Orosomucoid As a Biomarker for Chronic Kidney Disease Associated with Sickle Cell Anemia
Authors: Taye Ammanuel , Smith Nathan , Afangbedji Nowah Kokou Apeadoufia , Niu Xiaomei , Taylor James G. , Jerebtsova Marina , Nekhai Sergei 
Published: October 2019

Abstract
BACKGROUND: Chronic kidney disease (CDK) is a serious and highly prevalent complication of Sickle cell anemia (SCA). Proteinuria and microalbuminuria can be under-detected in SCA because of the renal concentrating defect. Hence, complementary diagnostic biomarkers are necessary for early detection of CKD in patient with SCA. Our recent study identified alpha 1-acid glycoprotein (orosomucoid, ORM) (1) as potential biomarker of CKD. ORM is a major acute phase inflammatory protein synthesized by the liver and its presence in urine may indicate underlying inflammation in SCA patients.
OBJECTIVES: We aimed to validate ORM as biomarker for CKD in a cohort of SCA patients recruited at Howard University.
METHODS: We analyzed samples collected from patients enrolled in a sickle cell disease registry study at Center for Sickle Cell Disease, Howard University. Plasma and urine samples were collected and stored at -80°C. Plasma samples were assayed for creatinine and cystatin C. Urine samples were analyzed for albumin, creatinine and ORM. Multistix was used to determine protein, blood, pH and specific gravity (SG) in urine samples. Estimated GFR (eGFR) was calculated using CKD-EPI creatinine-cystatin equation. CKD stages were assigned according to the National Kidney Foundation, Kidney Disease Outcomes Quality Initiatives (K/DOQI) guidelines: stage 0 - eGFR>60 ml/min/1.73m2 and AL/CRE<30 mg/g; stage 1- eGFR>90 ml/min/1.73m2 and AL/CRE≥30 mg/g; stage 2 - eGFR 60-89 ml/min/1.73m2 and AL/CRE≥30 mg/g, stage 3 - eGFR 30 - 59 /1.73m2; stage 4 - eGFR 15 - 29 ml/min/1.73m2 and stage 5 - eGFR < 15 ml/min/1.73m2. Glomerular hyperfiltration was defined as eGFR>130 ml/min/1.73 m2 for females and eGFR>140 ml/min/1.73 m2 for males.
RESULTS: The Howard University registry study group consisted of 96 patients (mean age 38.1 years, range 18 - 67 years, 55% females) and included 65.6% HbSS, 21.8% HbSC, 7.2% HbS β+thalassemia, 4.1% HbS β0 thalassemia. Based on urinary dipstick test, proteinurea was detected in 16.5% patient. Hyperfiltration was detected in 45 patients (56.3%). CDK (combined stage 1-4) was detected in 35.8% patients. No difference was found between females and males in CKD prevalence. Prevalence of CKD in HbSC patients was 11% whereas it was 37% in HbSS patients. Prevalence of CDK increased with the age.
The overall urinary ORM/CRE levels demonstrated modest correlation with CKD stage (R2=0.22). Next we stratified samples based on the levels of ORM/CRE. The samples were separated into two groups: (i) samples with no or mild inflammation (ORM/CRE<10μg/mg) and (ii) samples that displayed significant inflammation (ORM/CRE>10 μg/mg). We observed strong inverse correlation between the percentage of samples without inflammation and CKD stage (R2=0.996). We also observed strong correlation of CDK progression with number of samples with significant inflammation (R2=0.996). Sensitivity of ORM/CRE was 53.8%, specificity 70.2% and odds ratio OR 2.72 for samples with CKD stages 1-4 compared to the samples without CKD (stage 1).
CONCLUSIONS: In our cohort, determination of proteinurea evaluated by dipstick (16%) grossly underestimated CKD prevalence (35%) determined by eGFR and AL/CRE ratios. Subjects with HbSC had 3-fold less prevalence of CKD comparing to the patients with HbSS. ORM/CRE levels correlated with the stages of CKD stages (OR 2.72). Further longitudinal study is needed to determine whether ORM/CRE ratio can be used as a prognostic marker of renal disease development.
ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. AT was supported by ASH MMSAP summer program. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.
REFERENCES: Jerebtsova, M., Saraf, S. L., Soni, S., Afangbedji, N., Lin, X., Raslan, R., Gordeuk, V. R., and Nekhai, S. (2018) Urinary orosomucoid is associated with progressive chronic kidney disease stage in patients with sickle cell anemia. American journal of hematology93, E107-E109

Disclosures
Taylor: Pfizer: Research Funding; NHLBI: Research Funding. Nekhai:NIAID, NIH: Research Funding; NIMHD, NIH: Research Funding; NHLBI, NIH: Research Funding.