Conditions: Diabetes Mellitus, Type 1 Nephropathy Diabetic Nephropathies Juvenile Diabetes Diabetes Mellitus Complication Autoimmune Diabetes Type 1 Diabetes Mellitus
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 50
Healthy volunteers: no

Brief summary:
Over 1.25 million Americans have type 1 diabetes (T1D), increasing risk for early death from cardiorenal disease. The strongest risk factor for cardiovascular disease (CVD) and mortality in T1D is diabetic kidney disease (DKD). Current treatments, such as control of hyperglycemia and hypertension, are beneficial, but only partially protect against DKD.

Hyperfiltration is common in youth with T1D, and predicts progressive DKD. Hyperfiltration is also associated with early changes in intrarenal hemodynamic function, including increased renal plasma flow (RPF) and glomerular pressure. Intrarenal hemodynamic function is strongly influenced by the renin-angiotensin-aldosterone system (RAAS), which is also considered a key player in the pathogenesis of DKD. Preliminary data demonstrate differences in intrarenal hemodynamic function and RAAS activation in early and advanced DKD in T1D. However, the pathophysiology contributing to the differences observed in RAAS activation and intrarenal hemodynamic function in T1D are poorly defined Animal research demonstrates that arginine vasopressin (AVP) acts directly to modify intrarenal hemodynamic function, but also indirectly by activating RAAS. Preliminary data suggest that elevated copeptin, a marker of AVP, which predicts DKD in T1D adults, independently of other risk factors. However, no human studies to date have examined how copeptin relates to intrarenal hemodynamic function in early DKD in T1D. A better understanding of this relationship is critical to inform development of new therapies targeting the AVP system in T1D. Accordingly, in this study, the investigators propose to define the relationship between copeptin and intrarenal hemodynamics in early stages of DKD, by studying copeptin levels, renal plasma flow, and glomerular filtration in youth (n=50) aged 12-21 y with T1D duration \< 10 y.

Detailed description:

Arms:
arm 1: All participants will undergo assessment of Glomerular Filtration Rate, (Iohexol Inj 300 mg/mL) and Effective Renal Plasma Flow (Aminohippurate Sodium Inj 20%). In addition, participants will undergo imaging assessment that includes Dual X-Ray Absorptiometry (DXA), renal Blood Oxygen Level Dependent (BOLD) and Arterial Spin Labeling (ASL) MRI.

Interventions: intervention 1: Aminohippurate Sodium Inj 20% intervention 2: Iohexol Inj 300 mg/mL
Intervention descriptions: intervention 1: Diagnostic aid/agent used to measure effective renal plasma flow (ERPF) intervention 2: Diagnostic aid/agent used to measure glomerular filtration rate (GFR)

Protocol document:
Prot_SAP_000.pdf: 
Copeptin in Adolescent Participants with Type 1 Diabetes and Early 
Renal Hemodynamic Function  
Protocol and Statistical Analysis Plan 
COMIRB: 17-0820 
NCT03618420 
8/5/2021 
 


Protocol 17-0820 
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COMIRB Protocol 
COLORADO MULTIPLE INSTITUTIONAL REVIEW BOARD 
CAMPUS BOX F-490    TELEPHONE:  303-724-1055  Fax:  303-724-0990 
 
 
Protocol #: 
 
17-0820 
 
Project Title:              CASPER Study: Copeptin in Adolescent Participants 
with Type 1 Diabetes and Early Renal Hemodynamic Function 
Principal Investigators:  
Petter Bjornstad, M.D.  
Co-Investigators: 
Kristen J. Nadeau, M.D., M.S., Richard J. Johnson, M.D., Marian J. Rewers, 
M.D., Ph.D., Bruce A. Perkins, M.D., M.P.H. and David Z. Cherney M.D., Ph.D. 
Version Date: 12/14/2018 
I. Hypotheses (H) and Specific Aims (SA):   
Over 1.25 million Americans have type 1 diabetes (T1D), increasing risk for early death from cardiorenal 
disease (1, 2). Despite advances in glycemic and blood pressure control, a child diagnosed with T1D is 
expected to live up to 17 years less than non-diabetic peers (3-6). The strongest risk factor for cardiovascular 
disease (CVD) and mortality in T1D is diabetic kidney disease (DKD) (7, 8). Current treatments, such as 
control of hyperglycemia and hypertension, are beneficial, but only partially protect against DKD. Clinical trials 
in DKD have yielded disappointing results (9-16), 
potentially due to the lack of interventions at early stages of 
disease when the benefit is most likely. In fact, the recently 
published results from the AdDIT trial demonstrated that 
use of angiotensin converting enzyme inhibitor (ACEi) and 
statin failed to change albumin excretion over time in youth 
with T1D (17). Thus, identifying new therapies to impede 
DKD remains a public health priority. 
 
Hyperfiltration is common in youth with T1D (18, 19), and 
predicts progressive DKD. Hyperfiltration is also associated 
with early changes in intrarenal hemodynamic function, including increased effective renal plasma flow (ERPF) 
and glomerular pressure. Figure 1 summarizes the changes in glomerular filtration rate (GFR) and ERPF, that 
we propose underlie the natural history of DKD in T1D. Intrarenal hemodynamic function is strongly influenced 
by the renin–angiotensin–aldosterone system (RAAS) system, which is also considered a key player in the 
pathogenesis of DKD. In particular, angiotensin II (Ang II), shows increased activity during DKD (20, 21). Our 
preliminary data demonstrate differences in intrarenal hemodynamic function and RAAS activation in early and 
advanced DKD in T1D (22,23). The pathophysiology contributing to the differences observed in RAAS 
activation and intrarenal hemodynamic function in T1D are poorly defined. 
 
Animal research demonstrates that arginine vasopressin (AVP) acts directly at the V2 receptor (V2R) to modify 
intrarenal hemodynamic function (24), but also indirectly by activating RAAS (22) (Fig 2). Our preliminary data 
suggest that elevated copeptin, a marker of AVP, predicts DKD in T1D adults, independently 
of other risk factors (25, 26). However, no human studies to date have examined how 
copeptin relates to intrarenal hemodynamic function in early DKD in T1D.  A better 
understanding of this relationship is critical to inform development of new therapies targeting 
the AVP system in T1D. Accordingly, in this application, we propose to define the 
relationship between copeptin and intrarenal hemodynamics in early stages of DKD, by 
studying youth aged 12-21 y with T1D duration < 10 y (Fig. 1). 
SA1: Define the relationship between copeptin and intrarenal hemodynamic function 
in T1D youth. 
H1.1: Copeptin will be higher in youth with T1D and hyperfiltration vs. those without 
hyperfiltration. 
H1.2: Copeptin will positively correlate with GFR, ERPF and intrarenal hemodynamics in short-standing T1D. 
 
SA2: To understand the effects of copeptin on renal oxygenation and perfusion in T1D youth.  
H2.1: Copeptin will correlate positively with renal oxygenation consumption and perfusion in T1D youth.  


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II. Background and Significance: Prior bench and translational research into the mechanisms of 
hyperglycemic injury and its modifiers (27) has to date not successfully 
translated into adjuvant therapeutics to supplement intensive insulin therapy in 
the prevention of DKD. Accordingly, there is an urgent need for novel 
modifiable risk factors. AVP has diverse actions in physiology which include 
direct effects on vascular hemodynamics, RAAS, volume, and osmolality 
regulation (28, 29). The actions of AVP are mediated by at least three distinct 
receptor subtypes: V1a, V1b, and V2. Experimental data strongly support a 
causal and direct role of AVP in the pathogenesis of kidney disease through 
V2R activation (30, 31) (Fig 3). In laboratory animals, elevated AVP directly 
leads to afferent arteriolar vasodilation by V2R stimulation (24) and indirectly to 
efferent arteriolar constriction by RAAS activation (28, 29). The net effects are 
increased ERPF with glomerular hypertension and hyperfiltration (Fig 3), in 
addition to albuminuria and tubulopathy (32-34). Supraphysiologic increases in 
ERPF, glomerular pressure and GFR are hallmarks of hyperfiltration (35), an 
early phenotype of DKD which predicts rapid GFR decline and impaired GFR in 
adults with T1D (36). Measuring AVP is cumbersome due to its small size and 
short half-life. Copeptin, a more stable peptide derived from the same precursor 
molecule as AVP, is thus recently recognized as a better method to determine 
AVP activity and hence will be used in this proposal  (33). 
 
