A Comparative Study between Serum Cystatin C and Microalbuminuria in the Diagnosis of Nephropathy in Type 2 Diabetes Mellitus

Abstract

Background:

Diabetic nephropathy is the most serious complication associated with diabetes mellitus and the diagnosis typically relies on the identification of albuminuria. However, there are cases of renal insufficiency without albuminuria reported in diabetic patients. This study aimed to evaluate the association between microalbuminuria and serum cystatin C, as well as to assess the clinical usefulness of cystatin C in predicting the renal impairment in diabetic patients.

Materials and Methods:

It was a cross-sectional study conducted on type 2 diabetic patients and healthy controls, who were categorized into four groups based on their albuminuria levels. All the participants were investigated for serum cystatin C, albuminuria, and traditional markers of nephropathy.

Results:

The level of cystatin C was significantly elevated in diabetic patients as compared to healthy individuals. The mean ± standard deviation progressively raised among the normoalbuminuric diabetic patients (1.32 ± 0.32 mg/L) and reached the peak in nephropathy patients (1.86 ± 0.25 mg/L). There was a significant positive correlation of cystatin C with albuminuria (P < 0.001). Receiver operating characteristic analysis of cystatin C showed an area under the curve of 0.881 (95% confidence interval, 0.832–0.930) with a sensitivity of 80.83% and a specificity of 86.67%, demonstrating the diagnostic efficacy of cystatin C (P < 0.001).

Conclusion:

Elevated serum cystatin C level was observed in type 2 diabetic patients before the development of notable albuminuria, and its concentration rises as nephropathy advances, indicating that cystatin C could serve as a potential and earlier marker of renal dysfunction compared to albuminuria.

INTRODUCTION

Diabetes mellitus (DM) is the most common metabolic disorder, characterized by hyperglycemia resulting from the defects in insulin action or insulin secretion.[1] Prolonged DM is linked to microvascular complications such as nephropathy, retinopathy, and neuropathy, as well as macrovascular complications including cardiomyopathy, vasculopathy, and atherosclerosis.[2] The most prevalent microvascular complication is diabetic nephropathy (DN), which is the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). The mortality rate associated with CKD is increasing globally, with diabetes recognized as major risk factor, contributing to half of the deaths from renal failure.[3] Timely diagnosis and the prompt initiation of treatment can potentially prevent the progression of nephropathy to CKD and total renal failure.

In accordance with the kidney disease outcomes quality initiative guidelines, DN is identified through the evaluation of albuminuria alongside the estimation of the glomerular filtration rate (GFR). Albuminuria is determined through the measurement of urine albumin/creatinine ratio (ACR). Microalbuminuria (MAU) is regarded as the earliest clinical indicator of DN and the level of ACR level ranges from 30 to 300 mg/g is identified as MAU.[4]

This approach is subject to scrutiny, as a moderate rise in albumin excretion can be linked to various other factors, such as obesity, physical activity, dietary habits, smoking, infections, and inflammation.[5] In addition, some patients may possess a genetic predisposition, particularly evident in podocytes, which heightens their risk of developing albuminuria.[6] Conversely, certain diabetic patients experience a decline in renal function without the presence of albuminuria, a condition referred to as nonproteinuric-DN, where kidney dysfunction does not remarkably lead to albuminuria.[7,8] In addition, a substantial number of individuals with MAU may revert to normoalbuminuria.[9] This indicates that the onset and advancement of nephropathy might take place independently of the development albuminuria.

The essential metric for forecasting overall kidney function is estimated GFR (eGFR), and the most commonly utilized biomarker for eGFR assessment is serum creatinine. Even though the reduction in eGFR indicates abnormal kidney function, it is not regarded as an early sign of diabetic kidney disease, as creatinine levels rises significantly only after more than 50% of kidney damage has occurred.[10] In addition, plasma creatinine level is influenced by various factors, including muscle mass, gender, ethnicity, and certain medications.[11] Consequently, both albuminuria and eGFR may not be sensitive markers for the early identification of DN. With the rising number of nephropathy patients, it is imperative to investigate reliable markers for the early prediction of renal impairment in individuals with type 2 DM (T2DM).

