Glycosuria-mediated urinary uric acid excretion in patients with

Articles in PresS. Am J Physiol Renal Physiol (November 5, 2014). doi:10.1152/ajprenal.00555.2014
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Glycosuria-mediated urinary uric acid excretion in patients with
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uncomplicated type 1 diabetes mellitus
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Yuliya Lytvyn, HBSc1,2, Marko Škrtić, MD PhD1, Gary K. Yang, PhD1, Paul M. Yip, PhD3,
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Bruce A. Perkins MD4, David Z. I. Cherney, MD, PhD1
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Toronto, Ontario, Canada
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Toronto
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Word Count: 3280 for main text and 247 for abstract; 1 table and 4 figures
Department of Medicine, Division of Nephrology, Toronto General Hospital, University of Toronto,
Department of Pharmacology and Toxicology, University of Toronto, Canada
University Health Network, Department of Laboratory Medicine and Pathobiology, University of
Department of Medicine, Division of Endocrinology, Mount Sinai Hospital, University of Toronto,
Toronto, Ontario, Canada
Running Title: Glycosuria causes uricosuria in type 1 diabetes
Please address correspondence to:
David Cherney, MD CM, PhD, FRCP(C)
Toronto General Hospital
585 University Ave, 8N-845
Toronto, Ontario,
M5G 2N2
Phone: 416.340.4151
Fax: 416.340.4999
Email: [email protected]
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Copyright © 2014 by the American Physiological Society.
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ABSTRACT:
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Objective: Plasma uric acid (PUA) is associated with metabolic, cardiovascular and renal
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abnormalities in patients with type 2 diabetes, but is less well understood in type 1 diabetes
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(T1D). Our aim was to compare PUA levels and fractional UA excretion (FEUA) in patients with
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T1D vs. healthy controls (HC) during euglycemia and hyperglycemia.
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Methods: PUA, FEUA, blood pressure (BP), glomerular filtration rate (GFR-inulin) and effective
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renal plasma flow (ERPF – paraaminohippurate) were evaluated in patients with T1D (n=66)
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during clamped euglycemia (glucose 4-6 mmol/L) and hyperglycemia (9-11 mmol/L), and in HC
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(n=41) during euglycemia. To separate the effects of hyperglycemia vs. increased glycosuria,
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parameters were evaluated during clamped euglycemia in a subset of T1D patients before and
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after SGLT2 inhibition for 8 weeks.
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Results: PUA was lower in T1D vs. HC (228±62 µmol/L vs. 305±75 µmol/L, p<0.0001). In
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T1D, hyperglycemia further decreased PUA (228±62 µmol/L to 199±65 µmol/L, p<0.0001),
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which was accompanied by an increase in FEUA (7.3±3.8 to 11.6±6.7, p<0.0001). In T1D, PUA
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levels correlated positively with SBP (p=0.029) and negatively with ERPF (p=0.031) and GFR
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(p=0.028). After induction of glycosuria with SGLT2 inhibition while maintaining clamped
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euglycemia, PUA decreased (p<0.0001) and FEUA increased (p<0.0001).
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Conclusions: PUA is lower in T1D vs. HC, and positively correlates with SBP and negatively
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with GFR and ERPF in T1D. Glycosuria rather than hyperglycemia increases uricosuria in T1D.
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Future studies examining the effect of UA lowering therapies should account for the impact of
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ambient glycemia, which causes an important uricosuric effect.
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INTRODUCTION:
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Humans have higher uric acid (UA) levels in comparison to other mammals due to
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mutational silencing of the enzyme uricase, which results in UA remaining the end product of
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purine metabolism (29). Additionally, about 90% of filtered UA is reabsorbed by the S1 segment
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of the proximal convoluted tubule, in a process regulated by intracellular anion transporters on
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the basolateral membrane, such as urate transporter 1 (URAT1) and the more recently discovered
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glucose transporter 9 (GLUT9) (29). The lack of uricase, combined with the high reabsorptive
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capacity in the kidney, predisposes humans to the development of hyperuricemia.