Scientific Premise: The relationships we and others have demonstrated between copeptin, DKD in T1D are 
based on epidemiologic data and limited to adults with advanced DKD (25, 26, 37). These studies have 
established that copeptin is significantly higher in adults with T1D, and that elevated levels of copeptin are 
associated with cardiorenal disease. Yet no human studies have examined the mechanisms whereby elevated 
AVP activity contributes to DKD, nor whether this relationship is different in early DKD. Intrarenal 
hemodynamic function is compromised in both early and advanced DKD (35, 38-40), and recent studies have 
also provided compelling evidence that intrarenal hemodynamic dysfunction can be partially restored with 
therapeutic agents in T1D (41-51). However, no data exist on the interaction between copeptin, intrarenal 
hemodynamic function in early DKD in T1D. Until this gap is addressed, our understanding of AVP as a risk 
factor and therapeutic target in DKD is limited. 
 
Our overall hypothesis is that elevated copeptin in T1D is associated with distinct changes in intrarenal 
hemodynamic function in early vs. advanced DKD, which includes increased ERPF and GFR in early DKD 
(e.g. hyperfiltration) and reduced ERPF and GFR in established DKD (e.g. impaired GFR). To test these 
hypotheses, we will carry out a cross-sectional study in youth with short-standing T1D as well as take 
advantage of unique mechanistic data already collected in adults with longstanding T1D, with and without 
advanced DKD (DKD and DKD Resistors) from the JDRF-funded Canadian Study of Longevity in Diabetes 
(Longevity).  
 
Impact: This proposal seeks to investigate the roles of AVP on intrarenal hemodynamic function in youth and 
adults with early and advanced DKD. By understanding the interplay between AVP and intrarenal 
hemodynamic function in T1D, we will provide a foundation for future mechanistic studies and clinical trials 
targeting the AVP system. Renal physiology studies are required to advance DKD research and direct the 
development of new therapies to improve cardiorenal health and mortality for the growing number of people 
afflicted by T1D (52). 
 
Our proposal is innovative because: 
1. We move beyond the traditional determinants of DKD (ABC risk factors; HbA1c, Blood pressure and LDL-
Cholesterol), to focus on elevated AVP activity. 
2. We can validate our epidemiologic findings in CACTI and the T1DX with mechanistic renal physiology data. 
3. Hyperfiltration is understudied. It is important to examine early changes in GFR, because albuminuria does 
not reliably reflect progressive DKD and commonly reverts to normoalbuminuria (53). Hyperfiltration is now 
recognized to be a common phenotype of early DKD in young adults with T1D (18, 19). Hyperfiltration 


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predisposes to progressive nephron injury by increasing glomerular pressure (54). We have shown that 
hyperfiltration precedes albuminuria and predicts rapid GFR decline and chronic DKD in adults (36). 
4. We propose innovative methods by a highly skilled, multi-disciplinary team to define intrarenal hemodynamic 
function, including gold standard measurements of GFR by iohexol clearance, ERPF by para-aminohippurate 
(PAH) clearance and calculations of intrarenal hemodynamic parameters by Gomez’ equations (55).  
5. There is the potential to translate the findings of these studies to a rational intervention strategy in T1D using 
selective V2R blockers (e.g. vaptans), which are FDA approved in adults with clinical hyponatremia. 
 
 
III. Preliminary Studies/Progress Report:   
1. Copeptin concentrations are greater in adults with T1D compared to non-diabetic controls: Copeptin was 
greater in participants with T1D compared to those without (geometric mean, 95% CI: 3.5 [2.3-3.8] vs. 2.8 [ 
2.7-3.1] pmol/L, p=0.003), and the difference remained significant after adjusting for age, sex and eGFR 
(p=0.0008) (26). 
 
2. Men with T1D and albuminuria have 3-fold higher copeptin levels than 
men with T1D and normoalbuminuria: In a case-control study from the 
T1DX Biobank registry, men with T1D and albuminuria (T1D DKD) had 
greater copeptin levels than men with normoalbuminuria (T1D N). The 
difference between T1D DKD and T1D N remained significant after 
adjusting for HbA1c, eGFR and ACEi / angiotensin II receptor blockers 
(ARB) use (Fig 4). Higher copeptin levels conferred greater odds of 
impaired GFR, independent of other important risk factors. In a 
generalized linear mixed model, one standard deviation (SD)  increase in 
copeptin predicted greater odds of impaired GFR (OR: 2.94, 95% CI 
1.39-6.21), and remained significant after multivariable adjustments 
(T1D duration, ACEi/ARB use, HbA1c and case/control status) (25). 
 
3. Copeptin is associated with cardio-renal disease in adults with T1D: 
In adults with T1D in in the CACTI study, elevated copeptin (>13pmol/L 
[>97.5th percentile for healthy adults]) was associated with greater odds 
of impaired eGFR (OR: 18.52, 95% CI 4.03-85.02), albuminuria (OR: 
10.55, 95% CI 2.24-49.62), and CAC scores ≥300 (OR: 6.24, 1.51-
25.90) adjusting for age, sex, HbA1c, systolic BP and LDL-C (26).  
 
4. Stimulation of the V2R with desmopressin is associated with 
glomerular and tubular injury: We showed that the administration of 
desmopressin (des), a V2 agonist, to mice resulted in glomerular and 
tubular damage. Urine albumin concentration was greater in mice 
exposed to des (Fig 5). Urinary NGAL excretion was also highest in 
groups receiving des (30). We next stained tissues for angiotensin 
converting enzyme (ACE) as a marker of the proximal tubular injury. A 
progressive loss of ACE was associated with the addition of des (Fig 6). 
These data support glomerular and tubular injury in response to V2R 
stimulation (30). 
 
5. Hyperfiltration is common in youth and adults with T1D, and predicts 
rapid GFR decline and impaired GFR: We demonstrated that the 
prevalence of hyperfiltration in youth with T1D can exceed 50% when 
GFR is measured by inulin clearance (56), and between 13-31 % when GFR is estimated by serum creatinine 
and serum cystatin C (19, 57). The discrepancy in prevalence is likely attributed to the inaccuracy of estimated 
GFR in the normal to elevated GFR range (44), hence we propose to measure GFR in this study. In adults with 
T1D (n=646) in CACTI, we also demonstrated that hyperfiltration predicted greater odds of rapid GFR decline 
over 6-years (OR: 5.00, p<0.0001), when adjusting for traditional risk factors including HbA1c, SBP, LDL-C, 
Fig 5. V2R Stimulation - Glomerulopathy  
Fig 6. V2 stimulation - Tubulopathy  


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sex, duration and albumin-to-creatinine ratio (ACR)  (58). Furthermore, over 12-year follow-up in CACTI, adults 
with T1D who experienced rapid GFR decline (annual GFR loss > 3mL/min/1.73m2) were more likely to 
demonstrate hyperfiltration at baseline (OR: 32.9, p<0.0001) and impaired GFR at 12-year follow-up (OR: 11.6, 
p=0.0003) (unpublished data). In a parallel analysis we conducted in CACTI and Pittsburgh Epidemiology of 
Diabetes Complications Study (EDC), elevated GFR at baseline was associated with greater odds of rapid 
GFR decline over 6 and 8-years respectively (CACTI: OR 2.62, p<0.0001, EDC: OR 2.16, p=0.0003) (59).  
 