Cystatin C in serum has recently been identified as a promising new biomarker for the diagnosis of kidney injury. This protein, which is nonglycosylated and has a low molecular weight of 13 kDa, belongs to the cystatin super family of cysteine protease inhibitors. It is produced consistently by all nucleated cells, is freely filtered at the glomerulus due to its small size and positive charge, and is subsequently reabsorbed and catabolized, but not secreted by proximal renal tubules.[12] Cystatin C serves as an alternative biomarker for estimating eGFR, as it is not influenced by muscle mass and remains relatively stable with age and gender.[13] The current study aimed to assess the potential role of serum cystatin C as an early indicator of kidney dysfunction and to explore the relationship between MAU and cystatin C in individuals with T2DM.

MATERIALS AND METHODS

Study design and study population

This cross-sectional study was conducted through the appropriate recruitment of subjects to assess the prevalence of nephropathy in the population. The research was performed at the laboratory of Clinical Biochemistry, Vinayaka Mission’s Medical College and Hospitals located in Salem, Tamil Nadu, India, from March 2024 to August 2024, involving 180 participants, which included 102 females and 78 males, aged between 30 and 75 years.

Ethical consideration

The study was granted approval by the Institutional Ethics Committee (Tracking No. VMKVMCandH/IEC/23/046 dated July 12, 2023) and was carried out in accordance with the ethical standards set forth in the Declaration of Helsinki, 2013. Informed written consent was obtained from all participants following a briefing on the objective of the study.

Inclusion criteria

The study population included T2DM patients serving as cases and nondiabetic healthy individuals as controls.

Exclusion criteria

Individuals diagnosed with malignancies, cardiovascular diseases, type 1 DM, and kidney diseases excluding DN, were not permitted to participate.

Diagnostic methods

Venous blood samples and spot urine samples were obtained after the selection of cases and controls. A portion of the whole blood is preserved in K₂ ethylene diamine tetraacetic acid tubes for the assessment of glycated hemoglobin A1c (HbA1c). The remaining blood samples were subjected to centrifugation to separate the serum. Subsequently both serum and urine samples were employed for biochemical analysis, with measurements performed on the same day as sample collection.

Demographic data including age, gender, height, weight, and body mass index (BMI), of all participants were recorded. In addition, both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured. The urine albumin level was evaluated by immuno turbidimetric method and urine creatinine by enzymatic method to compute the urine ACR. Serum cystatin C was assessed by immuno turbidimetric method and HbA1c using the particle-enhanced immunoturbidimetric method. The electrolytes were determined using the ion-selective electrode methods and the baseline parameters were analysed utilizing enzymatic techniques. The eGFR was calculated using the CKD-EPI-2021 equation based on serum creatinine concentrations.

Statistical analysis

The statistical analyses were performed using the statistical package for the social sciences (SPSS) version 26.0; Inc., Chicago IL, USA. The findings were reported as mean and standard deviation (SD), with P < 0.05 considered statistically significant. A one-way ANOVA was applied for the comparative analysis of the study subjects, whereas Pearson’s correlation was used for intergroup comparisons. In addition, receiver operating characteristic (ROC) curve was employed to assess the efficacy of the biomarker in predicting renal impairment.

RESULTS

In the study group, the participants were classified into four groups based on their albuminuria levels: Group I – nondiabetic healthy controls exhibiting normoalbuminuria; Group II – T2DM patients with normoalbuminuria; Group III – T2DM patients presenting with MAU; and Group IV – T2DM patients with macroalbuminuria. Among the groups analyzed, all the nondiabetic healthy participants were normoalbuminuric (n = 60), while from the 120 diabetic patients, 60 were normoalbuminuric, 45 were microalbuminuric, and 15 were macroalbuminuric. The evaluation of albuminuria was performed by measuring urinary ACR, expressed in milligrams of albumin per gram of creatinine.