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UA has recently emerged as an inflammatory factor that increases oxidative stress and
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promotes activation of the renin angiotensin aldosterone system (RAAS) (29). From a clinical
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perspective, higher UA levels are associated with metabolic abnormalities (insulin resistance,
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hyperglycemia), cardiovascular disease (hypertension, endothelial dysfunction, arterial stiffness,
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cardiac diastolic dysfunction) and kidney injury (28, 29) and thus could be involved in the onset
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and progression of diabetic nephropathy, a common microvascular complication of diabetes
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mellitus (DM). Plasma UA (PUA) levels could therefore serve as a biomarker and an effective
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therapeutic target to supplement current clinical targets such as hemoglobin A1c (HbA1c),
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cholesterol and blood pressure.
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Evidence from rodent models suggests an association between high UA levels and
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markers of high intraglomerular pressure such as hyperfiltration, and with subsequent increases
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in proteinuria, glomerular sclerosis and tubulointerstitial fibrosis, leading to chronic kidney
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disease (28). More recently, in an animal model of type 1 DM (T1D), UA lowering reduced
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proteinuria, preserved GFR and suppressed renal expression of inflammatory interleukins (37).
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In patients with T1D, UA is associated with impaired renal function, even when UA levels are in
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the normal range (28, 35). For example, in 355 T1D participants from the second Joslin Study on
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the Natural History of Microalbuminuria, baseline UA (within the normal range) showed a
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significant association with early GFR loss of more than 3.3% per year over a 6-year follow-up
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period (15). UA also increases the risk of developing proteinuria in T1D patients (23). For
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example, in 652 normoalbuminuric type 1 DM patients recruited into the Coronary Artery
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Calcification in Type 1 Diabetes Study (CACTI), each 60 μmol/L increment in UA from baseline
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increased the risk of micro- or macro-albuminuria by 80% after a 6-year follow-up period (23).
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Though observational associations between higher UA levels and renal outcomes show
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consistency among independent cohorts (29), UA levels are not clearly defined in the T1D
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populations.
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Accordingly, the first goal of this study was to compare PUA levels in healthy control
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patients (HC) with patients with T1D. It was hypothesized that even within the normal range,
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PUA levels would be higher in the T1D cohort and that higher PUA levels will be associated
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with deleterious hemodynamic profiles such as higher blood pressure and changes in renal
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hemodynamic function. The second goal was to examine the relationship between clamped
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hyperglycemia, hemodynamic parameters and PUA levels to determine if this acute
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physiological stimulus, which promotes deleterious hemodynamic effects such as increased
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blood pressure, influences PUA levels.
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RESEARCH DESIGN AND METHODS:
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Subject Inclusion Criteria and Study Preparation:
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Forty-one HC and 66 T1D patients underwent detailed physiological examinations. In
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brief, inclusion criteria were: 18-40 years of age, blood pressure <140/90, normoalbuminuria on
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a 24-hour urine collection, diabetes duration >1 year, no history of renal or cardiovascular
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complications and no intake of concomitant medications that would alter blood pressure or
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cardiovascular outcomes. Study visits were performed after a controlled diet for 7 days
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consisting of 150 mmol/day sodium and 1.5 g/kg/day protein. The sodium-replete diet was
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used to avoid circulating volume contraction, RAAS activation and between-subject
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heterogeneity. Pre-study protein intake was modest to avoid the hyperfiltration effect of high
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protein diets (24). All the studies were approved by the University Health Network Research
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Ethics Board and all subjects gave written informed consent.
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Experimental Procedures:
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Patients with T1D were studied on 2 consecutive days during euglycemia and
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hyperglycemia. Euglycemic (4-6 mmol/L) and hyperglycemic (9-11 mmol/L) conditions in T1D
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were maintained using a modified glucose clamp technique as previously described (8). Blood
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glucose levels were stable for at least 2 hours prior to the measurement of the study end points
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and were maintained 3 to 5 hours for the rest of the study day. HC were studied during
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normoglycemic conditions at the Renal Physiology Laboratory at the Toronto General Hospital.
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Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were estimated using
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inulin and paraaminohippurate (PAH) steady state infusion clearance techniques (5),
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respectively, using previously described methods (9). The results of the 2 clearance periods were
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averaged. Brachial artery blood pressure measurements were obtained at 30-minute intervals
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throughout the study days (Critikon, Tampa, Florida, USA).
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In a post-hoc analysis undertaken to understand the relative effects of hyperglycemia vs.
