Assessment of Subclinical Renal Glomerular and Tubular Dysfunction in Children with Beta Thalassemia Major

Background: A good survival rate among patients with beta thalassemia major (beta-TM) has led to the appearance of an unrecognized renal disease. Therefore, we aimed to assess the role of serum cystatin-C as a promising marker for the detection of renal glomerular dysfunction and N-acetyl beta-D-glucosaminidase (NAG) and kidney injury molecule 1 (KIM-1) as potential markers for the detection of renal tubular injury in beta-TM children. Methods: This case-control study was implemented on 100 beta-TM children receiving regular blood transfusions and undergoing iron chelation therapy and 100 healthy children as a control group. Detailed histories of complete physical and clinical examinations were recorded. All subjected children underwent blood and urinary investigations. Results: There was a significant increase in serum cystatin-C (p < 0.001) and a significant decrease in eGFR in patients with beta-TM compared with controls (p = 0.01). There was a significant increase in urinary NAG, KIM-1, UNAG/Cr, and UKIM-1/Cr (p < 0.001) among thalassemic children, with a significant positive correlation between serum cystatin-C, NAG and KIM-1 as regards serum ferritin, creatinine, and urea among thalassemic patients. A negative correlation between serum cystatin-C and urinary markers with eGFR was noted. Conclusion: Serum cystatin-C is a good marker for detection of glomerular dysfunction. NAG and KIM-1 may have a predictive role in the detection of kidney injury in beta-TM children.


Introduction
Beta thalassemia is considered the most common heterogeneous genetic disorder, resulting from reduced or absent beta globin synthesis, leading to globin chain imbalance [1]. Beta thalassemia is characterized by reduced hemoglobin production with excess α-goblins. This leads to microcytic hypochromic anemia, which is associated with oxidative stress and ineffective erythropoiesis, leading to chronic hemolytic anemia [2,3]. Beta thalassemia includes beta thalassemia major (beta-TM), beta thalassemia intermedia, or thalassemia minor [4]. Despite the increased survival of patients with thalassemia, some complications have been detected, such as renal injury or damage [5].
Hypoxia and chronic anemia lead to oxidative stress and lipid peroxidation, resulting in tubular cell function impairment [6]. Moreover, iron overload has an important role in for complete blood count. The second part was transferred into a plain vacutainer tube, left to clot, and then centrifuged to separate serum used for the assessment of kidney function tests, electrolytes, and ferritin levels. Complete blood count was determined using an automated Sysmex XN-1000 hematology analyzer. Kidney function tests were performed using an AU680 chemistry analyzer. A serum ferritin assay was performed using a mini VIDAS immune-analyzer. Serum cystatin-C was measured by a quantikine human cystatin-C immunosorbent assay (ELISA) kit (Cat. No. DSCTC0; R&D systems, Inc., Minneapolis, MN, USA).
Fresh first morning midstream urine samples were collected in a sterile polypropylene container for analysis of creatinine, total protein, calcium, sodium, potassium, uric acid, and urinary markers including NAG and KIM-1.
For urinary KIM-1, centrifugation occurred for 20 min at 2000-3000 r.p.m. twice with the removal of supernatant. If precipitation appeared again, the sample was analyzed by an available quantitative sandwich immunoassay technique (Sun Red Biotechnology Company, Shanghai, China). Normalized KIM-1 level was obtained by dividing it by urine creatinine.
Urinary sodium, potassium, and calcium were analyzed by an enzymatic colorimetric method using an Integra 800 device. The urinary calcium to creatinine ratio and uric acid were measured by spectrophotometry.

Sample Size Calculation
The sample size relied upon 95% CI with 80% power, using an unpaired t-test and assuming an α (two-sided) of 0.05. Based on a previous study, the mean of the urinary NAG of β-thalassemia major case group was 106.5, while that of the healthy control group was 66.3, and the SD was 101.5, with a group size ratio of 1/1 [5].

