Globally, chronic kidney disease (CKD) is recognized as an important public health concern as it significantly contributes to health care expenses, morbidity as well as mortality from non-communicable disease [1
]. Chronic kidney disease is defined as the existence of kidney impairment and diminished function [e.g., glomerular filtration rate (GFR) less than <60 mL/min/1.73 m2
or albumin excretion rate ≥ 30 mg/24 h] lasting greater than three months [2
]. Defining and staging chronic renal failure is contingent on assessing the GFR, and an estimate is provided by creatinine clearance. Serum creatinine, which is used to determine the creatinine clearance, is not a very sensitive indicator of renal disease and there are a number of limitations associated with obtaining an accurate measurement of the urine creatinine concentration [3
A practical approach in determining the GFR in clinical practice is the estimated glomerular filtration rate (eGFR) that is determined by utilizing equations and which provide a more accurate and precise assessment of renal function [4
]. These formula-based equations such as Modification of Diet in Renal Disease (MDRD) and Cockcroft-Gault are employed in the determination of eGFR and utilized patient-related parameters such as gender, age, body weight, ethnicity and serum creatinine concentrations [5
]. These GFR-based formulas, as well as the CKD-EPI equation, are endorsed by regulatory agencies and there are clinical practice guiding principles for the routine assessment of GFR. Measured GFR utilizing endogenous or exogenous markers is commended as a confirmatory test when more accurate evaluation is necessary [6
Cystatin C, an endogenous marker for measuring GFR is a 13 kD non-glycosylated simple protein that is synthesized and secreted by all nucleated cells at a continual rate. This endogenous serum biological marker possesses a cationic nature and thus is freely filtered via the glomerulus [7
]. It possesses other characteristics that are present in steady concentrations in the plasma, not secreted by tubular cells, and compared with creatinine, it is less affected by non-renal factor influences such as gender, age, muscle mass and analytical interfering substance during its measurement [8
]. Studies have demonstrated that serum cystatin C is a sensitive biomarker for detecting changes in GFR and identifying preclinical renal disease, particularly in diabetic patients with a normal serum creatinine concentration [9
]. Furthermore, cystatin C is a better discriminator between type 2 diabetic patients with normoalbuminuria or microalbuminuria and its quantification is useful in the early identification of diabetic nephropathy allowing for the appropriate intervention and management [10
There is increasing data that the synthesis of cystatin C could be activated by thyroid hormones and it may readily respond to minor alterations in the function of the thyroid glands [11
]. The kidneys play a vital role in the metabolism and elimination of thyroid hormones. In chronic renal disease, there is significant reduction in kidney function accompanied by iodine retention, metabolic acidosis and alteration of thyroid function [13
]. There are a number of observational studies that have reported various modifications of thyroid function test profile in chronic kidney disease patients [14
]. Thyroid dysfunction ensues in chronic kidney disease with a significant decrease in total and free T4, total and free T3 and elevated TSH that is associated with increasing renal impairment and reduced GFR [14
]. In chronic kidney disease patients that present with overt or subclinical hypothyroidism, there was a positive correlation between serum urea and creatinine concentrations with TSH, and negative correlation between these renal function biomarkers and FT4 and FT3 [14
]. However, there are studies that have reported unchanged TSH [18
] as well as normal TSH and FT4 [20
] in chronic kidney disease patients compared to controls. Notwithstanding these studies, thyroid status in patients with chronic renal disease remains inconclusive.