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increased glycosuria, the effect of sodium glucose co-transporter 2 (SGLT2) inhibition on PUA
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and urinary UA was examined using frozen, archived samples. The aim of this analysis was to
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induce glycosuria while maintaining euglycemia to determine whether effects on PUA were due
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to increased urinary UA excretion. For this analysis, we analysed urine and plasma samples
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(n=40) obtained during baseline clamped euglycemic conditions and at follow-up after treatment
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with empagliflozin 25mg QD for 8 weeks in the Adjunctive-To-Insulin and Renal MechAnistic
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pilot trial of empagliflozin in T1D (ATIRMA trial, ClinicalTrials.gov NCT01392560). The
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primary and secondary outcomes from this trial have been published (9).
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Sample Collection and Analytical Methods:
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After clamped euglycemia was achieved for at least 2 hours, blood was collected for
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measurements of inulin, PAH, sodium, PUA and RAAS mediators (angiotensinogen, plasma
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renin activity [PRA], aldosterone and angiotensin II) and urine samples were collected for UA,
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sodium, glucose and creatinine measurements.
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The blood samples were immediately centrifuged at 3000 rpm at 4ºC for 10 minutes.
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Plasma was extracted, placed on ice and stored at -70ºC. Inulin and PAH were measured in
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serum by colorimetric assays using anthrone and N- (1-naphthyl) ethylene-diamine respectively
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(16). All hemodynamic measurements were adjusted for body surface area. Filtration fraction
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(FF) represented the ratio of GFR to ERPF. Renal blood flow (RBF) was derived as ERPF / (1-
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hematocrit). Renal vascular resistance (RVR) was derived by dividing mean arterial pressure
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(MAP) by the RBF.
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Plasma and urine samples were measured for UA, sodium, creatinine, glucose and urea
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on the Architect c8000 Clinical Chemistry System using the manufacturer’s reagents (Abbott
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Diagnostics, Abbott Park, Illinois, USA). In addition, UA excretion was expressed as fractional
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excretion (FEUA), derived using (UUA×PCr)/(UCr×PUA)×100 where UUA, PCr, UCr and PUA are
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urinary UA, plasma creatinine, urinary creatinine and plasma UA concentrations respectively.
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Similarly, sodium excretion was expressed as fractional excretion (FENa), derived using
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(UNa×PCr)/(UCr×PNa)×100 where UNa and PNa are urinary sodium and plasma sodium
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concentrations, respectively.
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Aldosterone was measured using a Coat-A-Count radioimmunoassay system. PRA was
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measured using a radioimmunoassay kit (Diasorin, Stillwater, Minnesota, USA). HbA1c was
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measured by high-performance liquid chromatography with the Variant II system (Bio-Rad
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Laboratories, Hercules, California, USA).
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Statistical analysis:
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Data are presented as mean ± standard deviation (SD). To assess for between-
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group differences, analysis of variance with post-hoc Tukey’s test was used. To compare within-
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group differences (responses to hyperglycemia or SGLT2 inhibition) a paired student’s t-test was
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used. Linear regression analysis was used to determine correlations between responses and PUA
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levels. Statistical significance was defined as p<0.05. All statistical analyses were performed
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using SAS v9.1.3 and GraphPad Prism software (version 6.0).
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RESULTS:
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Baseline characteristics
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Baseline parameters were similar between HC and T1D patients (Table 1). Participants
were young, normotensive, normoalbuminuric and the two groups were similar in age and BMI.
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During euglycemia, heart rate was significantly higher, but still within the normal range,
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in the T1D versus HC and no significant differences in SBP or DBP were observed. During
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hyperglycemic conditions, SBP significantly increased and HR decreased compared to
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euglycemia in the T1D group. As expected, T1D participants had significantly lower levels of
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circulating RAAS mediators compared to HC (aldosterone, PRA and angiotensin II) (41). During
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hyperglycemia, aldosterone and PRA levels further decreased.
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As expected, T1D subjects exhibited higher GFR, ERPF, RBF and lower RVR compared
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to HC (p<0.0001 for all comparisons). Out of the 66 T1D patients, 29 exhibited normofiltration
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(44%) and 37 hyperfiltration (56%), where hyperfiltration was defined as GFR 135
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mL/min/1.73m2. In response to clamped hyperglycemia, GFR tended to increase in T1D (147±40
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to 159±39 mL/min/1.73m2, p=0.064) and RVR decreased (0.069±0.021 to 0.055±0.016
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mmHg/L/min, p<0.0001). No significant changes to ERPF, FF or RBF were observed in
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response to hyperglycemic conditions.
