Renal Transplant Injury and the Renin-Angiotensin System in Kids (RETASK)
Conditions: Renal Transplant, Renin-Angiotensin System, Rejection Acute Renal, Rejection Chronic Renal, Rejection of Renal Transplant
Sponsor: Wake Forest University Health Sciences

Renal Transplant Injury and the Renin-Angiotensin System in Kids 
(RETASK) 
Andrew M. South, PI 
 
Abstract 
Kidney disease is a major source of childhood morbidity and mortality. Kidney 
transplants improve quality of life and life expectancy. Renal graft injury is a 
significant problem and early identification is critical. No biomarkers exist to 
identify subclinical injury. Biopsies are the gold standard but are invasive and can 
miss early injury. The renin-angiotensin system (RAS) is activated in renal injury. 
The alternative RAS pathway ACE2/Ang-(1-7)/mas is important in counteracting 
these harmful effects. The components of these pathways are excreted in the 
urine and are altered in renal injury. The aims of this proposal are to develop 
noninvasive biomarkers of 1) early and 2) chronic subclinical graft injury. The 
project was an observational study with a convenience sample of children 
undergoing renal transplantation. The profile of these pathways in the urine will 
be compared to outcomes of 1) renal biopsy-confirmed early graft injury and 2) 
chronic graft injury using Cortical Fractional Interstitial Fibrosis Volume (VIntFib) on 
biopsy specimens. The components of this pathway are novel potential 
biomarkers and have not been previously evaluated in this population. The 
findings of this proposal may enable more prompt diagnosis and more effective 
treatment of renal graft injury in children. 
 
Specific Aims 
In pediatric kidney transplant patients, rejection, medication toxicity and ischemia 
cause early and chronic renal allograft injury, which reduces graft lifespan and 
patient survival1. Early identification of injury would facilitate prevention and 
treatment. The gold standard surveillance biopsy has limitations including 
delayed discovery of injury2,3. No noninvasive test identifies graft injury before it 
is clinically apparent4,5,6. This project’s goal is to develop a novel early marker of 
subclinical graft injury to facilitate prompt recognition and treatment. Kidney 
damage activates the renin-angiotensin system (RAS), and the ACE2/Ang-(1-
7)/mas pathway counteracts this damage.7 The balance, or ratio, between levels 
of RAS and the ACE2 pathway may be clinically important because the ACE2 
pathway counteracts RAS-mediated injury. An increase in ACE and Ang II 
expression and a decrease in ACE2 and Ang-(1-7) expression on tubular cells 
may promote renal injury. Tubular damage may increase urinary loss of 
protective ACE2 and Ang-(1-7), propagating renal damage by allowing ACE and 
Ang II to stimulate inflammation and fibrosis unopposed. 
 
Aim 1: Diagnose early subclinical graft injury in renal transplant patients with the 
ratios from ACE2 to ACE and from Ang-(1-7) to Ang II in urine. 
Hypothesis 1: A shift in the urinary ACE2 to ACE and Ang-(1-7) to Ang II ratios 
towards ACE2 and Ang-(1-7) predicts early subclinical graft injury diagnosed on 
renal biopsy. 
 

Aim 2: Diagnose chronic subclinical graft injury using the ratios of ACE2 to ACE 
and Ang-(1-7) to Ang II in the urine. 
Hypothesis 2: A shift in the ACE2 to ACE and Ang-(1-7) to Ang II ratios in the 
urine towards ACE2 and Ang-(1-7) predicts chronic subclinical graft injury on 
renal biopsy. 
 
Background/Significance 
Angiotensin-converting enzyme (ACE) cleaves angiotensin (Ang) I into Ang II, 
which operates through the Ang II type 1 receptor (AT1). The ACE2/Ang-(1-
7)/mas pathway is an alternative, less characterized pathway that operates 
through the mas receptor8. ACE2 cleaves Ang II into Ang-(1-7) (figure 1). The 
exact mechanism of action of Ang-(1-7) is unknown but most data support its 
anti-inflammatory, anti-fibrotic and immune modulatory effects9-17. ACE2, Ang-(1-
7) and mas expression is reciprocal to ACE, Ang II and AT1 expression10, 12. In 
the kidney, expression of ACE2 and Ang-(1-7) is greatest in the proximal tubules, 
especially the brush border membrane7,10, 12, 18, 19. The balance between the ACE 
and ACE2 pathways is critical in preventing renal fibrosis7,10,12. Ang-(1-7) directly 
blocks Ang II and Ang II’s downstream effects through the AT1 receptor10, 14, 18-27. 
ACE inhibitors (ACEi) and AT1 blockers (ARB) reduce proteinuria and graft injury 
and improve graft function, independent of blood pressure32, 37, 38. The ACE2 
pathway may be responsible for these effects because it is enhanced at the 
expense of the RAS32, 37-42. 
 