6. Unique intrarenal hemodynamic dysfunction in early vs. established DKD:   
a. Early DKD in young adults: We evaluated 
intrarenal hemodynamic function in healthy 
controls (n=30, mean age 27) and in 
normotensive, normoalbuminuric participants with 
T1D and either normofiltration (n=36, mean age 
24, GFR 90–134 ml/min/1.73m2) or hyperfiltration 
(n=32, mean age 23, GFR≥135 ml/min/1.73m2) 
during euglycemic and hyperglycemic conditions. 
We used Gomez’s equations to derive renal blood 
flow (RBF), efferent (RE) and afferent (RA) 
arteriolar resistances, glomerular hydrostatic 
pressure (PGLO) from inulin (GFR) and PAH 
(ERPF) clearances, plasma protein and estimated 
ultrafiltration coefficients (KFG). Participants with 
T1D and hyperfiltration (T1D-H), had significantly 
greater GFR, ERPF, RBF, and lower RA and 
higher PGLO compared to healthy controls (HC) (60) 
(Fig 7). These observations illustrate the potential 
of applying Gomez’ equations in DKD research. 
 
In summary, our preliminary epidemiologic data 
suggest that: 1) copeptin is higher in adults with T1D compared to their non-diabetic peers; 2) elevated 
copeptin is associated with DKD in the form of albuminuria and impaired GFR, as well as with coronary artery 
calcification (CAC) in adults with T1D; 3) men with T1D and albuminuria have 3-fold greater copeptin levels 
than men with T1D with normoalbuminuria; 4) stimulation of V2R with desmopressin (vasopressin agonist) 
induces tubular and glomerular injuries in murine models; 5) hyperfiltration is common in youth and adults with 
T1D and predicts rapid GFR decline and impaired GFR; 6) there are unique intrarenal hemodynamic 
dysfunction profiles in early DKD. We also present experimental data showing unique intrarenal hemodynamic 
profiles in early and advanced DKD in people with T1D, arguing for dedicated studies at different time points in 
the natural history of DKD. To understand the pathogenesis whereby elevated AVP activity contributes to DKD, 
we need carefully designed translational studies, like the one proposed in this application, to define the 
interplay between AVP activity and intrarenal hemodynamic dysfunction in early DKD. The proposed study is 
feasible in the current environment: 1) the Barbara Davis Center for 
Diabetes (BDC) and the Anschutz Medical Campus (AMC) provide 
ample study participants for a study in youth with short-standing 
T1D; 2) the experience and expertise of the investigator and 
collaborator team will ensure the success of the planned research.  
 
IV. Research Methods: 
Study design and study population: We propose to address SA1 
and SA2 with a cross-sectional observational study with 50 youth 
with short-standing T1D (50% female, 12-21 yr, <10 yr duration). 
The participants will be recruited from the BDC Clinic (>4000 
pediatric patients). Following a screening visit, eligible participants 
(Table 1) will be asked to refrain from strenuous physical activity for 
3 days prior to admission due to the impact of exercise on 
Table 1: Eligibility Criteria 
Inclusion criteria 
Exclusion criteria 
Antibody+ T1D with 
<10 yr duration 
Severe illness, recent 
DKA  
Age 12-21 years 
 
ACEi, ARB, diuretics, 
SGLT 2/1 blockers, 
atypical antipsychotics, 
steroids  
BMI ≥ 5%ile 
Macroalbuminuria 
(>300mg/g) or eGFR 
<60ml/min/1.73m2 
Weight<300 lbs. 
Pregnancy, nursing 
HbA1c <12% 
Anemia or allergy to 
shellfish or iodine 
BP < 140/90 mm Hg 
Fig 7. Intrarenal 
hemodynamics estimated by 
Gomez’s equations in normal 
healthy controls (HC), and 
patients with T1D, renal 
normofiltration (T1D-N) and 
hyperfiltration (T1D-H) 
according to sex. *P<0.001 
vs. HC, §P<0.001 vs T1D-N 


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intrarenal hemodynamic 
function. They will also be 
provided with a 3-day diet 
instructions (45% 
carbohydrates, 30% fat, 25% 
protein and a goal of 3.45 g 
salt/day), with females studied 
in the follicular phase of the 
menstrual cycle where 
possible to limit impacts of 
nutrient and menstrual 
hormone variations on IR and 
renal function, as previously 
described (61). Participants 
will ask to administer their long 
acting insulin (e.g. Lantus or Levemir) like they normally would. Patients on insulin pumps can remain on their 
basal rate throughout the study. If on a hybrid-closed loop (artificial pancreas), participants will be asked to 
open their loop for the duration of the study. The morning of the study, participants will be asked to check their 
blood sugar prior to arriving at the CTRC. If their blood sugar is >= 250 mg/dl, they will be asked to administer 
a bolus of short acting insulin using their home correction factor with a blood sugar target of 200 mg/dl. 
Participants will arrive the evening before, or present fasting in the 
morning and have 2 IVs placed and have their blood glucose 
concentrations clamped in the mild hyperglycemic range (details 
below) (Fig 8). Baseline blood will be drawn for serum copeptin, 
lipids, insulin, cystatin c, creatinine, sodium, hematocrit and total 
protein, and iohexol and PAH boluses given via IV (see below for 
details). GFR and ERPF will be measured by iohexol and PAH 
clearance in plasma. A 4 hr urine sample will be collected to calculate 
ACR and AER.  
 
Specific methods: Outcomes are summarized in Table 2. Detailed 
methods are listed below: 
 
Hyperglycemic insulin clamp: One IV will be placed in each arm to 
clamp the blood glucose concentrations between 170-190 mg/dL. One IV will be used to administer the 
boluses of iohexol and PAH in addition to a variable infusion of 20% dextrose and insulin, and the other IV will 
be used for blood draws. Blood glucoses will be measured every 10 minutes at the bedside, and glycemic 
control will be achieved using the modified and simplified insulin clamp methodology to remove glycemic 
effects on GFR and ERPF. The rate of insulin is adjusted based on blood specimens drawn every 10 minutes 
(example algorithm). Patient blood glucose (BG) levels will be checked at 8am, and adjusted to reach and 
maintain mild hyperglycemia up until 10am. If participant BG <70mg/dl, we will give 4 oz juice and re-test BG in 
15 min. If still <70mg/dl, another 4 oz juice will be given, and BG will be re-tested 15 min later. If still <70 mg/dl, 
study MD will be called, and IV D20 will be started at 100ml/hr. For BG 
between 70-180 mg/dL, a variable D20 infusion will be given on a sliding 
scale based on participant current BG and weight, as used in previous and 
current studies (COMIRB #13-0122, #16-1752, #18-0704). Once BG > 
190mg/dl, D20 will be discontinued. If BG is not responding to changes 
within 2 hours or if increases above 200 mg/dl, a study physician will be 
contacted. Each time blood is drawn during the clamp, blood will be drawn 
to a clear line in the syringe to ensure there will be no IV fluid dilution and 
will be re-infused into the subjects after obtaining the blood sample to 
minimize blood loss. A blood glucose range of 170-190mg/dL (mild 
hyperglycemia) was chosen to limit the acute effects of severe hyperglycemia on GFR and ERPF 
reproducibility, while allowing us to assess renal function in a glycemic milieu similar to the participants native 
Table 2 – Aims 1 and 2 
Primary Outcomes    Measurement Tool 
Copeptin 
ThermoFisher Kryptor Plus 
GFR  
Iohexol clearance 
ERPF 
PAH clearance 
Renal O2 
BOLD-MRI 
Renal perf. 
ASL-MRI 
Secondary Outcomes 
RVR 
MAP/RBF (Table 3) 
eGFR 
Creatinine, cystatin C 
Albumin 
excretion 
Albumin-to-creatinine 
ratio (ACR) 
Figure 8 Study Protocol for SA 1 
        
 
Insulin Algorithm 
BG 
Insulin dose 
<180 
-- 
181-190 
0.01   u/kg/hr 
191 - 200 
0.015 u/kg/hr  
201 - 250 
0.03   u/kg/hr  
251 - 300 
0.045 u/kg/hr  
>301 
0.06   u/kg/hr  


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pathophysiology. Furthermore, the clamp maintains an easily 
achieved and safe range of glycemia. Co-I Dr. Nadeau has 
extensive experience with normoglycemic, hyperinsulinemic, 
hyperglycemic and hypoglycemic clamps in T1D youth, which 
are well tolerated in the proposed ages. 
 
