Online First

2021 : Volume 1, Issue 1

Abstract

Background: Klotho is a trans membrane protein expressed in the renal tubules and serves as an obligatory co-receptor for fibroblast growth factor 23(FGF23) to aid in phosphorus excretion. Prior studies have shown FGF23 resistance in sickle cell disease (SCD). The purpose of the study is to investigate urinary klotho/creatinine (Ur Kl/Cr) in pediatric SCD with normal traditional markers of renal function (eGFR > 90 ml/min and no micro albuminuria) and to compare it with the healthy control population.

Methods: Cross-sectional observational study to compare Ur Kl/Cr in pediatric SCD and controls. To do a subgroup analysis among the study population to assess the effect of hydroxyl urea (HU) on Ur Kl/Cr.

Results: 20 controls and 22 pediatric SCD were enrolled. In the SCD group, 13 were on treatment with HU. The baseline characteristics of the study and control group were the same. Wilcoxon rank-sum test was used to compare the levels of Ur Kl/Cr ratio between SCD and control. For P value of 0.05, the levels of Ur Kl/Cr were statistically significantly higher in the sickle cell group (752.7 ± 1101.0) over the control group (216.8 ± 225.3). Subgroup analysis in the SCD group showed high Urinary Kl excretion in the non-HU group (1346.7 ± 1523.4) vs. non HU group (341.4 ± 355.3) but not statistically significant.

Conclusion: Children with SCD tend to have increased secretion of Ur Kl/Cr compared to control likely due to tubular receptor resistance. HU may reverse this phenomenon by its effect on preventing tubular damage.

Introduction

Sickle cell nephropathy (SCN) refers to a spectrum of renal abnormalities causing increased mortality and morbidity in patients with sickle cell disease (SCD) [1]. The tools available for early detection of SCN remain limited. Glomerular filtration rate (GFR) and albuminuria are two widely used clinical screening tools for early detection of SCN. Estimated glomerular filtration rate (eGFR) calculated by modified Schwartz formula [2] has been found to significantly overestimate GFR when compared to qualitative GFR measurements in the sickle cell population. [3] Albuminuria defined as urine albumin to creatinine ratio greater than 30 mg/g of creatinine is widely considered an early marker of glomerular damage. Albuminuria in SCD occurs in about 16 to 27% of patients [4-6] and is linked to widened inter podocyte radius and reduced podocyte numbers by the sickled red blood cells. [7] The typical age of onset of albuminuria is around 7 years of age though there are studies that show renal damage has occurred by the time proteinuria is detected. [8] There is a distinct need for novel biomarkers that could pick up early renal involvement in SCD over the currently used screening tools. Klotho is an anti-aging gene that encodes a single-pass trans membrane protein whose predominant expression is in the renal tubules. The extracellular domain of klotho protein is clipped off the cell surface and secreted into blood and urine by ectodomain shedding. [9] The membrane klotho acts as an obligatory co-receptor for bone hormone fibroblast growth factor 23 (FGF23) and plays an important role in urine phosphorus excretion. Secreted klotho acts as a humeral factor in regulating multiple ion channels and growth factors and plays a role in preventing oxidative damage to cells. Defects in Klotho or FGF23 expression have led to accelerated aging syndrome documented in animal studies [10] revealing an unexpected link between phosphate homeostasis and aging.

Urinary Klotho levels have been shown to decline with falling glomerular filtration rate (GFR) in chronic kidney disease (CKD) [11,12] Prior studies have documented irregularities in phosphate metabolism, decreased urine phosphorus excretion, and possible FGF23 resistance in children with sickle cell anemia [13]. The purpose of this study is to investigate urinary klotho/creatinine (Ur Kl/Cr) in children with sickle cell anemia with current clinical markers of normal renal glomerular function (eGFR > 90 ml/min and no microalbuminuria) and to compare it with the healthy control population.

Hypothesis

We hypothesize that renal damage in sickle cell anemia starts much sooner than noted by current traditional markers available (eGFR, micro albuminuria) due to recurrent vaso-occlusive episodes in the renal tubules. As such we anticipate urinary klotho abnormalities in these children even if traditional markers show no evidence of renal damage.

Method/Study Design

Cross-Sectional Observational Study

Children with sickle cell anemia who attend the sickle cell clinic for their regular care will be enrolled in the study after obtaining informed consent from their parents. These children will undergo a baseline evaluation including history and physical examination and will have their urine and blood samples collected the same day. The control population will include similar age and gender-matched children with no sickle cell anemia or renal disease.

Inclusion Criteria

Children (age<18 years) with SCD and normal renal function as documented by eGFR ? 90 ml/min/1.73 m2 by serum creatinine and no proteinuria/albuminuria were included in the study. Subgroups within the included children will be classified further into hydroxyl urea/non-hydroxy urea groups.

Exclusion Criteria

Children with SCD, who had other co-existent hemoglobinopathies, renal function by GFR<90ml/min/1.73 m2, presence of proteinuria or albuminuria were excluded. Also, children with SCD who had a known renal abnormality or with a history of acute renal injury in the last 3 months were excluded.

