Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2014  |  Volume : 27  |  Issue : 1  |  Page : 60--65

Evaluation of serum cystatin C as an indicator of early renal function decline in type 2 diabetes


Nabil A. El-Kafrawy1, Ahmed A. Shohaib1, Samar M. Kamal El-Deen2, Hani El Barbary1, Ameer S. Seleem1,  
1 Department of Internal Medicine, Faculty of Medicine, Menoufyia University, Shibin Elkom, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufyia University, Shibin Elkom, Egypt

Correspondence Address:
Ameer S. Seleem
MBBCh, Shopra qas, El-Santa, Gharibyia
Egypt

Abstract

Objectives This study was conducted to evaluate the clinical usefulness of cystatin C levels in the serum in predicting renal impairment in patients with type 2 diabetes. Background In clinical practice, the glomerular filtration rate is often estimated from plasma creatinine levels. Several studies have shown cystatin C to be a better marker for the diagnosis of impaired renal function. Materials and methods Plasma samples were obtained from 20 healthy individuals and 40 patients with diabetes mellitus type 2 for the determination of the levels of creatinine and cystatin. In addition, we classified all participants according to the urine albumin/creatinine ratio. Results Patients were categorized into the following groups depending on their urine albumin/creatinine ratio (mg/g creatinine): macroalbuminuric, microalbuminuric, and normoalbuminuric. There were no significant differences in age and sex between the three groups. However, the estimated glomerular filtration rate was significantly lower in the macroalbuminuric group compared with the microalbuminuric and normoalbuminuric groups, and cystatin C showed a highly significant difference in detecting early decline in diabetic patients. Conclusion From this study, we concluded that estimation of the serum cystatin C level is a useful, practical, and noninvasive tool for early detection of renal impairment in the course of diabetes.



How to cite this article:
El-Kafrawy NA, Shohaib AA, Kamal El-Deen SM, Barbary HE, Seleem AS. Evaluation of serum cystatin C as an indicator of early renal function decline in type 2 diabetes.Menoufia Med J 2014;27:60-65


How to cite this URL:
El-Kafrawy NA, Shohaib AA, Kamal El-Deen SM, Barbary HE, Seleem AS. Evaluation of serum cystatin C as an indicator of early renal function decline in type 2 diabetes. Menoufia Med J [serial online] 2014 [cited 2024 Mar 28 ];27:60-65
Available from: http://www.mmj.eg.net/text.asp?2014/27/1/60/132748


Full Text

 Introduction



Diabetes mellitus (DM) is a syndrome associated with chronic hyperglycemia due to relative insulin deficiency, resistance, or both. It affects more than 120 million people worldwide, and it is estimated that it will affect 370 million people by the year 2030. Diabetes is usually irreversible; its late complications result in reduced life expectancy and have major health costs. These include macrovascular disease, leading to an increased prevalence of coronary artery disease, peripheral vascular disease, and stroke, and microvascular damage, causing diabetic retinopathy and nephropathy [1].

Chronic kidney disease involves progressive and irreversible loss of renal function, which begins with the appearance of proteinuria and elevated serum creatinine levels, representing a decrease in the glomerular filtration rate (GFR), and finally results in complete loss of kidney function, that is, end-stage renal disease [2].

A higher proportion of individuals with type 2 diabetes are found to have diabetic nephropathy shortly after the diagnosis of their diabetes, because of the prevalence of diabetes for many years before its diagnosis [3].

GFR is considered to be the best marker of renal function, and serum creatinine level is the most commonly used biochemical parameter to estimate GFR in routine practice. However, there are some shortcomings to the use of this parameter. Factors such as sex, age, muscle mass, and protein intake can influence serum creatinine levels, leading to inaccurate estimation of GFR. Normal serum creatinine levels may be observed in individuals with significantly impaired GFR [4].

The presence of albuminuria may be less specific to the presence of diabetic nephropathy; about 20-40% of type 2 diabetic patients with microalbuminuria develop overt nephropathy, and most patients show progression to end-stage renal disease [3].

Cystatin C is a small 13-kDa protein that is a member of the cysteine proteinase inhibitor family; it is produced at a constant rate by all nucleated cells. Because of its small size, it is freely filtered by the glomerulus, and it is not secreted but is fully reabsorbed and broken down by the renal tubules. This means that the primary determinate of blood cystatin C levels is the rate at which it is filtered at the glomerulus, making it an excellent GFR marker. A recent meta-analysis demonstrated that serum cystatin C is a better marker of GFR than serum creatinine [5].

Cystatin C is an alternative and more sensitive endogenous marker for the estimation of GFR than serum creatinine [6].