Iohexol clearance (GFR) and PAH clearance (ERPF): A bolus of iohexol (5mL of 300 mg/ml iohexol 
[Omnipaque 300, GE Healthcare]) will be given slowly over 2 min followed by a 10 ml normal saline flush, 
which will be time 0 for the iohexol clearance. 120 min will be allowed for equilibration of iohexol per or local 
protocol (62, 63). Blood for iohexol clearance will be drawn at +120, +150, +180, +210 and +240 min  (62, 63). 
PAH (weight/75 x 4.2 ml) [University of Minnesota] will be given slowly over 5 minutes followed by a 10 ml 
normal saline flush. An infusion mixture of 8 mL of PAH and 42 mL of normal saline will be prepared in a 60mL 
syringe and infused in Syringe Pump at a rate of 24mL/hr, which will be time 0 of the PAH clearance. Ninety 
min will be allowed for equilibration of PAH per co-investigator Dr. Cherney’s protocol (62, 63). Blood for PAH 
clearance will be drawn at +90 and +120 min (33-36) (Fig 9). We 
will use Gomez’ equations to calculate parameters of intrarenal 
hemodynamics (56, 64) (Table 2, pg. 5). Hyperfiltration by 
measured GFR (iohexol) will be defined as ≥135ml/min/1.73m2 
(23, 65-67).  
 
The iohexol and PAH infusion period lasts 240 minutes and will 
occur concurrently with the mildly hyperglycemic clamp during 
which time the subject will rest in bed in the pediatric CTRC. 
During the entire 240-minute glucose/insulin infusion period, a 
pediatric CTRC nurse will remain at the bedside, and a Study 
Physician or Pediatric Nurse Practitioner or Pediatric Physician 
Assistant will also remain at the bedside to minimize any risks 
associated with iohexol and PAH infusion. The IV site will be 
continuously monitored to minimize risk of IV infiltration.   
 
De-identified coded blood samples will be sent to collaborator Dr. 
Robert G. Nelson’s lab at NIH/NIDDK who will run iohexol and 
PAH clearance on a HPLC platform.  
 
RVR and Gomez equations (Table 3):  RVR will be calculated as 
RVR = MAP/RBF. To derive additional information about intrarenal 
hemodynamic function we will use mathematical equations by Gomez et al (55). By using measurements of 
GFR, RBF, ERPF, renal vascular resistance, hematocrit and serum protein, we can calculate afferent and 
efferent arteriolar resistance (RA and RE), glomerular pressure (PGLO) and filtration pressure (Table 3). 
 
Albumin excretion: ACR and AER be determined from 4 hr. urine collection.  
 
Copeptin: Copeptin will be measured by ultrasensitive assays on KRYPTOR Compact Plus analyzers using the 
commercial sandwich immunoluminometric assays (Thermo Fisher Scientific, Waltham, MA). The copeptin 
assay has a lower limit of detection of 0.9 pmol/L, and a sensitivity of <2pmol/L. Elevated copeptin will be 
defined as >13pmol/L, which is >97.5th percentile for healthy adults (68). 
 
Estimated GFR: As an exploratory aim, we will evaluate the agreement between measured GFR (iohexol) with 
estimated GFR by serum creatinine and cystatin C. Due to the expected normal to elevated GFR for youth with 
T1D, we will calculate eGFR by Zappitelli equation (eGFR = 25.38 * (1/serum cystatin C)0.331 * (1/serum 
creatinine)0.602 * (1.88 * height)) (69-71).  
 
Table 3 Measures 
Definition: 
Filtration fraction 
(FF) 
FF = GFR / RPF 
(renal plasma flow) 
Renal blood flow 
(RBF) 
RBF = RPF (1-Hct) 
Renal vascular 
resistance (RVR) 
RVR = MAP/RBF 
Filtration pressure 
∆PF 
∆PF = GFR/KFG, KFG: 
filtration coefficient 
Mean plasma 
protein (CM) 
CM = total protein / 
FF x natural log 
(1/1-FF) 
Glomerular oncotic 
pressure (πG) 
πG = 5 x (CM – 2) 
Glomerular pressure 
(PGLO) 
PGLO = ∆PF + PBOW + 
πG, PBOW is 10 mmHg 
Afferent arteriolar 
resistance (RA) 
RA = [(MAP-
PGLO)/RBF] x 1328  
Efferent arteriolar 
resistance (RE) 
RE = [(GFR/KFG x 
(RBF-GFR))] x 1328 
9 am 
4 hr iohexol GFR 
1 pm 
hyperglycemic clamp 
2 hr PAH RPF 
Fig 9 


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Blood pressure: After participants have been laying supine for a minimum of 5 minutes, blood pressure 
measurements will be obtained using an automatic blood pressure machine and 3 measurements will be 
averaged. MAP will be calculated from the blood pressure readings. 
 
Metabolomics: We will measure serum metabolomics by Q Exactive UHPLC-MS Analysis at the Metabolomics 
Core Facility Services at University of Colorado (Dr. Angelo D’Alessandro, PhD).  
 
Urine and serum osmolality:  We will directly measure urine and serum osmolality using freezing point method 
with Micro-Osomometer Model 3300 (Advanced Instruments, Massachusetts, USA) at Johnson’s Lab.  
 
Fractional excretion of sodium (FeNa): We will measure serum and urine sodium and creatinine to calculate 
fractional excretion of sodium.  
 
DXA: Participants will also undergo DXA by standard methods on a Hologic device (Waltham, MA) to 
determine lean and fat mass (72). 
 
Renal oxygenation and renal perfusion by MRI: The primary outcomes of Aim 2 are renal oxygenation and 
perfusion (Table 2). We will perform BOLD MRI (73) to quantify renal oxygenation, whereas renal perfusion will 
be measured by ASL (74)(Table 2). These studies will be performed at the UCD Research Imaging Center (3T 
Siemens Skyra scanner) which already has an active renal BOLD-MRI protocol as part of the multi-center 
COMBINE (CKD Optimal Management with BInders and NicotinamidE) study (Table 4) (75), for which our 
collaborator Dr. Prasad directs the CORE for trouble-shooting and analyzing the renal MRIs. Dr. Prasad is an 
international leader in advanced MR research and will provide the techniques for BOLD and ASL MRI 
acquisition and analyses. 
 
Table 4. MRI protocol_3T Skyra  
 
 
Series  
Localizer 
BOLD 
ASL 
Plane  
2 Plane  
Coronal  
Axial  
Coronal  
Coronal 
Pulse sequence  
gre  
gre  
gre  
gre  
Custom 
TR/ms  
7  
7  
7  
61  
3000 
TE/ms  
2.66  
2.66  
2.66  
4.92, 9.84, 14.76, 
19.68, 24.6, 29.52, 
34.44, 39.36  
1.92 
Read out mode  
n/a  
n/a  
n/a  
Bipolar  
n/a 
In line mapping  
n/a  
n/a  
n/a  
T2* (VB 17)/R2* (VD 
13) Map  
n/a 
BW Hz/pixel  
290  
290  
290  
260  
501 
FOV mm  
400  
400  
400  
400  
400 
Slices  
3 ax, 3 cor  
5  
5  
5  
1 
Concatenation  
6  
5  
5  
5  
1 
Slice Thickness / 
mm  
8  
8  
8  
5  
8 
Skip  
50%  
50%  
50%  
0  
n/a 
Fat suppressed  
No  
No  
No  
Fat Sat  
n/a 
Parallel Imaging 
acceleration 
factor  
None  
None  
None  
None  
None 
Diffusion mode  
n/a  
n/a  
n/a  
n/a  
n/a 
B value s/mm2  
n/a  
n/a  
n/a  
n/a  
n/a 
Diff. gradients  
n/a  
n/a  
n/a  
n/a  
n/a 
NEX  
2  
2  
2  
1  
50 
Acq-matrix  
154x256  
154x256  
154x256  
192x256 
128x128 
Flip 
angle/degrees  
20  
20  
20  
30  
60 
Typ. Scan time 
m:s  
0:14  
0:07  
0:07  
0:30-1:00 (5 slices)  
0:05 


Protocol 17-0820 
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Options/comme
nts  
Site preference  
Instruct subjects to 
not to take deep 
breaths.  
Changing FOV and/or 
matrix size not preferred  
Instruct subjects to not 
to take deep breaths. 
Abbreviations: BOLD: Blood oxygen level dependent, SSFP: steady state free precession, VLA: vertical long axis view, 
LVOT: left-ventricular outflow tract view, GRE: gradient recalled echo. 
 