Statistical Analysis
Data were analyzed using IBM SPSS statistics version 20 (SPSS Inc., Chicago, IL). The chi-square test was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was done using either a Student's t-test or a Mann-Whitney test (non-parametric t-test), as appropriate. Pearson's correlation coefficient or the Spearman-rho method (as appropriate) was used to test the correlation between numerical variables. Multiple linear regression was applied to determine the relationship between dependent and independent variables. A p-value < 0.05 was considered significant.

Results
Demographic and clinical features are given in Table 1. No significant difference in age and sex were found between the two groups. There were significant differences in hemoglobin (Hb), hematocrit (HCT), and mean corpuscular volume (MCV) levels of two groups, as they were lower in patients with beta-TM than controls (p < 0.001). Serum ferritin was significantly higher in patients with beta-TM than in the controls (p < 0.001).
Twenty-three percent of the studied patients underwent splenectomy ( Table 1). Analysis of kidney function tests showed a significant increase in serum urea and creatinine in beta-TM patients compared with the controls (p < 0.001), but they were within the normal range for both groups, and a significant decrease in eGFR in patients with beta-TM compared with the controls (p = 0.01). Eleven percent of beta-TM had a low eGFR < 90. Moreover, there was no significant difference in serum sodium and calcium between the two groups, and there was a significant increase in serum potassium in patients with beta-TM than in the controls, but it was still within the normal range. In addition, analysis of urinary electrolytes and urinary protein whereby creatinine was divided, there was a significant increase in urinary uric acid/Cr, UCa/Cr, UProtein/Cr, and UK/Cr in patients with beta-TM than in the controls ( Table 2). There was a significant increase in serum csytatin-C and urinary NAG, KIM-1, UNAG/Cr, and UKIM-1/Cr among thalassemic patients compared with the controls (p < 0.001) ( Table 3). There was a significant positive correlation between serum cytatin C, UNAG, and UKIM-1 as regards blood transfusion index per year, serum ferritin, serum creatinine, and serum urea among patients with beta-TM (p < 0.001). In addition, there was a negative correlation between cystatin C, urinary markers and eGFR. Moreover, there was a significant positive correlation between UNAG, UKIM-1, UNAG/Cr, and UKIM-1/Cr as regards urinary protein/Cr, UCa/Cr, UNa/Cr, UK/Cr and Uuric acid/Cr (p <0.001). No significant correlation was found between renal markers as regards age, body weight and the time from diagnosis with beta-TM. There was a significant positive correlation between UKIM-1 and the iron chelation therapy with deferasirox (p < 0.001). Multiple linear regression revealed that serum cystatin-C, UNAG, and KIM-1 were independent risk factors for the occurrence of renal affection among beta-TM patients (Tables 4 and 5). There was a significant increase in UNAG, UNAG/Cr, UKIM-1/Cr, serum creatinine, Uprotein/Cr, and UCa/Cr and a significant decrease in eGFR in beta-TM patients who underwent splenectomy compared to those who had splenomegaly, as shown in Table 6.