Methods

Hematologic parameters measured included complete blood count (CBC), reticulocyte count, lactate dehydrogenase (LDH) levels, and 25 hydroxy Vit D levels. Renal function (eGFR) was calculated using the modified Schwartz formula from serum creatinine measurements. A random clean catch urine sample was collected for measurement of protein, albumin, and creatinine. Both urine and serum creatinine were measured by the standard enzymatic method. Urine samples were thawed and centrifuged at 1500 xg for 10 minutes at 4°C to pellet the debris and cells [14]. The supernatant was carefully transferred to another tube and was used for all the assays.

Assays

?-Klotho was measured using a solid-phase sandwich ELISA (Immuno-Biological Laboratories Co. Ltd, Gunma, Japan). This assay has a sensitivity of 6.15 pg/ml. Micro-albumin was measured using a competitive ELISA (Orgentec Diagnostika, Mainz, Germany). The sensitivity for this kit is 0.5 µg/ml. For detection of urinary creatinine, we used a standard picric acid-based colorimetric assay (Creatinine urinary detection kit, Invitrogen, Carlsbad, USA). The analytical sensitivity for this assay is 0.019 mg/dL. We estimated total protein using an improved pyrogallol red-molybdate protein dye-binding assay (Quantichrom Total protein assay kit, BioAssay Systems, Hayward, USA). This assay has a 1-20 mg/dL protein detection range. All the assays were performed strictly as per the manufacturer's instructions. Samples and standards in all the assays were analyzed in duplicate and the mean values were used for calculations.

Results

There were a total of 20 control and 22 children with sickle cell disease enrolled in the study. The baseline characteristics of the control group and sickle cell group did not differ significantly and are provided in Table 1. The mean age of the sickle cell cohort was 9.6 ± 4.8 years with 40% being males. GFR and urine pr/cr ratio were reported normal in both sickle cell and control groups. In the sickle cell group, 13/22 children were on hydroxyl urea. Four children, all in the non-hydroxy urea group were on a chronic transfusion protocol.

 

Control

Sickle cell

P value

N

20

22

 

Age (Mean ± SD)

11.9 ± 3.6

9.6 ± 4.8

0.09

Sex

9 (M) 11 (F)

9 (M) 13 (F)

 

GFR (Mean ± SD)

107 ± 20.1

111.2 ±18

0.87

Ur Pr/Cr (Mean ± SD)

0.07 ± 0.03

0.1 ± 0.06

0.66


Table 1: Baseline characteristics.

A descriptive analysis of the measured parameters in the sickle cell group is provided in Table 2. Patients were anemic as expected with mean Hb at 9.5 gms/dl. GFR, Urine Pr/Cr, and Urine MA/Cr ratio were all reported in the normal range. Vitamin D deficiency was noted in most of the study population with a mean value of 13.6 ng/ml.

Variables

N=22

Normal Value

Age (years)

Median (Min – Max)

9.5 (2.0 – 17)

Mean ± SD

9.6 ± 4.8

Median

9.5

Sex

Female

13 (59.1%)

Male

9 (40.9%)

GFR (ml/min/1.73 m2)

 

>90 ml/min/1.73 m2

Median (Min – Max)

108(91-165)

Mean ± SD

111.2± 18

Ur Pr/Cr

< 0.2

Median (Min – Max)

0.1(0.02 -0.2)

Mean ± SD

0.1 ± 0.06

Ur MA/Cr (mg/g of Cr)

< 30 mg/g of Cr

Median (Min – Max)

12.6( 4.5 -21.7)

Mean ± SD

14.8± 8.1

Hb (g/dl)

10.8-13.3 g/dl

Median (Min – Max)

9.5 (6.9 - 11.8)

Mean ± SD

9.5 ± 1.4

Hct (%)

34-40%

Median (Min – Max)

26.5 (20.4 - 32.3)

Mean ± SD

26.4 ± 3.8

Reti count (%)

1-1.9%

Median (Min – Max)

7.6 (2.8 - 23.9)

Mean ± SD

8.9 ± 5.7

Vit D (ng/ml)

30-100 ng/ml

Median (Min – Max)

12.0 (7.0 - 34.0)

Mean ± SD

13.6 ± 6.7

Serum Cr (mg/dl)

0.2-0.7 mg/dl

Median (Min – Max)

0.5 (0.4 - 0.7)

Mean ± SD

0.5 ± 0.1

Ferritin (ng/ml)

22-274 ng/ml

Median (Min – Max)

169.0 (9.0 - 522.0)

Mean ± SD

184.0 ± 136.1

Alpha_ Kl/Cr

Median (Min – Max)

380.7 (15.9 - 4115.3)

Mean ± SD

752.7 ± 1101.0


Table 2: Descriptive analysis of sickle cell cohort.