Recruitment: Ultimately 50 youth with T1D (50% male) will be enrolled (see power analysis). Recruitment will 
begin year 1 and will consist of adolescent patients with type 1 diabetes from the Barbara Davis Center for 
Childhood Diabetes (BDC), where principal investigator Dr. Bjornstad and co-investigator Drs. Nadeau and 
Rewers are medical providers and have weekly clinics. The investigator team has excelled when recruiting 
adolescent T1D patients. There are almost 4000 pediatric patients actively followed at BDC, and we do not 
foresee any difficulties with recruitment.  
 
Detailed Study Visit Descriptions: The study timeline for is illustrated in Fig 10, and details from each study 
visit is summarized below. Visit 1 is the screening visit and visit 2 and 3 will occur consecutively. 
 
 
 
 
 
 
 
 
 
 
Visit 1 (Screening visit): Prescreening will be done by phone or in conjunction with a clinical diabetes visit. 
The full screening visit will include the consent process, history, allergies confirmed, physical exam, and 
screening labs (HbA1c, hemoglobin and hematocrit [H&H], serum creatinine, urine microalbumin to creatinine 
ratio). All females will also have a pregnancy test and detailed menstrual history. The screening visit will take 
approximately 2 hours.  
 
Visit 2, part 1: Participants will present the evening before the study visit, or present fasting to the Pediatric 
CTRC in the morning, and have IV(s) placed.  Fasting blood will be drawn for serum copeptin, lipids, insulin, 
cystatin c, creatinine, sodium, hematocrit and total protein prior to the clamp. Participants will be escorted to 
the Brain Imaging Center or CHC Radiology for a BOLD and ASL MRI per local protocol. BOLD MRI 
measurements will be performed at baseline and following administration of a small dose of furosemide (20 mg 
IV) via the PIV. A study physician or nurse will be responsible for the furosemide administration. Total duration 
of part 1 will be approximately 1.5 hours. 
 
Visit 2, part 2: Participants return to pediatric CTRC and will have their blood glucose concentrations clamped 
in the hyperglycemic range. Participants will also undergo iohexol and PAH clearance studies, in addition to 4-
hour urine collection for album excretion rate (AER) and albumin to creatinine ratio (ACR). Upon completion of 
visit 2, participants will be provided with a study meal (lunch). After lunch, participants will undergo DXA scan 
for quantification of body composition. This concludes the study. Total duration 6 hours. 
 
 
 
 
 
 
 
 
 
 
Fig 10 Study Design 


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A. 
Outcome Measure(s):  
 
Aim 1: Measured GFR by iohexol clearance and RPF by PAH clearance 
 
Aim 2: Renal oxygenation by BOLD-MRI and renal perfusion by ASL-MRI 
B. 
Description of Population to be Enrolled:   
* Shortly after arrival to radiology unit, a MRI technician will meet with patient and/or guardian to provide 
preparation for the scan. This conversation will include a thorough overview including what the scanner looks 
like, a sample of the MRI sounds, the scan set up and the importance of lying still for the scan. The metal 
prescreening precaution procedure will be conducted for both patient and guardian.  
 
D. Description, Risks and Justification of Procedures and Data Collection Tools: 
 
1. Study Duration: The anticipated duration for the study is 2 years  
 
2. Duration of Participation for Each Subject: Subject participation consists of 2 visits (~ 11 hours) 
 
3. Sources of Research Material: The patient’s medical record will be reviewed for diabetes diagnosis, 
medications, allergies and other diagnoses that may disqualify patient from participation. 
Table 6: Data to be collected during study 
Blood and Urine Samples  
Blood pressures and HR 
Questionnaires 
Hyperglycemic clamps 
DXA 
Iohexol clearance 
Anthropometric measurements 
PAH clearance 
ASL and BOLD MRI 
Metabolomics 
 
4. Informed Consent Plan: Appropriately qualified and informed personnel who have completed the 
COMIRB and HIPPA course requirements will fully explain the study protocol and consent form to the 
subject and guardian verbally in the language they understand.  The explanation will be conducted in a 
quiet environment with adequate time given for the subject and guardian to review the study procedure 
before the commencement of the study.  Asking the subject to explain the study in their own words will 
assess the subject’s understanding.  If non-English speaking subjects are enrolled in the study, the 
investigators will adhere to Section 10C of the COMIRB Instructions for Clinical Investigators regarding the 
consent of these subjects.  The qualified personnel mentioned above will then obtain written consent from 
Table 1: Eligibility Criteria 
Inclusion criteria 
Exclusion criteria 
Antibody+ T1D with <10 yr duration 
Severe illness, recent DKA 
Age 12-21 years 
eGFR <60ml/min/1.73m2 or creatinine > 1.5mg/dl or history of 
ACR≥300mg/g 
BMI ≥ 5%ile 
ACE inhibitors, angiotensin receptor blockers (ARB), diuretics, 
sodium-glucose co-transport (SGLT) 2 or 1 blockers, daily 
NSAIDs or aspirin, sulfonamides, procaine, thiazolesulfone or 
probenecid, atypical antipsychotics and steroids 
Weight<350 lbs and > 57 lbs. 
No anemia 
Anemia or allergy to shellfish or iodine 
HbA1c <12% 
Pregnancy or nursing 
MRI scanning contraindications (claustrophobia, implantable 
devices, >350 lbs) 


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the guardian and assent from the subject, co-signed on the consent form, or in subjects who are 18 years 
or older, direct consent. The subject and guardian will be provided a copy of the consent form for better 
understanding and record purposes.   
 
5. Special Consent/Assent Plan: Consent will be obtained from all participants in the study.  Following 
explanation, all subjects below 18 years old will co-sign the consent form in addition to the parents signing 
the consent form.  All subjects age 18 or older will sign the standard consent form. 
 
6. Participant Compensation: Subjects will be paid $350 upon completion of all procedures in this study 
(Table 7 below). If the subject withdraws before the start of visit 2 (and all successive visit parts), they will 
still receive a $25 gift card for completion of the screening visit. Subjects will be paid for all subsequent 
completed visit parts as outlined below. These payments are similar to the payments being made for each 
visit type in our currently ongoing adolescent studies. 
       Table 7 Subject payment schedule 
 
 
 
 
 
7. Potential Risks to Subjects:  
Blood Samples: The collection of blood samples may result in temporary discomfort, bruising, bleeding, 
and on rare occasions, infection. EMLA cream helps to prevent discomfort, and sterile technique helps to 
minimize these risks. Nationally, the NIH Clinical Center has a guideline of 9mL/kg in 6-8 weeks for 
pediatric studies. Certain studies at our institution do draws over 7mL/kg in 6 weeks, or up to 7 mL/kg in a 
single draw but include iron supplementation. The screening visit (visit 1) will include 5 ml or less of blood 
(1 tsp) (HbA1c, H/E). The clamp and renal infusion studies (visit 2) includes 66mL of blood, which includes 
48 mL from the clamp and 18 mL from the iohexol and PAH clearance studies. Thus, the clamp and renal 
infusion studies are within the more conservative pediatric CTRC guidelines of 2.5mL/kg for a single draw 
for pediatric studies for subjects 26 kg or greater. This is highly unlikely to exclude many subjects, as we 
are recruiting adolescents between ages 12 and 18 and excluding, and subjects with BMI<5% (due to 
potential undiagnosed illness), those groups likely to be the lightest in weight. Therefore, we will limit 
recruitment to subjects 26 kg or greater. In addition, by study design, subjects <5% for weight are excluded 
as are subjects with anemia, screened by our baseline H/H, further increasing the safety of the study 
regarding blood draws.  
 
IV risks: There is temporary discomfort when the needle goes in and 10% of the time there is a small 
amount of bleeding under the skin that may produce a bruise. Rarely, there is a risk of a blood clot 
formation, infection or infiltration. EMLA cream helps to prevent discomfort and sterile technique by 
experienced pediatric nurses help to minimize these risks.  
 