New immunoassay methods from several different manufacturers measure cystatin C levels, and this has made the estimation of GFR more practical and easier. These methods are automated and results can be rapidly obtained. Standardization of testing by clinical laboratories is important to derive accurate GFR estimates [7].

 Materials and methods



The present study was carried out on 20 healthy individuals and 40 patients with type 2 DM; these included individuals attending the outpatient clinic and those who were inpatients of the Internal Medicine Department of El-mogama El-Tebby Hospital during the period between 2012 and 2013.

The participants were classified into the following groups:

Control group: included the 20 healthy individuals (14 male and six female). The patients were classified according to the urinary albumin/creatinine ratio (A/C) into the following groups:Normoalbuminuria group: included 16 patients (12 male, four female) with normoalbuminuria - that is, urinary A/C of 30 mg/g or lower.Microalbuminuria group: included 10 patients (seven male, three female) with microalbuminuria - that is, urinary A/C of 30-300 mg/g.Macroalbuminuria group: included 14 patients (five male, nine female) with macroalbuminuria - that is urinary A/C of 300 mg/g or higher.

The following patients were excluded from this study:

Patients with urinary tract infections.Patients with malignancies.Patients with liver disease.Patients with thyroid gland dysfunction.Patients with congestive heart failure.

All patients and controls were subjected to the following:

Full history taking, including age, sex, previous medications, and duration of DM.Clinical examination, with emphasis on blood pressure, neurological and cardiac examination, electrocardiography, and abdominal ultrasound examination.Abdominal ultrasonography, for detection of kidney abnormalities.Laboratory investigations, including the following:

Estimation of the A/C ratio.Estimation of fasting and postprandial blood glucose levels.Complete urine analysis. Estimation of glycated hemoglobin levels (HbA1c).Estimation of complete blood count.Estimation of serum urea levels.Estimation of serum creatinine levels (modified rate Jaffe method).Estimation of serum cystatin C levels (mg/dl) using the ELISA technique.Measurement of GFR using the following equation:

[INLINE:1]

Determination of serum cystatin C concentration

Principle of the test

This assay is based on the quantitative sandwich enzyme immunoassay technique (ELISA technique). The surface of the wells of the microtiter plate was coated with polyclonal anti-human cystatin C-specific antibodies. Diluted standards, diluted quality controls, and diluted samples were pipetted into the wells. Any human cystatin C present was captured by the immobilized antibodies, and unbound protein was washed away after the first incubation period. Thereafter, horseradish peroxidase-conjugated polyclonal anti-human cystatin C antibodies were added to the wells and incubated. This was followed by another washing step to remove the unbound antibody-horseradish peroxidase conjugates. A substrate solution (H202) was added to the wells. The enzymatic reaction yielded a blue product that turned yellow when the acidic stop solution was added.

The intensity of the color was measured spectrophotochemically at 450 nm and was directly proportional to the amount of human cystatin C bound in the initial step.

Concentrations of unknown samples were then obtained from the standard curve, which was constructed by plotting the absorbance values against each respective human cystatin C standard level using a four-parameter function.

Preparation of reagents

All reagents were brought to room temperature before the assay.

Assay reagents

Conjugate solution: the working conjugate solution was prepared by adding one part of conjugate solution concentrate (50×) to 49 parts of conjugate diluent.Wash solution: 100 ml of wash solution concentrate (l0×) was diluted with 900 ml distilled water.Dilution buffer: 10 ml of dilution buffer concentrate (l0×) was diluted with 90 ml distilled water to prepare 100 ml of dilution buffer (1×).Human cystatin C standards: each concentration of standard (400×) was diluted in two steps (l0× and 40×) as follows: 10 μl standard was added to 90 μl dilution buffer (dilution l0×) and mixed well; thereafter, 10 μl of the l0× dilution was added to 390 μl of dilution buffer to yield a final volume of 400 μl (40×) and mixed well.

Quality controls and samples

High-quality and low-quality controls and samples were diluted just before the assay at the same ratio as standards.

Procedures

Reagents, diluted standards, controls, and samples were prepared as directed in the previous sections.Diluted standards, quality controls, samples, and dilution buffer (blank) were pipetted into appropriate wells at a volume of 100 μl.The plate was incubated at room temperature for 30 min on an orbital microplate shaker (300 rpm).The wells were washed three times with wash solution (0.35 ml/well), and the plate was inverted and blotted against paper towels to remove the remaining wash solution.A volume of 100 μl of conjugate solution was added into each well.The plate was incubated at room temperature for 30 min on an orbital microplate shaker (300 rpm).The wells were washed three times with wash solution (0.35 ml/well), and the plate was then inverted and blotted against paper towels to remove the remaining wash solution.A volume of 100 μl of substrate solution was added to the wells and the plate was protected from light (the plate was covered with aluminum foil).The plate was incubated (without shaking) at room temperature for 10 min.The development of color was stopped by adding 100 μl of stop solution.The optical density was determined by reading absorbances at 450 nm (within 15 min after step 10).

