Associations of Hyper-filtration and Proteinuria with Clinical Severity Score among Adult Sudanese Sickle anemia patients

Background: Sickle Cell Anemia (SCA) is an autosomal recessive haemoglobinopathy with high morbidity and mortality. Global survival of sickle patients is increased and subsequently, prevalence of chronic complications including renal manifestations due to advances in management. Therefore, early detection and management of these complications become mandatory. This study aimed to investigate the estimated Glomerular Filtration Rate (eGFR), proteinuria and serum uric acid as markers of renal involvement in Sudanese sickle adults and association between these parameters and clinical severity score of sickle cell disease. Methods: Cross-sectional hospital-based study included thirty-two adult Sudanese patients diagnosed with SCA and twenty-three controls. Written informed consent was obtained. Blood and urine samples were collected. Severity score was calculated using Bios online calculator and eGFR was calculated using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula without adjustment for ethnicity. Results: Protein/Creatinine Ratio (PCR) was significantly higher (p-value < 0.001) in sickle cell anaemia group compared to controls. Hyper-filtration and Hyperuricemia were manifested in 75% and 6.3% of SCA group respectively. There was no association between the severity score and renal manifestations in the SCA group. Conclusions: Hyper-filtration and proteinuria were the most prevalent renal manifestations in SCA group. Further studies are recommended to determine the predictors of renal complications and early management of them.

high mortality rates even in developed countries where the average survival age is 40 to 50 years (5).
In Africa an accurate data is inadequate, but the median survival is far lower and most of the children die before completing their first decade (5).
Sickling of the RBCs, hemolysis, increase viscosity, vaso-occlusion, hypoxia, and ischemia-reperfusion injury are the hallmarks of the SCD(6). Survival age increases in patients with SCA due to advances in management leading to widespread tissue necrosis and end organ damage (5). Renal impairment predominates organs' failure and it was documented among 40% of SCA patients who died (5).
Previous study revealed that the median age of diagnosis of renal failure in SCD patients is 23.1 years and median survival after diagnosis of End Stage Renal Disease (ESRD) in SCD was only 4 years despite hemodialysis (7).
This study aimed to measure proteinuria, uric acid level, and estimated GFR as markers of renal function in steady state SCA adult patients, and to identify the associations between these parameters and the clinical severity score of SCA. Steady state refers to a point of time where SCA patient does not experience acute infection, crisis or needs hospital admission or blood transfusion in the previous three months preceding data collection (8). Studying renal parameters in SCA enables early detection and management of renal complications to decrease morbidity, mortality and improve quality of life (QOL). Best to our knowledge this first study investigated renal parameters and their association with the severity score among sickle cell anemia patients.

Methods:
This study is a cross sectional hospital-based study held in Omdurman Military Hospital, which is the one of the biggest tertiary hospitals in the Sudan. A total sample of 32 SCA adult patients (13 males, 19 females) in a steady state, who attended the hematology referred clinic in the hospital from October to December 2017. Control group (11 males, 12 females) were healthy students recruited from Faculty of Medicine, Al Neelain University. Diabetics, Chronic Kidney Disease (CKD) patients due to other causes, patients with acute illness and pregnant women were excluded.
Blood pressure (BP) measured for all participants using mercury sphygmomanometer and stethoscope. Participants were classified according to the last American Heart Association classification of hypertension in to: normal BP, elevated BP, stage 1 and stage 2 hypertension; if their BP ˂ 120/80, 120-129/80, 130-140 /80-90 and > 140/90 respectively (9). Weight was measured in Kilogram using digital weight scale. Height was measured by meter using wall mounted scale. BMI was calculated from the height and weight using this equation: BMI = Weight in Kg/ (height in meter) 2 Patients were classified according to the WHO classification of BMI available at (http://apps.who.int/bmi/index) to underweight, obese, overweight and normal weight when their BMI: Random spot urine collected in clean, dry, closed, wide neck urine container and sent to the laboratory for PCR using colorimetric method. Study population classified according to their PCR using NICE Guidelines to group with significant proteinuria if PCR ≥ 50 mg/mmol and another with nonsignificant proteinuria when PCR ˂ 50 mg/mmol (10).
GFR calculated using CKD-EPI formula without adjustment for ethnicity which is the best GFR formula for SCD (11). This formula was proved by France prospective observational cohort study conducted in Sub-Saharan and French West Indies native populations; after the comparison between measured GFR using Iohexol plasma clearance method with estimated GFR by using formulas (MDRD, Cockcroft-Gault, CKD-EPI, CKD-EPI and MDRD without adjustment for ethnicity) (11). GFR < 60 ml/min/1.73 m 2 regarded as CKD. Despite there is no consensus about the cut-off point of hyper-filtration; more than 130 ml/min/1.73 m 2 in females and > 140 in males is regarded as glomerular hyper-filtration based on previous reported literature (12).
Severity score is estimation of risk of death among SCD was calculated using a validated online clinical severity score calculator based on study made by Sebastian et al (13) and developed by Bioinformatics group called Bios in the University of Granada in Spain and made it available freely online at http:/bios.ugr.es/dss-calculator. Severity score includes clinical and laboratory characteristics to estimate 5-year-risk of death among sickle cell anaemia patients. The range of the score is from zero to one; where zero means 0% and one means 100% risk of death in the next five years. Fifteen out of sixteen clinical and laboratory characteristics were entered to the online calculator. Severity Score Variables include: Stroke, blood transfusion times, pain, priapism, acute coronary syndrome, avascular necrosis, systolic blood pressure, Hemoglobin, Hb genotype, MCV, TWBCs, Reticulocyte count, and Serum bilirubin beside age and gender. Patients were classified in to high, moderate and low risk of death if they score > 0.72, (0.55-0.72) and < 0.55 respectively according to Guilhreme et al study (14).
Data were summarized and analysed using (SPSS) version 23. We used frequency tables, means and standard deviation (SD) to describe the values. One-sample Kolmogorov-Smirnov Test was used to test the distributions of variables. Unpaired T. Test was used for normally distributed variables and Mann Whitney U test for non-normally distributed ones. A correlation analysis was applied to investigate the relation between study variables. p-value ˂ 0.05 was considered as statistically significant cut off point.
Study was ethically approved from Institutional Review Board (IRB) of Al Neelain University. Informed written consents were obtained from participants after explaining the purpose, procedures and measurement needed for the study.
Results: Characteristics of study population: Most of our participants were < 25 years. According to WHO classification 71.9% of SCA group were underweight and only 3.1% were overweight. In control group 21.7% were underweight and 56.6% had normal BMI (Table 1).   The mean severity score of SCA group was 0.69. SCA patients were stratified according to their severity score in to: Mild, moderate and severe according to Guilhreme et al study (14). (Table 4 ) shows severity score grading of SCA group. There is no correlation observed between the severity score and renal parameters in this study, but there is positive correlation between PCR, age and systolic BP. (Table 5) shows the correlation between renal parameters, age, BP and severity score.