Hyperglycemic Clamp: To minimize the risk of any complications with the clamp, the clamp is only 
performed in the inpatient pediatric Clinical Translational Research Center (CTRC), by experienced 
personnel. To minimize this risk further, IV access is obtained prior to glucose and insulin administration, 
and blood sugars are monitored approximately every 10 minutes throughout the procedure and maintained 
at 190 mg/dL. Two IV’s will be in place, in case there is a problem with one IV. If the blood glucose level is 
decreasing, dextrose will be given to stabilize and increase blood glucose levels. IV access will also be left 
in place until blood glucose values are stable after the study is completed. During the clamp, as with any IV 
fluids, there is also a risk of infiltration of the IV solution, which could lead to skin burn or tissue damage. 
During the entire clamp procedure, a pediatric CTRC nurse will remain at the bedside, and a Pediatric 
Visit 1 
Visit 2, Part 1Part 1 
Visit 2, Part 2Part 2 
Total 
$25.00 
$175.00 
$150.00 
$350.00 


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Study Physician or Pediatric Nurse Practitioner will also remain at the bedside. The IV site will be 
continuously monitored, and the IV integrity will be noted on a check sheet. 
o Glucose injection and infusion:  Irritation of a vein resulting in phlebitis can occur with administration of 
concentrated glucose solutions. The risk will be decreased by minimizing the use of concentrated 
solutions, monitoring the veins closely during the infusion and selecting large veins for infusion of 
solutions. 
 
Iohexol infusion: Iohexol (Omnipaque 300, GE Healthcare, Chicago, IL) is a nonionic, low osmolar contrast 
agent with a history of extensive use in Radiology practice (for contrast x-rays) that shares many qualities 
of an ideal GFR marker, like inulin, such as not being absorbed, metabolized, or secreted by the kidney. 
Low toxicity is reported in Radiology practice in children and adults in which doses that are 10-50 times 
higher than for GFR determination are used. In fact, for over 20 years, publications have argued that 
iohexol is a gold-standard measure of GFR. In contrast to some agents used to measure kidney function it 
is not radioactive (e.g. iothalamate renography at CHCO). The dose to be used in this study is 10-50 times 
less than that used in radiology studies. Our protocol has been adapted from that used by the RASS study 
where they reported only minor allergic reactions in over 1,200 iohexol studies performed in over 200 
adults with type 1 diabetes in that study. Principal investigator, Dr. Bjornstad, has performed iohexol 
studies in adolescents with type 1 diabetes at Barbara Davis Center for Diabetes (62) which was well 
tolerated without any reactions. Furthermore, collaborator Dr. Cherney has at least 10-year experience with 
iohexol studies in both pediatrics and adults with and without type 1 diabetes. To further limit risk of allergic 
reactions, patients with an allergy to iodine, seafood or iohexol will not be allowed to participate. Rare 
adverse effects associated with iohexol in radiology studies include cardiac arrhythmias (an irregular 
heartbeat), headaches, blurred vision, and nausea. To reduce risk of contamination, single use vials will be 
used. 
 
PAH infusion: PAH (University of Minnesota) has been used to measure RPF in human research for 
decades and is very well tolerated and generally recognized as safe with low toxicity. Collaborator, Dr. 
Cherney has infused PAH in children and adults with and without T1D for the last 10 years without any 
significant reactions (38, 55, 60, 62, 64, 79-81). PAH was previously approved by the FDA as the gold 
standard to quantify RPF. As the prior manufacturer, Merck, no longer supplies PAH, Dr. Bjornstad has 
submitted an IND application to use PAH produced by University of Minnesota in this study as an 
investigational product (IND #140129). To minimize risk, a sterile preparation is used and prepared by the 
pharmacy using sterile techniques. Furthermore, single use vials will be used to reduce risk of 
contamination. However, as with any infusion, there is the possibility of infection, and the possibility of an 
allergic reaction. In the event of an allergic reaction, the study will be discontinued, and the participant will 
be treated.   
 
Study Diet: There is a small risk that blood sugars will be higher or lower than normal in subjects with 
diabetes at home while eating the recommended study diet as it may differ from the subject’s usual diet. To 
avoid this risk, subjects will be asked to monitor their blood sugars frequently at home and will be called 
daily to review blood sugars. Carbohydrate content will also be provided for each food item to assist with 
carbohydrate counting. 
 
DXA Scan: There is no pain with this procedure. For females, a urine pregnancy test will be checked prior 
to the test to avoid any x-ray exposure to a pregnant female. This procedure involves the use of X-ray 
radiography. The amount of radiation exposure during the DXA test is approximately 15 mSv which is 7 
times the level of background radiation in Colorado or approximately equal to the amount of radiation a 
subject would receive being outdoors in Denver for one day.  
 
Metabolomics: Metabolomics is run on serum. The samples will be obtained off the IV so this assumes the 
same risk as the collection of blood samples: may result in temporary discomfort, bruising, bleeding and on 
rare occasions, infection.  
 


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Magnetic Resonance Imaging (MRI): The MRI is a non-invasive scan of the renal arteries. MRI uses a 
magnet and there is no radiation involved with the MRI. The scan may be loud; therefore, the subject is 
provided with audio protection and optional television. Due to the magnet, subjects with implanted metal 
devices will be excluded. Some people feel claustrophobic in small spaces, and if this occurs the MRI will 
be stopped. Incidental findings on MRI will be relayed to ordering physician and/or PCP. 
 
A small single dose of furosemide will be injected intravenously to obtain BOLD MRI before and after 
injection. The dose of furosemide (20mg) is below the typical 1-2 mg/kg dose used clinically in pediatrics. 
Potential side effects include excessive diuresis (increased discharge of urine), low potassium (leading to 
symptoms like dry mouth, excessive thirst, weak or irregular heartbeat, muscle pain or cramps), stomach 
upset, dizziness, muscle weakness, low blood pressure with change of position, hyperglycemia, jaundice, 
rash, sensitivity to the light, ringing or buzzing noises in the ears. All these while considered possible are 
very rare following a single low dose of 20 mg. After the Furosemide injection, participants will receive IV 
fluids during the clamp and will remain well hydrated. All participants will be asked to drink 500mL of water 
with their study lunch, and therefore ensure adequate hydration of all subjects. A study physician and/or 
nurse will be present during the injection.  
 
Physical Exam, urine samples and anthropomorphic measures: There are no known risks associated with 
the physical exam, urine samples, or anthropomorphic measures.  
 
Confidentiality: Violation of privacy and loss of confidentiality are both risks to which research participants 
are exposed. The possibility of these risks increases when protected health information is collected. Every 
effort will be made to decrease this risk by limiting access to protected health information, storing this 
information in a password protected database, and identifying subjects only by a unique identifier that is 
kept in a separate location in a locked container, traceable only by study personnel. All tests involve risk of 
identifying asymptomatic/subclinical abnormalities. The study may also include risks that are unknown at 
this time of examination.  
 
8. Plan to minimize risk / protection against risk:  
 
The hyperglycemic clamps, iohexol and PAH infusions are standard procedures used in several research 
studies and settings, including our RENAL-HEIR protocol (COMIRB #16-1752) and IMPROVE-T2D 
protocol (COMIRB #18-0704). Adverse events are uncommon when the procedure is done by experienced 
personnel in an appropriate setting. There have been no Serious Adverse Events (SAE) in the research 
group’s experience in the Pediatric Clinical and Translational Research Center with insulin clamps, iohexol 
or PAH clearance studies. Therefore, we do not anticipate encountering SAEs. However, we have defined 
the following as possible SAEs for the purposes of monitoring: infection related to blood draw or IV 
placement. In addition, allergic reactions to iohexol or PAH requiring intervention (e.g. oral Benadryl) would 
also be considered a SAE. The PI will report serious adverse events, and any decision to suspend or halt 
the protocol to the CTRC and COMIRB immediately. The subject will also be instructed to report the event 
to their PCP.  
All investigators are currently certified in the Colorado IRB and HIPAA regulatory courses required to 
perform human subject research at the University of Colorado Denver and the Children’s Hospital Colorado 
and will be required to maintain such certification throughout the study. No protected health information will 
be collected until the appropriate HIPAA forms are completed. This information will be accessible only by 
the study investigators, Federal agencies overseeing human subject research, the Colorado Multiple 
Institutional Review Board, regulatory officials from the institution where the research is being conducted to 
monitor safety and compliance with policies. Every effort will be made to decrease the risk of loss of 
confidentiality by limiting access to protected health information, storing this information in a password 
protected database, and identifying subjects only by a unique identifier that is kept in a separate location in 
a locked container, traceable only by study personnel. Electronic Data will be stored in a password 
protected database and file servers. Paper files will be stored in a locked cabinet in the office of the 
Principal Investigator, accessible only to study investigators.  