 Results



The present study was carried out on 40 patients with type 2 DM and 20 apparently healthy individuals.

The participants were classified into the following groups:

Control group: included the 20 apparently healthy individuals (14 male and six female), with a mean age of 51.4 ± 12.4 years and a mean weight of 77.2 ± 10.7 kg.

The patients (24 male and 16 female) were classified according to A/C into the following groups:Normoalbuminuria group: included 16 patients (12 male, four females) with normoalbuminuria and an A/C of 30 mg/g or lower (range, 8-28 mg/g). The mean age was 51.7 ± 8.7 years and the mean weight was 100.1 ± 28.5 kg. The group included 10 patients with a GFR of 90 ml/min or more and six patients with a GFR of 89-60 ml/min.Microalbuminuria group: included 10 patients (seven male, three female) with microalbuminuria and an A/C of 30-299 mg/g (range, 50-292). The mean age was 53.9 ± 5.8 years and the mean weight was 90 ± 16.6 kg. The group included four patients with a GFR of 90 ml/min or higher, four patients with a GFR of 89-60 ml/min, and two patients with a GFR of 60 ml/min or lower.Macroalbuminuria group: included 14 patients (five male, nine female) with macroalbuminuria and an A/C of 300 mg/g or higher (range, 352-1240). The mean age was 54.1 ± 6 years and the mean weight was 84.4 ± 24 kg. They group included four patients with a GFR of 90 ml/min or higher, two patients with a GFR of 89-60 ml/min, and eight patients with a GFR of less than 60 ml/min.

There was a positive correlation between cystatin C level and age, DM duration, A/C, and levels of HbA1c, FBS, 2HPP, serum urea, and serum creatinine [Table 1].{Table 1}

There was negative correlation between cystatin C level and weight and GFR. There was a positive correlation between serum creatinine level and A/C; levels of HbA1c, FBS, and 2HPP; weight; and DM duration. There was a negative correlation between serum creatinine level and age and GFR [Table 2].{Table 2}

 Discussion



Diabetic nephropathy is the leading cause of chronic renal disease in patients starting renal replacement therapy; it is associated with increased cardiovascular mortality [8].

DM has been classically defined by increased protein excretion in urine and decreased GFR thereafter. GFR has been expected to decrease when proteinuria is established [9].

GFR is considered the most accurate measurement of kidney disease and is reduced before the onset of clinical symptoms; it is measured or predicted using different methods [10]. There is no simple and practical way to measure GFR directly; hence, it is estimated. To estimate the GFR, an endogenous substance in the blood that is cleared by the kidney is used; this substance is currently serum creatinine. The Cockcroft-Gault and Modification of Diet in Renal Disease Study equations are serum creatinine-based equations that are used to estimate GFR. GFR determinations using creatinine-based equations are not precise; hence, other substances, such as cystatin C, are being explored to estimate GFR [11].

The primary limitation of using creatinine level is that the level is determined not only by GFR but also by muscle mass and dietary intake. Lower serum creatinine levels may less reliably detect impaired GFR in patients with certain characteristics like older age, female sex, chronic illness with muscle wasting, amputation, or a vegetarian diet; higher serum creatinine levels are associated with African American race, muscular body habitus, and a high protein diet. Although estimating equations attempt to adjust for these factors, the result is not precise. Different patients can have the same serum creatinine level with very different GFRs [12].

Estimation of creatinine clearance requires urine sample collection after a 24-h period. A blood sample is drawn during the 24-h period and creatinine clearance can then be calculated. There are several factors that may interfere with the accuracy of the test such as incomplete urine collection, pregnancy, vigorous exercise, and drugs such as cimetidine, trimethoprim, and those that can damage the kidneys; therefore, results may be inaccurate [13].

Several new biochemical markers have the potential to be markers of chronic kidney disease progression. These new markers might reflect the early diminished GFR compared with traditional markers; these include the following: N-acetyl-β-glucosaminidase, β2-microglobulin, ͍1-microglobulin, retinol-binding protein, human neutrophil gelatinase-associated lipocalin, interleukin-18, clusterin, fatty acid binding protein, and cystatin C [14].

The aim of this study was to evaluate serum cystatin C as an early marker of renal dysfunction in type 2 DM.