Wilcoxon rank-sum test was used to compare the levels of urinary Kl/Cr ratio between Sickle cell and control group. At a significance level of 0.05, the levels of urine klotho to creatinine ratio are statistically significantly higher in the sickle cell group over the control group. (Table 3) In the sickle cell group, the difference between Hydroxy urea vs. no Hydroxy urea subgroup analysis showed no statistically significant difference using a significance level of 0.05 (Table 4).

Variables

Control  N=20

Sickle  N=22

P Value

Alpha Kl/Cr

 

 

0.045 W

N

20

22

 

Median

134.7

380.7

 

Mean ± SD

216.8 ± 225.3

752.7 ± 1101.0

 

Missing

0

0

 

The effect size is 0.31, which indicates a medium difference between sickle group and control group.


Table 3: Urinary alpha Kl/Cr ratio compared between control and sickle cell group.

Variables

Hydroxyurea  N=13

Non- Hydroxyurea N=9

P Value

Alpha Kl/cr

0.051 W

N

13

9

Median

140.4

938.9

Mean ± SD

341.4 ± 355.3

1346.7 ± 1523.4

Missing

0

0

The effect size is 0.41, which indicates a medium difference between sickle group and control group.


Table 4: Sub group analysis of urinary Alpha Kl/Cr ratio among sickle cell cohort.

Discussion

Sickle cell disease (SCD) is the most common inherited hemoglobinopathy affecting about 300,000 newborns annually worldwide. [15] Renal manifestations of SCD start early in life and present as disorders of both tubular and glomerular dysfunction progressing to chronic kidney disease and End Stage renal disease. [16] Tubular dysfunction with impaired urinary concentrating ability is the earliest sign of renal involvement and is present universally in most SCD patients. [17] The unique environment of the renal medulla with the low oxygen tension, hyperosmolar environment, and low pH is a perfect setup for recurrent sickling episodes causing ischemic injury to the vascular architecture. The vaso-occlusion and micro infarction of the renal medulla play a central role in early tubular dysfunction and eventual glomerular hyper filtration and injury. [18,19] Glomerular involvement in SCD is characterized by an early increase of GFR associated with micro- macro albuminuria progressing to loss of GFR and chronic renal failure [20-22]. Albuminuria remains the best available early clinical marker for renal involvement in SCD.

Hydroxy urea remains the cornerstone of SCD therapy in preventing recurrent vaso-occlusive events and chronic organ damage. Hydroxy urea has been shown to decrease albuminuria in patients with SCD. [23] Early initiation of hydroxyl urea has also shown to increase urine-concentrating ability in infants [24] with some studies claiming a decrease in glomerular hyperfiltration [25]. Klotho was initially identified as an anti-aging factor. [26] Since then, more research has been done and its role in renal protection against ischemia-induced reperfusion injury has been documented. [27,28] Membrane Klotho serves as a receptor for FGF23 while the secreted Klotho acts as a paracrine and endocrine factor affecting multiple organs including bones, kidneys, and endothelium. [29,30] Experimental models have shown that overexpression of klotho can reverse acute kidney injury (AKI) caused by nephrotoxins [31]. Urine klotho levels have been investigated as early markers of acute kidney injury in post-cardiac surgery patients in adult literature [32,33]. Urine klotho levels in these studies tend to increase quickly in the group that ended with AKI. Klotho protein is known to be expressed in the brush border of the proximal tubules and early loss and shedding of the brush border cells in urine could have contributed to the acute increase. As AKI progress and with renal tubular necrosis, urine klotho levels continue to rise. With progression to CKD and declining GFR, studies have shown a decrease in urine klotho excretion [34,35].

This cross-sectional study showed that the urinary Kl/Cr ratio was higher in children with sickle cell when compared to the control population. This is likely due to tubular damage and end organ resistance, which could explain the increased klotho production and urinary secretion in the sickle cell group. Interestingly the sub-group analysis shows that children on hydroxyl urea treatment in the sickle cell arm have urinary klotho levels comparable to the control group. This is suggestive of a renal protective mechanism of hydroxyl urea likely by preventing the repetitive vaso-occlusive episodes in the renal medulla. The small number of subjects enrolled likely contributed to the non-statistical difference between the hydroxyl urea and non-hydroxy urea groups.

The limitations of our study include small sample size and the cross-sectional nature of the study. Further longitudinal clinical trials can provide more information on the usefulness of urine Kl/Cr ratio in SCD.

Conclusion

Traditional markers such as GFR, proteinuria, and albuminuria lag behind actual renal involvement in sickle cell anemia. In our study, we have shown that children with SCD with normal traditional markers of glomerular function tend to have increased secretion of alpha klotho in urine when compared to a control population. To our knowledge, this is the first reported data of urine klotho in the pediatric literature. Hydroxy urea may reverse this phenomenon likely related to preventing, modifying tubular damage. Further longitudinal studies could help establish the findings from this pilot study.

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CORRESPONDENCE & COPYRIGHT

Corresponding Author: Vimal Master Sankar Raj, Division of Pediatric Nephrology, University of Illinois College of Medicine, USA.

Copyright: © 2021 All copyrights are reserved by Vimal Master Sankar Raj, published by Coalesce Research Group. This This work is licensed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

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