The box and whisker plot [Figure 1] demonstrate the distribution of cystatin C within each group. A progressive rise in serum cystatin C levels is observed in relation to albuminuria. The overall characteristics of biochemical parameters and anthropometric variables in both diabetic and nondiabetic groups, as detailed in Table 1, provided a thorough overview of the sample. A significant difference was identified among the groups concerning the mean age, SBP, DBP, and BMI for anthropometric measurements (P < 0.001). The mean ± SD for glucose, HbA1c, creatinine, and C-reactive protein (CRP) exhibited a gradual increase from healthy controls to patients with nephropathy (P < 0.001). A significant difference in triglyceride level was also noted between group I and group IV (P < 0.001). Diabetic patients with nephropathy exhibited an elevated urinary ACR (408.33 ± 77.15 mg/g) in contrast to patients without nephropathy (15.43 ± 6.22 mg/g) and the control subjects (8.16 ± 7.6 mg/g). As albuminuria progresses from group I to group IV, there is a corresponding decline in serum total protein, albumin, and eGFR, which is significantly reduced in group IV, with all comparisons resulting in P < 0.001.

Figure 1.

Distribution of serum cystatin C among diabetic and nondiabetic participants

Table 1.

Mean±standard deviation of biochemical parameters and anthropometric variables among diabetic and nondiabetic groups

Parameters	Group I	Group II	Group III	Group IV	Test statistic	P
Age (years)	47.47±9.36	58.37±8.74	57.66±7.35	60.56±8.77	45.422	<0.001
Systolic BP (mm Hg)	122.73±6.55	125.68±4.81	126.48±5.52	132.94±3.97	31.78	<0.001
Diastolic BP (mm Hg)	83.32±3.92	86.88±4.73	88.52±3.96	91.56±3.05	51.15	<0.001
BMI (kg/m2)	23.72±1.52	25.30±1.81	25.18±1.63	25.16±1.90	31.03	<0.001
Glucose (mg/dL)	102.58±11.31	221.40±67.07	250.31±74.48	327.13±123.15	126.07	<0.001
HbA1c (%)	6.60±2.39	8.13±2.21	8.49±2.22	9.93±2.84	41.66	<0.001
Total protein (g/dl)	7.26±0.44	7.03±0.45	6.76±0.37	5.96±0.28	42.37	<0.001
Serum albumin (g/dL)	4.29±0.50	4.04±0.31	3.97±0.30	3.20±0.21	51.17	<0.001
Urea (mg/dL)	22.63±7.82	23.43±11.90	24.94±6.28	26.00±12.79	3.72	0.118
Creatinine (mg/dL)	0.738±0.128	0.822±0.216	0.94±0.381	1.075±0.21	27.75	<0.001
Uric acid (mg/dl)	5.245±1.715	4.852±1.569	5.336±2.045	4.594±0.942	3.66	0.2725
CRP (mg/L)	3.182±2.348	5.439±4.857	8.195±9.044	6.620±4.141	26.708	0.001
Na (mmol/L)	137.08±5.00	135.88±4.10	136.05±4.99	136.13±4.66	0.827	0.517
K (mmol/L)	4.207±0.377	4.292±0.551	4.341±0.710	4.189±0.341	1.14	0.5544
Cl (mmol/L)	106.20±2.96	104.78±3.39	104.98±3.26	105.06±3.43	7.31	0.0284
TC (mg/dL)	188.22±42.68	191.88±35.98	194.14±41.62	194.19±48.94	0.987	0.8863
TG (mg/dL)	162.72±67.71	229.53±143.25	221.47±107.84	274.33±150.78	18.949	<0.001
HDL (mg/dL)	44.12±7.61	47.08±10.97	43.86±6.95	44.06±11.68	1.29	0.2125
LDL (mg/dL)	110.23±29.79	104.02±32.98	112.20±29.12	103.75±34.99	1.38	0.4991
eGFR (ml/min/1.73m2)	100.22±11.90	87.10±10.26	72.99±14.90	63.76±8.77	101.046	<0.001
ACR (mg/g)	8.16±7.6	15.43±6.22	71.36±37.52	408.33±77.15	136.27	<0.001
Cystatin C (mg/L)	1.01±0.19	1.32±0.32	1.56±029	1.86±0.25	93.96	<0.001