Cystatin C is produced at a constant rate by all nucleated cells. Because of its small size, it is freely filtered by the glomerulus and is not secreted but is fully reabsorbed and broken down by the renal tubules. This means that the primary determinate of blood cystatin C levels is the rate at which it is filtered in the glomerulus, making it an excellent GFR marker [5].

Cystatin C may detect mild-to-moderate decreases in GFR that are not evident with serum creatinine-based measurements. Cystatin C-based estimates of GFR are better than creatinine-based estimates of GFR [12].

Most reports confirm that serum cystatin C concentrations are uninfluenced by age, sex, or muscle mass [15]. However, just recently, a study alleged that serum cystatin C levels appear to be influenced by nonrenal factors such as age, sex, weight, height, current cigarette smoking, and C-reactive protein levels [16].

In our work, we found that there was a positive correlation between cystatin C level and age, and there was no significant correlation between cystatin C level and sex.

A/C is considered to have a useful monitoring role in diabetes with respect to detecting kidney disease progression and evaluating treatment effects [17].

In this study, we found that there was positive correlation between cystatin C level and age, DM duration, A/C, and levels of HbA1c, FBS, 2HPP, serum urea, and serum creatinine; however, there was a negative correlation between cystatin C level and weight and GFR.

In addition, there was positive correlation between serum creatinine level and A/C; levels of serum urea, HbA1c, FBS, and 2HPP; weight; and DM duration; however, there was a negative correlation between serum creatinine level and age and GFR.

Jeon et al. [18] showed that there was no significant difference in age or in sex between the studied groups, and this was in accordance with our study.

In this study, we found that microalbuminuric and macroalbuminuric groups showed higher levels of HbA1c when compared with the normoalbuminuric group. This was supported by the findings of the study by Shehnaz and colleagues, according to which microalbuminuria had a highly significant correlation with duration of diabetes and a high HbA1c level [16].

In this study, we found a significantly higher concentration of serum creatinine and serum cystatin C in the macroalbuminuric group compared with the normoalbuminuric and microalbuminuric groups, and all groups had higher levels compared with the control group. Herget-Rosenthal et al. stated that cystatin C levels can help detect the reduced GFR with higher sensitivity (97 vs. 83%) and higher negative predictive value (96 vs. 87%) compared with creatinine levels; in parallel, sensitivity of cystatin C was significantly higher (P < 0.05) [19].

From all of the above, we found that cystatin C is a better indicator of renal functions than creatinine in patients with impaired renal functions; this is because of the unique properties of cystatin C such as constant production independent of age, sex, and muscle mass, and not being secreted or reabsorbed by the renal tubules. The major advantage of cystatin C over creatinine is its ability to detect a mild reduction in GFR, to which creatinine is insensitive. As there are no specific therapies, early detection of impaired renal functions is crucial to prevent the progression of renal disease and to improve patient outcome; the main disadvantage of cystatin C is the high cost of its immunoassay. Although all the studies reviewed here have demonstrated the distinct advantage of cystatin C over creatinine, it is important to document the advantages of cystatin C to improve patient outcome. Replacement of creatinine, which is the most widely used marker, by a new marker, cystatin C, ultimately depends on the results of patient outcome studies.

 Conclusion



Measurement of GFR is considered very helpful for the assessment of renal function, especially in patients with type 2 diabetes. Of the patients with type 2 diabetes, 20-40% will eventually develop diabetic nephropathy, and they will eventually need to receive renal replacement therapy.

It is well known that serum creatinine level is an insensitive measure of renal function Moreover, serum creatinine level is not an accurate reflection of GFR as it is influenced by many factors, including muscle mass, sex, diet, liver function, age, and tubular secretion, which can result in an overstatement of GFR up to 20%; hence, it is better to look for an alternative noninvasive measure, particularly for diabetic nephropathy.

From this study, we can conclude that serum cystatin C is a better marker for GFR than is serum creatinine in type 2 diabetes patients with reduced GFR, especially in the 'creatinine-blind area'. Cystatin C can detect mild-to-moderate decreases in GFR that are not evident with serum creatinine-based measurements.

New immunoassay methods from several different manufacturers measure cystatin C levels, and this has made estimation of GFR more practical and easier. These methods are automated and results can be rapidly obtained; hence, they may be recommended in routine investigations for the assessment of GFR in diabetic patients.

Finally, measurement of renal function on the basis of cystatin C levels will optimize early detection, prevention, and treatment strategies for diabetic nephropathy. Estimation of serum cystatin C levels is practical and can help detect early decline of renal function in type 2 DM.

 Acknowledgements



Conflicts of interests

None declared.

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