The thyroid gland may be affected via immune-mediated developments as well as vitamin D impeding the dose-dependent uptake of iodide stimulated by thyroid stimulating hormone [21
]. Vitamin D is a fat-soluble steroid prohormone with a cytosolic receptor produced from 7-dehydrocholesterol in the skin following sunlight exposure with the subsequent synthesis of vitamin D3 [22
]. Vitamin D3 is transported to liver where metabolic activation involves hydroxylation at position 25 to form 25-hydroxyvitamin D, which is consequently converted to the biologically active 1,25 dihydroxyvitamin D by the catalytic activity of 1α-hydroxylase present in the kidney’s proximal tubular epithelial cells [23
]. Vitamin D insufficiency or deficiency is very prevalent amongst chronic kidney disease patients and there appears to be an inverse association between serum concentrations of 25-hydroxyvitamin D and renal function [24
]. The causes of vitamin D deficiency is due to a number of factors including: (i) increased fibroblast growth factor 23 (FGF23) levels that decrease the expression and activity of 1α-hydroxylase and stimulate 24-hydroxylase that degrades 1,25 dihydroxyvitamin D [25
] (ii) increase phosphate retention that inhibit the activity of 1α-hydroxylase (iii) reduced renal mass and less proximal tubular cells with 1α-hydroxylase activity and (iv) renal loss of loss of 25-hydroxyvitamin D and its binding, particularly in chronic kidney disease patients with proteinuria [26
Circulating 25-hydroxyvitamin D levels is regarded as a sensitive determination of vitamin D status [27
] and the prevalence of vitamin D insufficiency and deficiency was found to be higher in type 2 diabetic patients with chronic kidney disease and albuminuria compared to those without albuminuria [28
]. In a recent study, lower 25-hydroxyvitamin D concentration was associated with reduced GFR in type 2 diabetic patients, but there was no difference between patients with elevated urinary albumin excretion compared to those with normoalbuminuria [29
This study aimed to determine and compare the levels of serum cystatin C in patients with chronic kidney disease (stages 1–5) based on creatinine clearance values and association with serum creatinine. This study is also aimed at determining the prevalence of vitamin D deficiency or insufficiency and thyroid dysfunction in these patients.
2. Materials and Methods
2.1. Patient Population and Ethical Approval
Patients from across Jamaica, and sometimes other Caribbean islands, were referred to the renal clinic at the University Hospital of the West Indies (UHWI) for management of CKD. This was a cross-sectional study conducted between February 2016 and May 2016. All patients attending the renal clinic were approached. After explaining the aims of the study, patients who agreed to participate were recruited. They were assigned data entry numbers in order to maintain confidentiality.
We received approval from the University of the West Indies/University Hospital of the West Indies Faculty of Medical Sciences Ethics Committee and the protocol for the conduct of research outlined were followed. Informed consent was obtained after which the patients were given directives on the proper collection of a 24 h urine sample. They were asked to repeat the instructions to ensure comprehension.
Fisher’s z test was used to estimate the sample size for a one-sample correlation test. The estimated sample size was 259 (using alpha = 0.05, power = 0.9, delta = 0.2, r° = 0 and ra = 0.2). Between February and May 2016, 140 patients from 18 to 97 years of age were recruited. The study included cases from all Jamaica encompassing those from western parishes, St. James, Hanover, Westmoreland and St. Elizabeth, who were more likely to visit the Cornwall Regional Hospital in St. James due to proximity.
2.2. Demographic Data Collection
Demographic data, including age, gender, date of birth, height, weight, marital status, education level and employment status were collected. Height and weight were measured in the renal clinic. The date of CKD diagnosis, age, stage and cause of renal impairment at diagnosis as well as any comorbid conditions were ascertained from the patients themselves and/or confirmed by medical record search.
2.3. Sample Collection and Preparation
Samples were processed within three hours of receipt in the Chemical Pathology Laboratory at the Department of Pathology, The University of the West Indies. Specimens were stored at −70 °C for assays not completed within 24 h. Serum and urine biochemistry tests with the exception of urine protein was performed on the cobas 6000 (Roche/Hitachi, Roche Diagnostics, Indianapolis, IN, USA) analyser.
2.4. Serum Assays Used to Determine Analytes
Seven (7) mL of blood was obtained from the patient by venipuncture from the antecubital fossa or another convenient site. Samples were obtained using a vacutainer system. Samples were allowed to clot for 30 min, separated by centrifugation at 3500 rpm for 5 min then the serum was aliquoted.
Analytes measured were serum creatinine, urea, electrolytes, albumin, uric acid, calcium, phosphorus, cystatin C, 25-hydroxyvitamin D [25-(OH)D], free thyroxine (FT4) and thyroid stimulating hormone (TSH).