Discussion:
This study proposed to investigate renal characteristics (proteinuria, eGFR and uric acid level) among sickle cell anemia patients and their association with severity score. Hyper-filtration and glomerular hypertrophy dominate early onset renal involvement in Sickle Cell Anemia ; as the disease progress with time, eGFR decreases due to increased fibrosis and glomerulosclerosis (15). Low eGFR was not reported in this study among SCA patients unlike other studies (15)(16)(17); this may be due to the low BMI and younger age of the patients. In contrast, all control group had normal eGFR ( Table 3).
Significant proteinuria was detected in 12.5% of SCA patients while none of the control group had significant proteinuria; this might have resulted from tubular damage, increase glomerular permeability related to repeated vaso-occlusive crisis and poor management of SCA (6). A retrospective study conducted in inpatients adult Sudanese with SCD, reported proteinuria in 81.8% of patients (18); this may indicate a higher levels of proteinuria in SCA patients during crisis.
Proteinuria is a prevalent complication of SCD, it was detected in variable proportions in many previous studies for example: Jamaican's (65.3%) (16), North American's (42.8% (17) and 65.6%) (15) and Nigerian's(51%) (19), this variation may result from specific nucleotide polymorphism (20). Further studies are necessary in this area to identify if there are specific genetic polymorphisms in Sudanese SCA patients related to proteinuria. In this study, proteinuria grade was determined through a spot urine PCR as an acceptable alternative for ACR (21).
More than 50% of SCA patients in this study scored a high risk of death (severity score > 0.72) ( Table 4). Using the same calculator a previous investigation conducted in referral center of hemoglobinopathies in St George's hospital in London found that only 12% of SCA had severe risk of death (22). This high severity score among Sudanese study population requires early preventive measures to decrease morbidity and mortality among adults Sudanese SCA patients.
Significant reduction in diastolic Blood Pressure (BP) was noted (p value 0.02) in SCA group compared to the control group. Other studies also showed similar observations (15,23,24). This may be due to loss of sodium and water related to medullary tonicity defects (25). In addition to, high levels prostaglandin, decrease vascular reactivity and systemic vaso-dilatation response to micro-vascular dysfunction (6) or might be due to chronic activation of Nitric Oxide system (NO) (15,25).
Positive correlation was observed in the current study between SBP and PCR among SCA patients (P value = 0.013) ( Table 3). This correlation was documented previously among hypertensive patients which also stated that both proteinuria and hypertension can increase cardiovascular and renal risks independently (26). There was also positive correlation between SBP and age which can be explained by aging process and chronic depletion of nitric oxide due to its consumption in SCA patients (27).
Uric acid was used as a marker of renal function (28). In SCA high uric acid level may result from increased haemolysis and rapid turnover of the RBCs and decreased uric acid clearance by the kidneys (29). No significant difference regarding UA between sickle and control group (Table 3), this finding is similar to former study among Sudanese population (24). In this study hyperuricemia is reported only in 6.3% of SCA group; this likely due to the compensation of the high GFR (high UA clearance) among those young group; as age increases, GFR decreases and they may develop higher rates of hyperuricemia (30). Hyper-filtration, proteinuria, high UA levels and lower BP were also reported in similar study conducted in Congo SCD pediatrics populations (31); which indicated the early onset of renal pathophysiological processes in SCD patients.
This report demonstrated that patients with SCA in spite having normal urea, creatinine, UA levels and lower diastolic BP in compere to control group, but they suffered from hyper-filtration and proteinuria. Eventually they may end up with CKD, hypertension and hyperuricemia. Therefore, caring physician of SCA patients must consider those early signs of SCN and manage them appropriately to prevent further complications. In spite of lack of association of severity score with each individual renal parameter but we observed that both impaired renal parameters and severity score were high among our SCA group; so we may need modified severity score to address these renal manifestations among sickle cell anemia patients .
In conclusion, Proteinuria and hyper-filtration were the most prevalent renal manifestation in SCA patients. SCA patient had higher UA, levels and lower BP compared to control group. There was lack of association between the clinical severity score and renal manifestations of SCA. Central Institutional Review Board at Al -Neelain University, approved the study. Written informed consent was obtained from each participant prior to enrollment.

Consent for publication:
Principal investigator obtained informed consent from each participant to publish the data without breaching confidentiality.

Availability of data and material:
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.