0.21 6 0.05 0.1064 RBF (mL/min/1.73 m2) 1,058 6 202 1,035 6 178 0.4044 1,051 6 219 1,063 6 185 0.6300 1,071 6 209 1,052 6 203 0.3956 RVR (mmHg/L/min) 0.077 6 0.015 0.077 6 0.015 0.6896 0.081 6 0.022 0.077 6 0.014 0.1327 0.080 6 0.016 0.082 6 0.020 0.4171 Intraglomerular hemodynamic parameters PGLO (mmHg) 48.9 6 2.7 49.4 6 2.5 0.2275 54.4 6 4.1 53.4 6 3.2 0.0497 54.9 6 4.0 56.1 6 4.1 0.0664 25 RA (dyne $ s$ cm ) 2,246 6 640 2,208 6 704 0.6845 2,167 6 885 2,010 6 662 0.1905 2,170 6 721 2,132 6 815 0.7373 25 RE (dyne $ s$ cm ) 994 6 263 1,028 6 197 0.3931 1,690 6 424 1,604 6 331 0.1299 1,871 6 423 1,997 6 573 0.0829 RA/RE ratio 2.39 6 0.86 2.20 6 0.76 0.1504 1.31 6 0.52 1.29 6 0.47 0.6641 1.21 6 0.44 1.11 6 0.43 0.1667 Systemic hemodynamic function HR (bpm) 62 6 8616 9 0.4042 67 6 11 66 6 10 0.8605 64 6 11 64 6 12 0.7484 SBP (mmHg) 107 6 91066 8 0.3267 112 6 10 109 6 9 0.0491 113 6 9 112 6 9 0.4405 DBP (mmHg) 64 6 6646 7 0.6106 67 6 6666 7 0.2823 69 6 7696 7 0.6546 Vascular parameters Aortic AIX (%) 27.7 6 9.7 29.6 6 8.1 0.4353 23.0 6 11.5 24.1 6 11.8 0.4247 21.8 6 14.3 21.5 6 12.5 0.8605 Carotid AIX (%) 23.6 6 13.6 24.3 6 14.1 0.7529 0.5 6 15.0 1.6 6 14.7 0.4740 4.4 6 15.7 2.5 6 14.9 0.1723 Carotid radial PWV (m/s) 7.1 6 1.0 6.8 6 1.1 0.2340 7.3 6 1.1 7.2 6 1.1 0.7043 7.6 6 1.0 7.3 6 1.3 0.0728 Carotid femoral PWV (m/s) 5.5 6 1.1 5.3 6 1.0 0.5332 5.8 6 1.0 5.6 6 1.2 0.2979 5.8 6 0.9 5.6 6 1.1 0.0972 FMD (%) 4.2 6 3.1 5.0 6 3.4 0.2428 4.1 6 4.4 5.3 6 3.4 0.8251 4.2 6 4.1 4.8 6 3.6 0.1733 FMD/ﬂow 0.047 6 0.038 0.073 6 0.076 0.1616 0.051 6 0.056 0.050 6 0.048 0.9343 0.056 6 0.041 0.061 6 0.050 0.5058 GMD (%) 15.5 6 6.0 15.9 6 4.7 0.6629 11.9 6 5.1 11.1 6 6.3 0.2777 13.0 6 5.7 12.6 6 5.2 0.3596 HR variability RMSSD (ms) 64.1 6 29.1 80.9 6 46.9 0.0293 63.5 6 42.6 63.6 6 43.8 0.9796 67.8 6 44.2 66.5 6 47.3 0.7555 SDNN (ms) 77.4 6 22.8 88.2 6 37.8 0.0769 76.2 6 33.9 78.9 6 36.9 0.4579 79.0 6 35.9 77.4 6 39.0 0.6982 Values are reported as the mean 6 SD, unless otherwise indicated. n, number of participants. diabetes.diabetesjournals.org 1946 Cardiorenal Effects of Uric Acid Lowering in T1D Diabetes Volume 66, July 2017