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E.   Potential Scientific Problems and Feasibility:   
Overall feasibility: While an ambitious study, PI and co-investigators have performed numerous observational 
studies and clinical trials in youth and adults with T1D (41, 82, 83) and T2D (84), including the NIH TODAY 
(85, 86), RISE (87), and JDRF studies in T1D youth and adults, including Canadian Study of Longevity in 
Diabetes (Longevity), Coronary Artery Calcification in Type 1 Diabetes Study (CACTI), and Bromocriptine 
Quick Release (BCQR) as Adjunct Therapy in Type 1 Diabetes (NCT02544321) with renal measures similar to 
those now proposed (36, 41, 88-101). The proposed study in this application will be completed in a single 
research visit, limiting concerns for drop-out and retention. By recruiting adolescents (12-18 y) we expect that 
30-50% will have hyperfiltration at baseline per our previous studies in this age group (23, 54, 65, 66, 81, 102-
104). Drs. Nadeau and Prasad have extensive experience with MR imaging in youth (73-75, 105-110). Drs. 
Bjornstad, Johnson and Cherney are experienced in renal physiology measures (38, 55, 60, 62, 64, 79-81). Dr. 
Prasad is an international leader in renal MRI BOLD and ASL. Finally, our proposal is based on strong 
preliminary data (41, 55, 64, 111-113) and a track record of successful collaborations (16, 36, 55, 60, 86, 92, 
93, 95, 111, 112, 114-120).  We are therefore the ideal group to perform the important research proposed. 
 
Potential scientific problems: In any clinical study, unforeseen human factors provide challenges. We have 
experience with hyperglycemic clamps, iohexol and PAH clearance studies and MRI methods in youth with 
T2D, and have already obtained preliminary data. Expected outcomes and limitations for each aim are 
summarized below:  
 
Aim 1 - Expected outcomes and limitations: 
We expect copeptin to be greater in T1D youth with hyperfiltration vs. those without hyperfiltration. In youth 
with short-standing T1D and hyperfiltration, we anticipate that copeptin concentrations will positively correlate 
with GFR, ERPF, RVR, glomerular pressure and albumin excretion. We theorize that elevated AVP results in 
afferent arteriolar vasodilation with resultant increased ERPF and glomerular pressure which leads to 
hyperfiltration (Fig 10). Furthermore, a mild hyperglycemic clamp allows us to quantify intrarenal hemodynamic 
function in a glycemic milieu similar to their native pathophysiology. In contrast, a hyperinsulinemic-euglycemic 
clamp would render the participants completely normoglycemic, which does not reflect their usual glycemic 
environment, and expose them to high concentrations of insulin which may interfere with the assessment of 
their intrarenal hemodynamic function. Most studies focus on the classical definition of hyperfiltration which 
disregards ERPF and PGLO. Our proposed project addresses these concerns and others in regard to scientific 
rigor, by also examining hyperfiltration defined as FF (GFR/ERPF) > 17.7% and calculating PGLO by Gomez’ 
equations (67, 121). The sustained hypermetabolism of hyperfiltration is thought to initiate the development of 
nephropathy as the energy profile of T1D cannot accommodate the increased energy demand which leads to 
progressive nephron loss (122-127). We hypothesize that the elevated AVP activity in T1D may play a role in 
initiating hyperfiltration through vasodilation of the afferent arterioles with increased ERPF and glomerular 
hypertension (Fig 10).  
 


Protocol 17-0820 
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Control for potential confounders influencing intrarenal hemodynamic function includes: renal measures 
obtained during mild hyperglycemia, macronutrient and salt-controlled diet, limiting strenuous exercise for 3 
days prior to visit, and control for stage of menstrual cycle. Our preliminary data demonstrate the feasibility of 
the proposal in young adults with T1D and our unique ability to perform the highly specialized intrarenal 
hemodynamic measures.  
 
Aim 2 - Expected outcomes and limitations: 
We expect increased renal perfusion and renal O2 consumption in T1D youth with hyperfiltration. We also 
anticipate a strong correlation between copeptin, renal perfusion and renal O2. Based on our preliminary work, 
we expect the 2 renal sequences (BOLD and ALS) to be completed within 60 min. Dr. Nadeau’s NIH R56, 
JDRF and ADA studies had adolescents with T1D, T2D and without diabetes undergo similar MRI procedures 
lasting 120 min, indicating that recruiting and tolerability in youth for such procedures are feasible. Our 
research MRI is equipped with in-MRI movie-viewing with noise-cancelling headphones and thus is very 
tolerable to youth. The participant will be monitored by heart rate, blood pressure, and oxygen saturation 
measurements, and will be able to communicate with the MRI technician and investigator immediately outside 
of the scanner. As BOLD is potentially influenced by O2 delivery (ERPF) and O2 consumption (tubular Na 
transport accounts a majority of renal O2 consumption (128)) we will control for ERPF and fractional excretion 
of sodium (FeNa). We will perform the MRI fasting in the morning to remove the effects of acute glycemic 
variations on RPF and the impact of changes in hydration status of renal oxygenation. Participants on 
medications that might alter O2 delivery and/or consumption, e.g. angiotensin-converting enzyme inhibitors 
(ACE-I), angiotensin receptor blockers (ARBs) and diuretics will be excluded. 
 
Expected outcomes and limitations:  
 
F.    Potential Benefits: 
 
Evidence of Direct Benefit to Subjects: This study is designed to learn more about the vasopressinergic 
system (copeptin) and how it relates to intrarenal hemodynamic function and renal energetics in early DKD in 
T1D. The goal is to identify strategies that can be implemented to impede the development of DKD to reduce 
future risk of cardiovascular morbidity and mortality. No intervention is being proposed in this study, therefore 
participants will not directly gain a medical benefit from participating.  
 
Information gained by conducting this study is expected to yield novel, important findings regarding the 
mechanisms underlying early DKD in T1D. These data will contribute to better understanding of the role of 
vasopressinergic system in the development of DKD. Participants will also benefit from being in close contact 
Fig 10. Proposed Mechanism of Vasopressin Over-activity and Hyperfiltration 
 
Normal AVP activity at V2R at the afferent arteriole in T1D (a) is associated with normal tone (b), glomerular 
pressure and GFR. Elevated AVP activity at V2R at the afferent arteriole in T1D (a) leads to vasodilation (b) with 
increased RBF, increased glomerular pressure and hyperfiltration.  


Protocol 17-0820 
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with study staff, blood sugar monitoring and learning more about their renal health. All lab and procedure 
results with clinical relevance obtained as part of the study will be shared with participants. 
 