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Sodium, Glucose and UA Handling at Baseline
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During clamped euglycemic conditions, PUA levels were lower in the T1D group vs. HC
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(228±62 vs 305±75 µmol/L, p<0.0001) (Table 1, Figure 1). PUA negatively correlated with
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FEUA in T1D patients (r=-0.60, p<0.0001). Uglucose excretion levels were also greater in T1D vs.
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HC during clamped euglycemia, but there was no significant difference in urine uric
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acid/creatinine ratio, FENa or FEUA between HC and T1D.
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Compared to levels during clamped euglycemia, PUA decreased further in response to
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clamped hyperglycemia (228±62 µmol/L to 199±65 µmol/L, p<0.0001) (Table 1, Figure 1). The
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decline in PUA levels in T1D patients during hyperglycemia was accompanied by significant
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increases in urine UA levels (257±121 to 339±161 umol/L, p=0.0007) and FEUA (7.3±3.8 to
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11.6±6.7, p<0.0001). PUA was negatively correlated with FEUA during clamped hyperglycemia
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(r=-0.50, p<0.0001). The increase in UA excretion during hyperglycemia was accompanied by
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significant increases in Uglucose (1.4±3.2 to 9.8±10.4 mmol/L, p<0.0001) and FENa (0.87±0.56 to
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1.63±0.89, p<0.001).
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UA Correlations with Hemodynamic Parameters
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PUA levels were positively correlated with SBP in T1D (r=0.27, p=0.029) under
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euglycemic conditions, but not during hyperglycemia (Figure 4). During euglycemia, PUA levels
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negatively correlated with ERPF (r=-0.27, p=0.031), and FEUA positively correlated with ERPF
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(r=0.30, p=0.017) in T1D patients. During hyperglycemia, PUA negatively correlated with GFR
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in T1D (r=-0.27, p=0.028).
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Sodium, Glucose and Uric Acid Handling Upon Empagliflozin SGLT2 Inhibition
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SGLT2 inhibitors are a new class of agents for the treatment of T2D that block proximal
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renal tubular glucose reabsorption, leading to increased glucose excretion. Therapeutically, this
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translates into important plasma glucose lowering effects (6). Trials with SGLT2 inhibitors in
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patients with T2D have reported consistent and clinically relevant decreases in PUA levels (14,
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40); however the mechanisms responsible were never clearly elucidated. Accordingly, to better
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understand whether PUA lowering with hyperglycemia is due to systemic effects leading to
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decreased production or renal effects causing increased uricosuria, plasma and urine UA levels
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were measured before and after SGTL2 inhibition while maintaining clamped euglycemia in 40
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T1D patients.
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During clamped euglycemic conditions, after empagliflozin treatment, the anticipated
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increase in Uglucose/creatinine (1.3±3.2 to 42.9±17.8, p<0.0001) was accompanied by a decline in
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PUA (225±65 to 191±62 mmol/L, p<0.0001) and increases in UUA/creatinine (290±110 to 327±103
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mmol/mmol, p=0.0075) and FEUA (8.2±3.6 to 11.1±5.1, p<0.0001) (Figure 2).
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DISCUSSION:
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Observational associations between higher UA levels and metabolic abnormalities,
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cardiovascular disease and kidney dysfunction show consistency among independent healthy and
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disease state cohorts, in both animals and humans (29). The potential renal protective effects of
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UA lowering in T1D patients are being studied as part of the NIH funded Protecting Early Renal
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Function Loss or “PERL” study (NCT02017171) (30), highlighting the promising future role for
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UA-based therapies in T1D. However UA levels during euglycemia compared to hyperglycemia
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have not been clearly defined in otherwise healthy T1D patients. Our first goal was to compare
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PUA levels in HC with levels in patients with T1D. Our second goal was to determine if acute
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clamped hyperglycemia, which promotes deleterious hemodynamic effects such as increased
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blood pressure, influences PUA levels.
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Due to the strong association between PUA levels and cardiovascular and renal
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abnormalities, especially in the context of diabetes, it was initially hypothesized that T1D
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patients would have higher PUA levels compared to HC. Our first major observation, however,
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was that T1D patients had lower PUA levels under euglycemic conditions compared to HC, in
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conjunction with increased urinary glucose that did not correlate with the degree of UA
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excretion. Hyperglycemia in T1D patients was associated with a significant increase in urinary
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sodium, glucose and UA excretion and thus a further PUA decrease, highlighting an important
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physiological link between renal handling of UA, glucose and sodium (6). Furthermore, the
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negative correlation between PUA and FEUA during euglycemia and hyperglycemia suggests that
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PUA decreased as a result of increased renal excretion. The lack of elevated UA excretion in
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T1D compared to HC under euglycemic conditions may suggest that T1D patients produce less
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UA in plasma or consume less UA-containing products, although the similar protein intake based
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on urine urea excretion in these groups suggests that differences in intake of UA-containing
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foods were not relevant to our findings.