Discussion
In beta thalassemia syndromes, iron overload due to regular blood transfusion leads to iron deposition in renal proximal tubules, glomeruli, and interstitium, resulting in tubular atrophy, glomerulosclerosis, and interstitial fibrosis [16].
Analytical interpretation of the current study results showed a significant elevation of serum ferritin and serum creatinine in beta thalassemia major patients. Our patients are on regular deferasirox as an iron chelation therapy. Ponticelli et al. [17] demonstrated that renal involvement in children with beta-TM was caused by chronic anemia, increased iron deposition, and iron chelator toxicity. Vichinsky [18] found that deferasirox increases proteinuria and serum creatinine and may cause renal failure and found a reversible non-progressive elevation of serum creatinine in about 14% of beta-TM patients.
The current study results showed decreased estimated GFR in eleven percent of thalassemic patients (n = 11). Glomerular capillary wall stretching, and subsequent endothelial and epithelial injury, can induce the transudation of macromolecules into the mesangium and glomerular dysfunction, which may cause a progressive decline in GFR [19]. Bekhit et al. [20] showed that the serum creatinine was higher in 40% of thalassemic compared with controls, but within a normal range, while the GFR was significantly lower in thalassemic patients compared with the controls. In addition, other studies found classical kidney function impairment (elevated serum creatinine and decreased eGFR) and early glomerular function impairment markers in children with beta-TM compared with controls [21,22]. On the other hand, some studies did not illustrate any difference in kidney function and GFR [23,24].
The present study showed hypercalciuria and an elevated urinary uric acid/creatinine ratio and urinary protein/creatinine ratio in children with beta-TM. Many studies have reported hypercalciuria, which was tested by the urine calcium to creatinine ratio, which is consistent with proximal tubulopathy and correlates with blood transfusion burden and deferasirox as iron chelation therapy [25][26][27]. Aldudak et al. [28] illustrated an increased level of urinary protein/creatinine ratio in children with beta-TM. Bekhit et al. [20] demonstrated increased urinary calcium in 26% of patients and increased urinary uric acid in 38% of patients. This may be explained by a decreased reabsorption of filtered uric acid from damaged renal tubules, combined with rapid erythrocyte turnover and excess uric acid urinary excretion [21,28].
Analytical interpretation of urinary kidney markers of the current results showed a significant increase in UNAG, UNAG/Cr, UKIM-1, and UKIM-1/Cr. Tantawy et al. [29] reported that 58.1% of patients with beta thalassemia had elevated levels of urinary NAG.
Smolkin et al. [30] showed an increased urinary NAG and UNAG/Cr ratio in patients with β thalassemia major and intermedia. In addition, some studies have demonstrated an increase in urinary NAG [31,32].Şen et al. [5] showed a significant increase in UNAG/CR ratio in patients with beta thalassemia compared with controls, but no significant differences were reported in urinary KIM-1-to-creatinine (UKIM-1/Cr).
The current study showed a significant positive correlation between UNAG, UKIM-1, UNAG/Cr, and UKIM-1/Cr as regards serum ferritin, serum creatinine, and serum urea among patients with beta-TM. In addition, there was a negative correlation between urinary markers and eGFR. Moreover, there was a significant positive correlation between uNAG, uNAG/Cr, and uKIM-1/Cr as regards urinary protein/Cr, U Ca/Cr, UNa/Cr, and UK/Cr. Sen et al. [5] illustrated a significant positive correlation between urinary biomarkers as regards the UCa/Cr, UNa/Cr, UK/Cr and Uuric acid/Cr as urinary markers and urinary solutes represent the renal tubular functions. Nafea et al. [33] reported that the UKIM-1/Cr level was significantly higher in thalassemic patients on deferasirox therapy than patients on deferoxamine and deferiprone therapy. Dou et al. [34] demonstrated that thalassemic patients who took deferasirox were more likely to have increased serum creatinine. Alkhabori et al. [35] illustrated the nephrotoxicity of deferasirox in 10% of children with beta-TM patients who discontinued deferasirox, as it caused a persistent elevation in serum creatinine. Deferasirox nephrotoxicity was observed in 1 of 10 patients. Nephrotoxicity may be in the form of tubulopathy, glomerulonephritis, interstitial nephritis, and even renal failure [36]. Sánchez-González et al. [37] reported that deferasirox administration leads to partial necrosis in renal tubules and increased UKIM-1, protein, and glucose secretion. Martin-Sanchez et al. [38] illustrated that