BP=Blood pressure, BMI=Body mass index, HbA1c=Glycated hemoglobin, CRP=C-reactive protein, TC=Total cholesterol, TG=Triglycerides, HDL=High-density lipoprotein, LDL=Low-density lipoprotein, eGFR=Estimated glomerular filtration rate, ACR=Albumin-to-creatinine ratio

Diabetic patients diagnosed with nephropathy in group IV show significantly elevated serum cystatin C levels (1.86 ± 0.25 mg/L) relative to those in group III (1.56 ± 0.29 mg/L). The normoalbuminuric diabetic subjects in group II demonstrated a considerably higher serum cystatin C levels (1.32 ± 0.32 mg/L) than the normoalbuminuric nondiabetic control group which exhibited lowest concentrations (1.01 ± 0.19 mg/L).

The correlation analysis performed with Pearson’s correlation coefficient shown in Table 2, revealed a significant positive correlation between cystatin C and ACR, glucose, HbA1c, creatinine, triglycerides, as well as SBP and DBP. A strong inverse relationship was noted with eGFR, serum total protein, and albumin. Figure 2 depicts the linear regression analysis of cystatin C in relation to ACR, glucose, HbA1c, and eGFR, which illustrates the correlation.

Table 2.

Correlation of cystatin C with other parameters

Parameters	Control		Cases
	r	P	r	P
ACR (mg/g)	−0.097	0.46	0.537	<0.001
Systolic BP	0.336	0.009	0.29	0.001
Diastolic BP	0.377	0.003	0.366	<0.001
Glucose (mg/dL)	0.282	0.029	0.405	<0.001
HbA1c (%)	0.299	0.02	0.257	0.005
Total protein (g/dL)	0.044	0.741	−0.325	<0.001
Serum albumin (g/dL)	0.071	0.588	−0.248	0.006
Creatinine (mg/dL)	0.005	0.968	0.219	0.016
TG	0.114	0.384	0.25	0.006
eGFR	−0.191	0.143	−0.397	<0.001

BP=Blood pressure, HbA1c=Glycated hemoglobin, ACR=Albumin-to-creatinine ratio, TG=Triglycerides, eGFR=Estimated glomerular filtration rate

Figure 2.

Correlation of cystatin C with other parameters (P < 0.05 was considered statistically significant)

The ROC curve analysis was performed to evaluate the diagnostic accuracy of serum cystatin C as an early indicator of DN, as illustrated in Figure 3. Cystatin C showed a sensitivity of 80.83% and a specificity of 86.67%, with an area under the curve (AUC) of 0.881 (95% confidence interval [CI]: 0.832–0.930 and cut-off value 1.186 mg/L). In comparison, urinary ACR displayed an AUC of 0.839 (95% CI: 0.783–0.895 and cut-off value 25.27 mg/g), with a sensitivity of 56.67% and a specificity of 98.33%. This finding suggests that the serum cystatin C level is increased before the development of MAU and it is statistically significant (P < 0.001).

Figure 3.

Receiver operating characteristic plots to assess the diagnostic efficiency of serum cystatin C and urinary albumin/creatinine ratio

DISCUSSION

There is an urgent requirement for precise and sensitive indicators to forecast DN, as the decline in renal function may initially be clinically silent and can advance to ESRD. In this research, the serum cystatin C was assessed to determine its efficacy in the early detection of renal dysfunction in diabetic patients. The results indicated a statistically significant relationship between cystatin C levels and the prognostic stages of nephropathy in patients with T2DM, aligning with previous studies.[14] Patients diagnosed with T2DM exhibited a greater average level of cystatin C in comparison to nondiabetic individuals. The level of cystatin C rises in diabetic patients, with a more pronounced increase observed in DN patients, supporting results from another research.[15]