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Our observations support several studies showing an increase in UA excretion in
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response to intravenous D-glucose infusion (4, 36). More recently, an association was found
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between lower PUA and poor glycemic control (11, 18, 22). Previous studies have shown that
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insulin levels are positively correlated with PUA and insulin administration decreases UA
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excretion (33). However, it is not known whether this is a direct effect of insulin or the result of
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insulin-mediated normalization of glycemia, leading to reduced glycosuria. Worsening glycemic
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control resulting in hyperglycemia and glycosuria has been correlated with a decrease in PUA
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(20, 22). Thus, it is perhaps not surprising that epidemiological studies have shown a decreased
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risk of UA-related conditions, such as gout, in diabetic compared to non-diabetic individuals –
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especially in the context of T1D (34). The mechanisms behind the glucose-mediated PUA
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lowering effects have been explained by osmotic diuresis caused by increased plasma glucose
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levels (36), proximal tubule alterations (18) or the effect of glucose on renal UA handling (20,
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36).
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Our second aim was to determine whether PUA lowering with hyperglycemia in T1D
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was due to systemic hyperglycemia causing decreased UA production or renal glycosuria
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causing increased UA excretion. SGLT2 inhibition with empagliflozin under clamped
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euglycemic conditions was used to increase urinary glucose excretion to determine if glycosuria
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during euglycemia results in a persistent decrease in PUA through increased renal UA excretion.
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Previous trials with SGLT2 inhibitors in patients with T2D have reported consistent and
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clinically relevant decreases in PUA levels, however urine UA excretion was not measured and
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the mechanisms responsible have not, to our knowledge, been clearly elucidated (14, 40). Our
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post-hoc analysis demonstrated a decline in PUA during euglycemia with glycosuria induced
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with SGLT2 inhibition, an effect that was accompanied by an increase in UA excretion.
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Consistent with our observations, in a recent study using healthy controls, SGLT2 inhibition with
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luseogliflozin resulted in a positive correlation between urine UA and urine glucose excretion
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(10). The results of the present study provide the first evidence in the T1D population suggesting
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that hyperglycemia-mediated uricosuria is likely due to renal glycosuria rather than a direct
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effect of systemic hyperglycemia. PUA lowering effects reported with SGLT2 inhibition may be
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of clinical relevance, since this may in part explain the potential protective renal and
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cardiovascular physiological profile that has been linked with this emerging drug class (6).
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The molecular mechanisms responsible for the uricosuric effect of glucose are not clear.
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PUA levels depend on the exogenous pool which varies with dietary intake, while the
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endogenous pool is mainly regulated by hepatic production, intestinal secretion and renal
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excretion (29). Approximately 70% of UA is excreted into urine, but is easily filtered into the
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renal tubule and about 90% of filtered UA is reabsorbed by the S1 segment of the proximal
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convoluted tubule (29). Approximately 10% of filtered UA is excreted (29). Accordingly, our
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HC showed a FEUA of 6.1±4.1% and T1D during euglycemia 7.3±3.8%. UA reabsorption occurs
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by intracellular anion transporters on the basolateral membrane – mainly by URAT1 and a more
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recently discovered GLUT9 isoform 2 (1, 2), and on the apical membrane OAT4 and OAT10 (3,
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21). Recently, transport experiments in Xenopus oocytes showed that none of the transporters
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involved in UA reabsorption were influenced by luseogliflozin (10). GLUT9 isoform 2 is a
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facilitative glucose transporter mostly expressed in the kidney and the liver, located on the apical
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membrane (2). GLUT9 isoform 2 secretes UA in exchange for glucose at 10mM (1).