Tubulointerstitial damage decreases ACE2 expression, enhancing ACE and 
propagating injury43,44. Urine ACE2 protein is elevated in renal transplant 
patients, independent of serum levels45,46. These findings suggest that tubular 
damage causes shedding of ACE2 and Ang-(1-7) from the apical membrane of 
tubular epithelial cells into the tubular lumen and urine. Urinary excretion of 
ACE2 and Ang-(1-7) is altered in relation to ACE and Ang II. The ratios of 
ACE2/ACE and Ang-(1-7)/Ang II in urine could be noninvasive markers of 
subclinical graft injury. It is important to evaluate the balance between the ACE 
and ACE2 pathways because increased ACE2 could be due to increased 
expression of ACE2 or decreased ACE. ACE2/Ang-(1-7)/mas directly alters renal 
inflammation, making it an intriguing biomarker and therapeutic target. 
 
Approach/Methods 
This was an observational study with a convenience sample of patients recruited 
from Lucile Packard Children’s Hospital’s kidney transplant evaluation clinic from 
2013 to 2015; 29 subjects completed the study. The recipient ages ranged 1 to 
20 years and 34% were living donors. Stanford University School of Medicine 
IRB approval was obtained (IRB 5136) and informed consent and assent were 
obtained. Data was collected from information gathered per standard of care. 
Baseline and post-transplant data included age, sex, self-reported race (Black, 
White, Hispanic, Asian, or Other), presence of obesity (body mass index ≥95th 
percentile for age and sex), primary cause of end stage renal disease, dialysis 
modality, time on dialysis, presence of hypertension, use of ACEi/ARB, presence 

of left ventricular hypertrophy, sensitization at transplant, creatinine, estimated 
glomerular filtration rate (eGFR) by modified Schwartz equation and urine 
creatinine and protein. Post-transplant data included deceased versus living 
donor transplant, antigen match, cold ischemia time, immunosuppression 
including drug levels, and protocol, and indication biopsy results. Specific to the 
study, plasma and urine protein ACE, ACE2, Ang II, and Ang-(1-7) were 
collected and analyzed. Data was collected at baseline and post-operatively at 
12 hours, 24 hours, daily for 1 week, weekly for 1 month and monthly until 6 
months post-transplant. Samples were collected at any clinically significant event 
concerning for graft injury, including a rise in creatinine, rejection, medication 
toxicity, and infection. 
 
Subjects underwent a six-month surveillance protocol biopsy and some received 
three-month surveillance protocol or indication biopsies per standard of care. For 
the study, existing biopsy tissue was stained with Sirius Red, providing a 
measure of fibrosis called (VIntFib) that was quantified by computerized image 
analysis47. Fibrosis is an established measurement of chronic graft injury. ACE, 
ACE2, Ang II, and Ang-(1-7) protein were measured by radioimmunoassays at 
the Hypertension and Vascular Research Biomarker Analytic Core at Wake 
Forest School of Medicine. All data, including protected health information, was 
stored securely on a HIPAA-compliant REDCap Database hosted by Stanford 
University School of Medicine. Summary statistics, including frequencies for 
categorical variables and mean with standard deviation (SD) or median with 
interquartile range for continuous variables will describe clinical and demographic 
characteristics. Graphical exploration will assess the data and quantile plots will 
evaluate the need for log transformation. Measurements below the lower limit of 
detection of the laboratory will be assigned a value of the lower limit of detection 
divided by the square root of two. Spearman rank correlation coefficients will be 
used to assess the correlation between ACE2, ACE, Ang-(1-7), Ang II and the 
continuous covariates and outcomes. We will estimate sensitivity, specificity, and 
the area under the receiver operator characteristics curve to assess the clinical 
predictive value of the biomarkers. 
 
Primary outcome for Aim 1: Early subclinical graft injury. 
Cox regression will be used to estimate hazard ratios for the association of 
biomarker levels with the outcome biopsy-proven graft injury, adjusting for 
potential confounders. Robust standard errors and clustering by subject ID for 
patients diagnosed with more than one episode of graft injury during the study 
period will be used. The time axis will be six months, starting at the time of 
transplant or time of recovery from the preceding acute injury event, and ending 
at six months post-transplant. Observations will be right-censored at the time of 
graft loss, loss to follow up, or withdrawal from the study. The model will be 
compared to other models that include the time dependent-covariates serum 
creatinine and eGFR. 
 
Primary outcome for Aim 2: Chronic subclinical graft injury. 

Cox regression will be utilized to estimate hazard ratios for the association of 
biomarker levels with the outcome biopsy-proven graft fibrosis, adjusting for 
potential confounders. The time axis will begin at the time of transplant and end 
at six months post-transplant. Observations will be right-censored at the time of 
graft loss, loss to follow up, or withdrawal from the study. The model will be 
compared to other models that include the time dependent-covariates serum 
creatinine and eGFR. 
 
Missing Data 
Subjects may miss lab collections or may be lost to follow up. However the 
reasons for missing data are presumed to be independent of the values of the 
predictors ACE, ACE2, Ang II, and Ang-(1-7). Clinical data including covariates 
and outcomes will be recorded even if a subject is lost to follow up. Therefore 
missing data will be presumed to be missing completely at random. We chose to 
handle missing covariate values by multiple imputations due to the longitudinal 
data and high rate of missing data (23%). 
 
Power 
The study was estimated to enroll 30 subjects (final enrollment 29), based on 
anticipated subjects and estimated incidence of kidney injury (10% in first six 
months post-transplant). The study was powered to detect a Pearson correlation 
of 0.56 with a two-sided p value of 0.025. 
 
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