Figure 2—GFR response during a euglycemic clamp day at baseline and after 8 weeks of treatment with FBX in individual patients with T1D and T1D-N (GFR <135 mL/min/1.73 m2, n = 42) (A)andT1D-H(GFR$135 mL/min/1.73 m2, n =6)(B).

as a result of the decreased PUA range after FBX treatment, ANG II, endothelial-dependent, or endothelial-independent which limited our power to detect significant correlations. function. Given that FMD is a measure of NO bioavailability Existing data therefore suggest that long-term PUA expo- and that PUA lowering has been shown not to have an sure does not cause hyperfiltration but that short-term impact on FMD in other cohorts, it is unlikely that the lowering of PUA may normalize hyperfiltration through as BP-lowering effect of FBX is mediated by significant yet undefined mechanisms. Our study does not clearly in- changes in NO bioavailability (36). Future studies may dicate whether PUA is a cause or a consequence of altered consider examining the role of inflammation and other renal hemodynamic function in patients with T1D and thus mechanisms in FBX-mediated BP-lowering effects. requires further investigation. Whether PUA influences the vascular response to Consistent with previous studies reporting allopurinol- clamped hyperglycemia is unknown in patients with un- related effects on BP (13–15), our second observation was complicated T1D. Our third major observation, contrary to the modest decrease in SBP observed in T1D patients under what we expected, was that PUA lowering enhanced the euglycemic conditions in response to FBX treatment. In our renal FF response to clamped hyperglycemia through an cohort, FBX treatment did not alter the following previously increase in RE. Constriction of the efferent renal arteriole is reported FBX-mediated BP-lowering effects (33–35): arte- a known mechanism of hyperfiltration mediated by intra- rial stiffness, HR variability, levels of plasma RAAS markers, renal RAAS activation in T1D (10). Moreover, PUA is pos- or the hemodynamic responses to an exogenous infusion of itively associated with plasma renin activity in humans,

Figure 3—GFR (A), ERPF (B), FF (C), RA (D), RE (E), and PGLO (F) responses to clamped hyperglycemia in patients with T1D at baseline and after 8 weeks of treatment with FBX. T1D group, n = 48. D in each outcome represents the difference between the outcome measured at hyperglycemic clamp day and euglycemic clamp day. RA,RE,andPGLO in patients with T1D calculated by Gomez equations (assumption: PGLO of 56.4 mmHg in patients with T1D). Values are reported as the mean 6 SD. diabetes.diabetesjournals.org Lytvyn and Associates 1947

Figure 4—GFR (A), ERPF (B), FF (C), RA (D), RE (E), and PGLO (F) responses to ANG II infusion (1 and 3 ng/kg/min) during a euglycemic clamp day in patients with T1D at baseline and after 8 weeks of treatment with FBX. T1D group, n = 48. D in each outcome represents the difference between the outcome measured after and before the 3 ng/kg/min ANG II infusion during a euglycemic clamp day. RA,RE,andPGLO in patients with T1D calculated by Gomez equations (assumption: PGLO of 56.4 mmHg in patients with T1D). Values are reported as the mean 6 SD.

, even when PUA levels are within the normal range ( 450 in RE and IL-18 but without impacting the RAAS or NO mmol/L) (34,37). The association between PUA and RAAS levels, as measured by the FMD and urinary nitrate and activation has been further strengthened by previous find- nitrite levels. PUA may therefore augment hemodynamic ings demonstrating a negative correlation between PUA and mechanisms that mediate the renal response to hypergly- the renal hemodynamic response to an ANG II infusion in a cemia at the efferent arteriole and may be an important cohort of individuals with a wide range of PUA values (100– anti-inflammatory mechanism required to maintain the re- 595 mmol/L) (38). Contrary to these previous observations, nal hemodynamic response to hyperglycemia. Our findings ANG II infusion hemodynamic responses and plasma renin suggest the need for an optimal PUA balance, whereby PUA and aldosterone levels did not change after FBX treatment levels are high enough to limit the renal vasoconstrictive in our cohort. Interestingly, PUA lowering with FBX in response to hyperglycemia but are not excessively elevated patients with T1D led to an increase in levels of a proin- in a range that lowers renal function and increases BP. Such flammatory cytokine, IL-18, which has been reported to observation are consistent with recent findings by Uedono play a pivotal role in the pathogenesis of diabetic nephrop- et al. (40), where a U-shaped relationship between PUA and athy (39). Therefore, PUA lowering may enhance the renal renal hemodynamic parameters was observed, with mild FF response to clamped hyperglycemia through an increase hyperuricemia and mild hypouricemia being significantly