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Additionally, GLUT9 isoform 2 is expressed in the collecting ducts where it plays a role in the
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reabsorption of UA (27). Plasma glucose is mostly filtered in the glomerulus and is concentrated
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in the proximal tubule. It is possible that during euglycemia the concentration needed for GLUT9
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stimulation is not reached in the proximal tubule and the lower PUA in T1D during euglycemia
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vs. HC could occur by mechanisms other than glycosuria- mediated uricosuria. Based on these
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findings, the results of our study could be explained as follows: glycosuria during SGLT2
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inhibition stimulates excretion of UA by GLUT9 isoform 2 on the apical membrane of the
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proximal tubule and possibly inhibits reabsorption of UA in the collecting ducts (Figure 3). Our
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conclusion may reflect recent in vitro data showing that stimulation of Xenopus oocytes
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expressing GLUT9 isoform 2 with 10mM D-glucose resulted in UA efflux and stimulation of the
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oocytes with 100mM D-glucose - thought to be the concentration in the collecting ducts -
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inhibited the uptake of UA (10). Finally, increased glycosuria and uricosuria could, in the
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appropriate context, suggest the presence of more generalized, “Fanconi-like” proximal tubular
299
dysfunction. Since SGLT2 inhibition causes minor but statistically significant increases, rather
300
than deceases, in serum potassium, phosphate and bicarbonate, a proximal tubulopathy with this
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class of agents is very unlikely and has not been reported (38).
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To examine the functional role of PUA in this otherwise healthy cohort of T1D patients,
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we correlated PUA with blood pressure and renal hemodynamic function. We found a significant
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positive correlation between PUA and SBP, negative correlations between PUA and ERPF and
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PUA and GFR in T1D. In contrast, PUA did not correlate with any of these measures in HC.
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These observations in T1D patients are consistent with the vasoconstrictive phenotype, as
13
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suggested by observational studies. For example, the independent association between PUA and
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blood pressure has been reported in various cohorts, including a subset of the Framingham Heart
309
Study (12, 13). The deleterious effect of PUA on cardiovascular function may be worsened by
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the hypertensive effect of hyperglycemia in T1D patients (7, 19, 31). Hyperglycemia induces
311
systemic vascular abnormalities such as endothelial dysfunction in humans (19, 31). As a result
312
of the effects of hyperglycemia and neurohormonal activation of the renin angiotensin
313
aldosterone system, UA levels are independently associated with endothelial dysfunction,
314
thereby promoting hypertension, even when UA levels are within the normal range (12, 26).
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Therefore, lower PUA levels in T1D patients, especially under hyperglycemic conditions, do not
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necessarily indicate that T1D patients are protected from the deleterious effects of UA. The
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effects of PUA may be exacerbated by hyperglycemia in T1D patients, leading to exaggerated
318
deleterious hemodynamic consequences despite lower absolute PUA levels. From a clinical
319
perspective, small trials have already started to show that lowering UA exerts anti-proteinuric
320
and antihypertensive effects and could prevent renal functional loss and vascular injury (13, 17,
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25, 29, 32, 39). Thus, despite the lower UA levels in T1D versus HC, which are further lowered
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during hyperglycemia, studying UA lowering agents in T1D patients could be a critical step
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towards preventing progression of diabetes-related complications.
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Our study has limitations. First, the study cohort consisted of a carefully selected group
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of patients with uncomplicated disease, limiting the generalizability of the data to populations
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outside of T1D, or to patients with existing complications. Additionally, although the similar
327
urine urea excretion and thus protein intake suggest that differences in dietary intake of high UA-
328
containing foods were unlikely, consumption of UA was not recorded. Fructose is another
329
exogenous source of UA, which was not recorded in this study, and should be considered in
14
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future analyses. Finally, while we propose a possible explanation for glycosuria-mediated
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uricosuria, we could not determine the mechanistic basis at the molecular level. Future studies
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are needed in order to confirm our hypothesis.
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In conclusion, glycosuria, rather than the direct effect of hyperglycemia, is responsible
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for increased uricosuria in T1D patients, and may be mediated by glucose-mediated activation of
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GLUT9 isoform 2 on the apical membrane of the proximal tubule. Since PUA lowering may lead
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to renal and vascular protective effects, our data suggest that PUA lowering by SGLT2 inhibition
337
via increased uricosuria may be clinically important. Finally, future studies examining the effect
338
of UA lowering therapies should account for the impact of ambient glucose levels, which cause a
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clinically relevant uricosuric and consequent PUA lowering effect.