deferasirox had a direct toxic effect on tubular cells and induced mitochondrial dysfunction. Balocco et al. [39] illustrated that the alternate use of deferasirox and deferiprone allowed for drug tolerability with no adverse effects and a similar efficacy in reducing serum ferritin. Ponticelli et al. [17] showed that the deferasirox dose should be decreased by 10 mg/kg if serum creatinine rises 33% above pretreatment values and above the age-appropriate upper limit of normal at two consecutive visits. On the other hand, Aldudak et al. [28] reported normal serum creatinine in patients receiving deferasirox therapy. Our study showed a significant positive correlation between serum cyatatin C, UNAG, UKIM-1 and the blood transfusion index per year among patients with beta-TM. Behairy et al. [40] found a significant positive correlation between serum cyatatin C and the frequency of blood transfusion per year in patients with beta-TM. We found no significant correlation between renal markers and body weight among patients with beta-TM. Baxmann et al. [41] showed no significant correlation between serum cystatin C as regards body weight, body muscle cell and fat-free mass.
The present study showed a significant increase in serum creatinine, Uprotein/Cr, UCa/Cr, UNAG, UNAG/Cr, and UKIM-1/Cr and a decrease in eGFR in β-TM patients who underwent splenctomy. Tantawy et al. [29] illustrated a prominent elevation of NAG in splenectomized patients and proved that splenectomy was an independent risk factor for renal tubular abnormalities. Ongazyooth et al. [42] found that tubular defects were more common in splenectomized patients. In addition, Bekhit et al. [20] reported higher levels of urinary NAG in splenectomized patients than in patients with splenomegaly. Belhoul et al. [43] stated that, after splenectomy, the transaminases were higher and serum albumin was lower compared with nonsplenectomized patients. This may be explained by the fact that the rates of iron-overload-related organ damage in splenectomized patients are higher than those in non-splenectomized patients, as the primary underlying pathology of red cell dysfunction persists after splenectomy. Ismail et al. [44] showed an elevated serum creatinine and Tantawy et al. [29] found an elevation of total urinary protein in splenectomized patients than in patients with splenomegaly.
Our results showed a significant increase in serum cystatin-C among beta-TM patients, with a significant positive correlation with serum ferritin and creatinine and a significant negative correlation with eGFR. Economou et al. [26] found that 36% of beta-TM patients had increased serum cystatin-C. Moreover, there was elevated serum cystatin-C in beta-TM patients with or without chelation therapy, and there was a significant positive correlation with serum creatinine and a negative correlation with eGFR [21]. Ali and Mahmoud [45] showed significantly higher levels of serum cystatin-C, with a significant positive correlation as regards serum creatinine and serum ferritin in children with beta-TM compared with a control group and a significant, strong negative correlation between serum cytatin-C and eGFR. Furthermore, Elbedewy et al. [46] showed a significant negative correlation between serum cystatin C and eGFR in adult patients with beta-TM.
Afshan et al. [47] found a significant moderate negative correlation between kidney T2* relaxation time by magnetic resonance imaging of kidneys (MRI) and the serum ferritin in patients with beta-TM. Therefore, MRI T2* may estimate the renal iron burden in thalassemic patients.
Strengths of the current study: we have analyzed the relationship between urinary kidney injury molecules and regular renal parameters such as serum ferritin, serum creatinine, and serum urea. Furthermore, we showed that an increase in UNAG, UNAG/Cr, UKIM-1/Cr, and Uprotein/Cr was found in beta-TM with splenectomy. Glomerular dysfunction was evaluated by serum cystatin-C, the glomerular filtration rate, serum creatinine, and the urinary protein creatinine ratio.
Recommendation: A serial assay of UNAG should be measured to evaluate its possible prognostic value in AKI in beta-TM patients.

Conclusions
Patients with beta thalassemia are at high risk of renal glomerular and tubular impairment, which are not found in routine renal investigations. Serum cystatin-C is a good predictive marker in the evaluation of glomerular dysfunction. Urine concentrations of NAG and KIM-1 represent sensitive, specific, and highly predictive early biomarkers for acute renal injury in patients with beta TM when subclinical kidney injury or dysfunction is expected before serum creatinine increases. Data Availability Statement: Data available on request due to restrictions e.g. privacy or ethical.