Figure 5—IL-5 (A), IL-9 (B), and IL-18 (C) response to clamped hyperglycemia in T1D at baseline and after 8 weeks of treatment with FBX. T1D group, n = 48. Values are reported as the mean 6 SD. 1948 Cardiorenal Effects of Uric Acid Lowering in T1D Diabetes Volume 66, July 2017 associated with lower GFR and ERPF in healthy Japanese thank M.L. Fritzler and Eve Technologies Corporation for performing the cytokine subjects. Hypouricemia has also been associated with endo- assays for this project. thelial dysfunction in otherwise healthy patients with a Funding. Y.L. was supported by a Heart & Stroke/Richard Lewar Centre of URAT1 loss-of-function mutation (41). Therefore, future Excellence Studentship, a Javenthey Soobiah Scholarship, a Queen Elizabeth II/Dr. Arnie Aberman Scholarship in Science and Technology, a University of studies should examine a range of FBX doses to find an Toronto Fellowship in the Department of Pharmacology and Toxicology, and a optimal level of PUA that maximizes cardiorenal protection. Canadian Diabetes Association Postdoctoral Fellowship. R.H. was supported Our study has limitations. To minimize the effects of the by Banting & Best Diabetes Centre Graduate Studentships (University of Toronto), relatively small sample size, we recruited homogenous study a Hilda and William Courtney Clayton Paediatric Research Fund Award, and an groups with careful prestudy preparation. As a result, our Institute of Medical Science Graduate Student Award. D.Z.I.C. was supported data are limited in generalizability to other populations, by a Kidney Foundation of Canada Scholarship, a Canadian Diabetes such as T1D patients with longer diabetes duration, degree Association-KRESCENT Program Joint New Investigator Award, and funding of proteinuria, nephropathy, and others. Moreover, all from the Canadian Institutes of Health Research and the Kidney Foundation of study participants were normouricemic, and, thus, our Canada. findings cannot be generalized to hyperuricemic patients. Duality of Interest. B.A.P. has received honoraria for continuing medical Therefore, it is possible that although the hemodynamic education events from Boehringer Ingelheim, Janssen, Medtronic, and Abbott. His research institute has received operating funds on his behalf changes we observed in normouricemic patients were from Medtronic, Novo Nordisk, and Boehringer Ingelheim. He has served as a modest, lowering PUA in patients with hyperuricemia fi consultant for Boehringer Ingelheim. D.Z.I.C. has received speaker/consul- might still yield bene ts around renal and cardiovascular tant honoraria from Boehringer Ingelheim, Eli Lilly, AstraZeneca, Sanofi, protection. Although endogenous production or exoge- Merck, Mitsubishi Tanabe, Abbvie, and Janssen and has received opera- nous consumption of uric acid in the form of purines and tional funding for clinical trials from Boehringer Ingelheim, Merck, Janssen, fructose were not recorded, we observed similar levels of and AstraZeneca. No other potential conflicts of interest relevant to this urinary urea excretion (as a surrogate marker of protein article were reported. intake) before FBX and after FBX treatment. Each study Author Contributions. Y.L., R.H., A.L., V.L., D.F., A.A., B.A.P., and D.Z.I.C. participant acted as his/her own control to decrease researched data, wrote the manuscript, contributed to discussion, and reviewed and possible intraindividual variability. Additionally, we were edited the manuscript. All authors have approved the final version of the manuscript. not able to study intracellular uric acid mechanisms because D.Z.I.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the of the inability to perform such measurements in human data analysis. studies. Next, we recognize that the Gomez equations are Prior Presentation. 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