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CONTRIBUTIONS:
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D.Z.I.C, B.A.P., Y.L., P.Y., researched data, wrote the manuscript, contributed to
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discussion, reviewed/edited manuscript. M.Š., G.K.Y., made contributions to data analysis and
344
interpretation, drafting and editing the manuscript. All authors have approved the final version of
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this manuscript.
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ACKNOWLEDGEMENTS:
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This work was supported by Boehringer Ingelheim and Eli Lilly (to D.Z.I.C. and B.A.P.)
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and operating support from the Canadian Institutes of Health Research and the Heart and Stroke
350
Foundation of Canada. D.Z.I.C. was also supported by a Kidney Foundation of Canada
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Scholarship and a Canadian Diabetes Association-KRESCENT Program Joint New Investigator
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Award. The authors would also like to thank Jenny Cheung-Hum for their invaluable assistance
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with biochemical assays included in this work. Finally, the authors are grateful to the study
354
participants whose time and effort are critical to the success of our research program.
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CONFLICT OF INTEREST:
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The results presented in this paper have not been published previously in whole or in part.
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D.Z.I.C. has received speaker honoraria from Boehringer Ingelheim and B.A.P received
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operational funding with D.Z.I.C. for this work.
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Table 1. Baseline subject characteristics and UA, sodium, glucose handling in HC and
patients with T1D during euglycemia and hyperglycemia (mean ± standard deviation).
24 hour protein intake: estimated by the formula ([urine urea X 0.18] + 14) / weight in kg. Values are
mean ± standard deviation. *p<0.05 for HC vs. T1D. †p<0.05 when comparing parameters of T1D
between hyperglycemia and euglycemia states. FEUA: fractional excretion of UA; FENa: fractional
excretion of sodium; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure;
ERPF: effective renal plasma flow; GFR: glomerular filtration rate; RBF: renal blood flow; RVR: renal
vascular resistance; PRA: plasma renin activity; HC: healthy controls; T1D: type 1 diabetic patients.
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512
513
Figure 1. PUA (A), FEUA (B) and urine glucose/creatinine (C) levels in HC (n=41), and T1D
(n=66) during euglycemic (EU) and hyperglycemic (HYP) conditions. The bars above
cohorts in each figure represent significance levels of p<0.05
514
515
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517
Figure 2. PUA (A), FEUA (B) and urine glucose/creatinine (C) levels in T1D (n=40) during
euglycemic conditions at baseline and after treatment with the SGLT2 inhibitor
empagliflozin (EMPA). The bars above cohorts in each figure represent significance levels of
p<0.05
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519
520
521
Figure 3. Proposed hypothesis for glycosuria mediated hyperuricemia in T1D patients,
supported by findings in this study and by Chino et al.(10). SGLT2i: Sodium glucose
transporter 2 inhibitor; GLUT9: Glucose transporter 9; URAT1: Urate Transporter 1; UA: Uric
Acid.
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523
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525
Figure 4. Linear regression analysis of PUA with SBP in T1D during euglycemia (A), with
ERPF in T1D during euglycemia (B), with GFR in T1D during hyperglycemia (C). T1D
n=40; SBP: systolic blood pressure; ERPF: effective renal plasma flow; GFR: glomerular
filtration rate.
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530
531
532
533
534
535
536
20
Table 1. Baseline subject characteristics and UA, sodium, glucose handling in HC and
patients with T1D during euglycemia and hyperglycemia (mean ± standard deviation).