The research indicates a notable positive correlation between serum cystatin C and various factors including glucose, HbA1c, creatinine, triglycerides, CRP, and albuminuria, as well as SBP, DBP and BMI (P < 0.001). This is consistent with traditional observational studies that have identified higher anthropometric measurements and blood pressure levels as being linked to an increased risk of DN.[16] The monitoring of glucose levels and the assessment of HbA1c supports the critical role of glycemic control in preventing the progression to nephropathy.[17] Chronic hyperglycemia is recognized as a significant risk factor for DN and the level of HbA1c acts as a predictor for diabetes-related complications, including retinopathy and nephropathy, which are associated with the harmful effects of advanced glycation end products. The individuals with diabetes often exhibit elevated HbA1c levels supports the findings of previous research.[18]

The elevated levels of cystatin C are positively correlated with creatinine and negatively correlated with eGFR in patients with kidney diseases.[19] The estimation of eGFR can be performed using a suitable mathematical formula that takes into account serum creatinine levels, as well as the age, gender, and ethnicity of patient. The research demonstrated that the increase in plasma creatinine was attributable to renal factors, which resulted in a decreased eGFR, establishing it as one of the most significant indicators of kidney function, as evidenced by earlier studies.[20] A similarly low concentration of serum protein and albumin is significantly linked to the advancement of DN. It is well established that individuals afflicted with DN experience a loss of serum albumin in their urine, caused by impaired renal filtration barrier, which plays a crucial role in the development of hypoalbuminemia. Our research confirmed this observation, revealing a higher occurrence of albuminuria in patients with diminished serum protein and albumin levels, suggesting a more progressed stage of renal impairment.[21]

High level of CRP is associated with the development of DN by aggravating glycemic control in DM populations as CRP is associated with insulin resistance and hyperglycemia. Prior researches have indicated that high glucose concentration stimulates the production of CRP, which is in connection with the activation of nuclear factors and transforming growth factors signalling pathways.[22] This activation results to renal inflammation and fibrosis in DM. The current study validates the findings of earlier research, confirming that CRP is related to nephropathy and emphasizing the significance of CRP in the inflammatory mechanisms that contribute to the advancement of DN.[23]

Both hyperkalemia and hypernatremia are fatal conditions associated with high mortality rate in CKD patients; however, it may present as normal due to dilution from fluid retention. Most of the patients in our research with DM exhibited normal sodium (Na) and potassium (K) levels. These findings align with those of previous studies.[24,25] In the current study, altered levels of chloride (Cl) were noted in numerous patients suffering from nephropathy. Chloride is the predominant anion found in extracellular body fluids and plays a vital role in tubuloglomerular feedback along with reabsorption in interstitium. Our findings support this assertion, as the prevalence patterns of sodium, potassium, and chloride are comparable.[26] The onset of nephropathy and its connection to dyslipidemia, particularly marked by increased triglyceride levels, has been the subject of extensive research. This observation is in agreement with previous studies that suggest lipid metabolism might hinder the advancement of nephron injury. Our results support this notion, indicating that dyslipidemia may act as a contributing factor to renal dysfunction in diabetic patients.[27]

In the current research, most of the patients were in the initial stages of nephropathy. However, cystatin C is found significantly higher in diabetic patients and its concentration in serum rises even prior to the detection of albumin in urine. This indicates the possible significance of this marker in diagnosing of nephropathy, which is in accordance with the previous studies.[28] Hence, cystatin C serves as an alternative biomarker for kidney damage, potentially identifying renal injury more promptly than albuminuria, owing to its lower molecular weight, constant production, and stability.

The ROC curves were employed to assess the diagnostic effectiveness of cystatin C, which demonstrated greater sensitivity and a higher AUC compared to albuminuria. The results were statistically significant (P < 0.001) with a 95% CI, suggesting that cystatin C is a reliable indicator that could serve as an excellent diagnostic marker.

CONCLUSION

The measurement of serum cystatin C serves as a valuable practical method for assessing renal failure earlier during the progression of diabetes, suggesting that it act as superior biomarker compared to microalbuminura for diagnosing DN. Consequently, we emphasize the regular use of this marker in monitoring the advancement of nephropathy in patients with diabetes. Such a discovery carries early intervention strategies aimed at preventing the onset of CKD and thus total renal failure in individuals diagnosed with T2DM.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.