An IDMS traceable Jaffé method, a kinetic colorimetric assay, was used to measure serum creatinine [30
]. Cystatin C was assayed by a particle enhanced immunoturbidimetric assay, the Tina-quant Cystatin C Gen. 2, which is standardized against ERM-DA471/IFCC (The International Federation for Clinical Chemistry and Laboratory Medicine) reference material. Anti-cystatin C-coated latex particles bind cystatin C in the specimen. The degree of turbidity at 546 nm is equivalent to the cystatin C concentration [31
Albumin was determined based on its binding to bromcresol green at pH 4.1 with the formation of a blue-green complex. The color intensity photometrically measured at 570 nm is equivalent to the level of albumin in the serum [32
]. The serum calcium was determined due to the reaction of calcium ions with 5-nitro-5’-methyl-BAPTA (NM-BAPTA) under alkaline conditions to form a complex, which is further complexed with ethylenediaminetetraacetic acid (EDTA) in a second reaction. The levels of calcium are determined by the difference in absorbance at 340 nm [33
The serum uric acid was determined as the analyte is cleaved by uricase to form allantoin and hydrogen peroxide. Peroxidase and N
-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS) catalyze the oxidation of 4-aminophenazone to produce a dye (quinone-diimine). The color intensity of the dye is proportionate to the uric acid concentration. The increasing absorbance at 546 nm is measured [34
Free thyroxine (FT4) was measured by an electrochemiluminescence immunoassay (ECLIA) (Roche Diagnostics, Indianapolis, IN, USA), which uses a specific anti-T4 antibody labelled with a ruthenium complex. A competitive method is employed and the concentration of T4 is inversely proportional to the chemiluminescent emission [35
]. Thyroid stimulating hormone (TSH) was measured by an assay that employs a sandwich principle. Two monoclonal antibodies specific for TSH, one biotin-labelled and the other ruthenium-labelled react with TSH in serum to form a sandwich complex. Streptavidin-coated micro-particles are added and binds biotin. Microparticles are bound magnetically to an electrode and a reaction is carried out. The concentration of TSH is proportional to the chemiluminescent emission [36
Total 25-hydroxyvitamin D [25-(OH)D] was measured by a competitive immunoassay that utilizes a vitamin D binding protein as a capture protein. Pretreatment reagents release hydroxylated vitamin D from vitamin D binding protein. With the addition of a ruthenium-labelled VDBP (VDBPr), a complex is formed with the hydroxylated vitamin D. Microparticles coated with streptavidin and biotin-labelled 25-(OH)D [25-(OH)Db] are added and attach to unbound VDBPr. A complex is formed. The micro-particles are bound electromagnetically and unbound substances are removed by washing. Chemiluminescence is induced by the application of a voltage. The emission is quantified by a photomultiplier and is inversely proportional to the concentration of 25-(OH)D [37
Patients were placed into the five stages of CKD determined by 24 h creatinine clearance (CrCl).
2.5. Statistical Analysis and Predictor Levels of Cystatin C, Vitamin D and Thyroid Status
The data analysis was conducted using the IBM Statistical Programme of the Social Science (SPSS) version 22 and Microsoft Excel. Demographic characteristics are presented as mean ± standard deviation (SD). The frequencies of different causes of CKD were determined. The Pearson coefficient (r) was used to assess the correlation of results by serum cystatin C and CrCl.
Cystatin C levels were divided into normal and abnormal based on the quoted normal range of 0.61–0.95 mg/L and compared to serum creatinine using the independent t-test. Vitamin D levels were (i) deficient < 20 ng/mL, (ii) insufficient 20–29 ng/mL and (iii) sufficient ≥ 30 ng/mL. Mean values according to gender and stages of kidney disease were ascertained. The association of hypovitaminosis D with diabetes mellitus was determined by the independent t-test. A p-value < 0.05 (two-tailed) indicated statistical significance.
The prevalence of thyroid disorders in the subjects was assessed. Patients were classified as (i) overt hyperthyroidism (TSH < 0.4 mU/L and FT4 > 1.9 ng/mL), (ii) sub-clinical hyperthyroidism (TSH < 0.4 mU/L and FT4 normal), (iii) overt hypothyroidism (TSH > 4.0 mU/L and FT4 < 0.8 ng/mL), and (iv) sub-clinical hypothyroidism (TSH > 4.0 mU/L and FT4 normal). The mean values of FT4 and TSH according to gender as well as the mean values according to CrCl compared. A p-value < 0.05 (two-tailed) indicated statistical significance.