Parameter
HC (n=41)
T1D (n=66)
Euglycemia
Hyperglycemia
Baseline parameters
Males
Age (years)
Diabetes duration (years)
Weight (kg)
Height (m)
Body mass index (kg/m2)
Hemoglobin A1c - mmol/mol (%)
19 (43%)
28.4±7.1
70.4±11.8
1.74±0.09
23.3±3.0
34.8±3.6 (5.3±0.3)
35 (53%)
25.0±6.0
17.0±6.6
73.9±13.7
1.73±0.09
24.8±3.9
66.7±16.1 (8.2±1.5) *
-
24 hour urine sodium (mmol/day)
24 hour protein intake (g/kg/day)
177±61
1.1±0.3
169±85
1.0±0.3
-
305±75
248±170
6.1±4.1
0.84±0.60
0.02±0.03
228±62*
257±121
7.3±3.8
0.87±0.56
1.4±3.2*
199±65†
339±161 †
11.6±6.7 †
1.63±0.89†
9.8±10.4 †
60±9
112±12
67±8
74±13*
115±10
66±6
72±11†
117±11†
66±8
Renal hemodynamic function
ERPF (mL/min/1.73m2)
GFR (mL/min/1.73m2)
Filtration fraction
RBF (mL/min/1.73m2)
RVR (mmHg/L/min)
653±157
116±12
0.19±0.04
1063±259
0.081±0.020
824±276*
147±40*
0.19±0.06
1310±434*
0.069±0.021*
853±253
159±39†
0.19±0.05
1305±419
0.055±0.016†
Circulating neurohormones
Aldosterone (ng/dL)
PRA (ng/mL/h)
Angiotensinogen (ng/mL)
Angiotensin II
245±254
1.34±1.14
1264±1000
11.6±8.4
45±31*
0.53±0.43*
1092±722
3.1±3.5*
31±10 †
0.35±0.27†
1076±742
2.2±2.2
Sodium, glucose, uric acid handling
Plasma uric acid (µmol/L)
Urine uric acid/creatinine ratio
FEUA (%)
FENa (%)
Urine glucose/creatinine ratio
Systemic hemodynamic function
HR (beats per minute)
SBP (mmHg)
DBP (mmHg)
24 hour protein intake: estimated by the formula ([urine urea X 0.18] + 14) / weight in kg. Values are
mean ± standard deviation. *p<0.05 for HC vs. T1D. †p<0.05 when comparing parameters of T1D
between hyperglycemia and euglycemia states. FEUA: fractional excretion of UA; FENa: fractional
excretion of sodium; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure;
ERPF: effective renal plasma flow; GFR: glomerular filtration rate; RBF: renal blood flow; RVR: renal
vascular resistance; PRA: plasma renin activity; HC: healthy controls; T1D: type 1 diabetic patients.
Figure 1. PUA (A), FEUA (B) and urine glucose/creatinine (C) levels in HC (n=41), and T1D
(n=66) during euglycemic (EU) and hyperglycemic (HYP) conditions.
B
Y
D
D
T
T
1
1
T
D
1
T
-
T
1
1
H
D
Y
-
P
E
E
U
C
H
D
0
R
R
P
-
T
1
H
D
Y
-
H
E
E
U
C
0
20
R
0
E
5
40
H
100
P
10
60
-
U A
200
U
(% )
15
80
C
300
E
20
F E
P U A ( P m o l/L )
U r in e G lu c o s e /C r e a tin in e
C
400
H
A
The bars above cohorts in each figure represent significance levels of p<0.05
Figure 2. PUA (A), FEUA (B) and urine glucose/creatinine (C) levels in T1D (n=40) during
euglycemic conditions at baseline and after treatment with the SGLT2 inhibitor
empagliflozin (EMPA).
300
15
U A
200
U r in e G lu c o s e /C r e a tin in e
20
(% )
400
10
F E
100
5
0
0
80
60
40
20
0
A
B
A
B
The bars above cohorts in each figure represent significance levels of p<0.05
1
A
P
E
M
IN
S
E
M
E
L
E
S
E
L
P
A
E
P
M
IN
L
B
A
S
E
IN
A
E
E
P U A ( P m o l/L )
C
B
A
Figure 3. Proposed hypothesis for glycosuria mediated hyperuricemia in T1D patients,
supported by findings in this study and by Chino et al.(10).
SGLT2i: Sodium glucose transporter 2 inhibitor; GLUT9: Glucose transporter 9; URAT1: Urate
Transporter 1; UA: Uric Acid.
Figure 4. Linear regression analysis of PUA with SBP in T1D during euglycemia (A), with
ERPF in T1D during euglycemia (B), with GFR in T1D during hyperglycemia (C).
B
C
400
r= 0 .2 7
300
200
100
0
80
100
120
140
S B P (m m H g )
160
500
r= -0 .2 7
p = 0 .0 2 9
P U A ( µ m o l/L )
P U A ( µ m o l/L )
400
p = 0 .0 3 1
300
200
100
0
P U A ( µ m o l/L )
A
r= -0 .2 7
p = 0 .0 2 8
400
300
200
100
0
0
500
1000
1500
E R P F ( m L /m in /1 .7 3 m 2 )
2000
0
100
200
300
G F R ( m L /m in /1 .7 3 m 2 )
T1D n=40; SBP: systolic blood pressure; ERPF: effective renal plasma flow; GFR: glomerular
filtration rate.
2