Evidence of Benefit to Society: The importance of the knowledge gained from this protocol is high, since 
DKD is very prevalent in T1D and increases the risk of death from CVD and shortens lifespan. In the United 
States, almost half of patients with renal failure have DKD. Despite the high prevalence and gravity of DKD in 
youth onset T1D, widely effective therapeutic options are lacking. Diabetes mellitus is the third most prevalent 
severe chronic disease of childhood. DKD remains a leading cause of morbidity and mortality, and a major risk 
factor for CVD in diabetes. Although data to guide care of DKD in adults with diabetes are limited, even less 
data exist regarding the antecedents of DKD in youth with diabetes.  Yet, these antecedents of adult DKD are 
present in children. As DKD is one of the leading causes of death in people with diabetes and the antecedents 
develop in childhood, data to inform clinicians as to treatment in this high-risk population are of great public 
health importance. In our novel and innovative proposal, we postulate that the inappropriate vasopressin 
activity (elevated copeptin) results in hypermetabolism of hyperfiltration with consequent increased oxygen 
consumption which cannot be met with the energy inefficiency of diabetes. A better understanding of the 
pathophysiology underlying early DKD, including hyperfiltration, and its strong relationship with the 
vasopressinergic system is critical to inform development of new therapeutics. Knowledge gained from this 
proposal will provide new information about the intrarenal hemodynamic changes associated with 
hyperfiltration, and the role of elevated vasopressin in early DKD development in T1D. The investigator team is 
also unique in being one the few groups skilled in performing both gold standard direct measures of GFR, RPF 
and advanced MRI evaluation in youth with T1D.  
 
 
G.   Data Analysis Plan (formulated with biostatistician Laura Pyle PhD):   
 
Aim 1 - Power calculations, statistical analysis, rigor and reproducibility: 
Primary outcomes for Aim 1 and study groups to be compared are summarized 
in Table 8. Based on our and others preliminary data we expect that 40% of 
youth with T1D (12-21 y) will demonstrate hyperfiltration (GFR 
≥135ml/min/1.73m2). Thus, a sample size of 50 youth with T1D provides 80% 
power to detect difference of 6.4 pmol/L in copeptin concentration between youth with T1D with and without 
hyperfiltration (H1.1), and 80% power to detect a correlation of 0.38 between copeptin, GFR and ERPF in 
youth with T1D (H1.2). Continuous variables will be checked for the distributional assumption of normality 
using plots. Variables that are skewed will be natural log-transformed for analyses. Descriptive statistics will be 
used to summarize baseline participant characteristics. Univariable and multivariable linear regression models 
will be built to examine the relationships between copeptin, GFR, ERPF and calculated intrarenal 
hemodynamic parameters (derived by Gomez’ equations). Variables considered for inclusion in the 
multivariable models will include: age, sex, ethnicity, pubertal staging, BMI and HbA1c. A P-value < 0.05 will be 
considered statistically significant.  Analyses will be performed in SAS (v9.4; SAS Institute, Cary, NC). 
 
Aim 2 – Power calculations, statistical analysis, rigor and reproducibility:  
Primary outcome for Aim 2 and study groups to be compared are summarized 
in Table 9. Our sample size (n=50, with 40% [n=20] anticipated to demonstrate 
hyperfiltration) will provide 80% power to detect an effect size of 0.8 SD for 
renal oxygenation and perfusion between participants with and without 
hyperfiltration. The analytic approach is as described in Aim 1. In our analyses, 
we will consider the following potential confounders for renal oxygenation and perfusion; age, sex, T1D 
duration, HbA1c, BP, LDL-C and FeNa. To avoid overadjusting our models, we will employ pared down models 
(e.g. model 1: univariable, model 2: age, sex, model 3: variables in model 2 + HbA1c, BP and LDL-C, model 4: 
variables in model 3 + FeNa). We will evaluate for collinearity before including both age and duration in the 
same model.   
Table 8 – Aim 1 
Primary Outcomes:      Groups: 
GFR and ERPF 
T1D w/ 
hyperfiltration 
vs. T2D w/o 
hyperfiltration 
Table 9 – Aims 2 
Primary Outcomes:     Groups: 
Renal O2 and 
perfusion 
T1D w/ 
hyperfiltration 
vs. T1D w/o 
hyperfiltration 


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Renal Volume:  Using 3D Slicer (SPL, Harvard Medical School, MA), the semi-automated segmentation of 
kidneys will be conducted initially with a thresholding procedure. Manual editing will then be performed to refine 
the segmentation of bilateral kidneys by placing straight lines at the convex level to separate the right kidney 
from liver and to remove bilateral ureters from the kidneys. This editing process will need to be performed on 
few contiguous slices with limited human interaction. The kidney volume will then be computed as the number 
of voxels within one kidney multiplied by the voxel size. 
 
BOLD MRI: Regions of interest (ROI) analysis for BOLD MRI will be performed on a Leonardo Workstation 
(Siemens Medical Systems, Germany).  Typically, 1 to 3 regions in each, cortex and medulla, per kidney per 
slice will be defined leading to a total of about 10 ROIs per region (cortex and medulla) per subject.  The mean 
and standard deviation of these 10 measurements will be used a R2* measurement for the region, for the 
subject and for that time point.  Additionally, two R2*s will be calculated as defined below:  
R2*(medulla, furosemide) = R2* (medulla, pre-furosemide) - R2* (medulla, post-furosemide); 
R2*(cortex, medulla) = Baseline R2* (medulla) – Baseline R2* (cortex).  
 
ASL MRI:  ROI analysis will be used to estimate M (difference in signal intensity between non-selective and 
selective inversion images).  Using the same ROI, M0 will be estimated from the proton density image.  T1 
measurements from the same ROI will be obtained by fitting the signal intensity vs. inversion time data as 
described previously (129) using XLFit (ID Business Solutions Ltd., UK) or T1 maps created using MRI Mapper 
(Beth Israel Deaconess Medical Center, Boston). Partition coefficient will be assumed to be 0.8 ml/gm (130, 
131).  These values will then be used to estimate regional blood flow.   
 
For data analyses, all MRI markers will be measured for each kidney (left and right) for each subject and these 
two measurements for each specific MRI marker would be averaged to generate a summary measurement per 
subject for further statistical analysis.  BOLD MRI measurements will be performed before and after 
administration of furosemide and the relative value (pre minus post) will be reported.  The final averaged value 
will be analyzed statistically. 
 
Summary of MRI Parameters: 
Renal Volume:                    Left kidney volume (LKV), Right kidney volume (RKV) 
Diffusion Imaging:  
ADCLeft (cortex), ADCLeft (medulla), ADCRight (cortex), ADCRight (medulla) 
BOLD MRI:  
R2*(cortex)preLeft, R2*(medulla) preLeft, R2*(cortex) preRight, R2*(medulla) preRight 
 
R2*(cortex)posLeft, R2*(medulla) posLeft, R2*(cortex) posRight, R2*(medulla) posRight 
 
Pre – pre-furosemide; pos – post-furosemide 
ASL MRI: 
flowLeft (cortex); flowRight(cortex) 
 
 
Aims 1-2 Data Management: 
 
Data Entry   
Data will be entered from paper forms. Once forms are completed, verified and corrected for inconsistencies, 
they will be manually entered using a HIPAA compliant, Red Cap database system.  
 
Edit Checks    
Computerized data validation routines will be used to enhance data quality and verify the accuracy of data 
within predefined value ranges. These checks include but are not limited to: (a) initial screening of data, using 
logic and range checks built into data entry screens; (b) cross-form functional and consistency checks; and (c) 
edits assessing the serial integrity of data. 
 


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Disaster Recovery    
Routine data backup will occur on data in conjunction with the children’s hospital secure server and Redcaps. 
 
Security and Confidentiality   
All hard copy forms will be de-identified with a study number and filed in a locked cabinet, to which only the 
investigators will have access.  Standard protection against computer hackers is implemented. Recovery from 
natural disasters (water, fire, or electrical) can occur through the ability to reconstruct both the database 
management system and the data from nightly backups.   
 
H.  Summarize Knowledge to be Gained:   
 
The innovative and novel proposal aims to define the intrarenal hemodynamic function and renal oxygenation 
in early DKD in adolescents with T1D and define how these parameters relate to vasopressin. Carefully 
designed human studies, like the one proposed in this application, are needed to advance our understanding 
of the pathophysiology driving early DKD and direct the development of new therapeutic strategies to impede 
the development of DKD.  
 
DKD outcomes have not changed significantly over the last couple of decades and continue to be a major 
concern for providers caring for patients with T1D (8, 132). Managing DKD in patients with T1D has been 
further complicated by disappointing results of clinical trials (9, 11-14). It is therefore important to identify novel 
and modifiable risk factors that contribute to the development and progression of early DKD (133). 
 
 
 
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