- Email: [email protected]
Cystatin levels in a rural South Indian population
APME-377; No. of Pages 5 apollo medicine xxx (2016) xxx–xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/apme
Research Article
Cystatin levels in a rural South Indian population Krishnan Swaminathan a,*, Ganesh Veerasekar b, Mangalakumar Veerasamy c, Sundaresan Mohanraj d, Kuppusamy Sujatha e, Ganesan Velmurugan f, Nalla G. Palaniswami g, on behalf of KMCH Research Foundation, Coimbatore, India a
Endocrinology, KMCH Research Foundation, India Clinical Epidemiologist, KMCH Research Foundation, India c Consultant Nephrologist, Kovai Medical Center & Hospital, India d Research Coordinator, KMCH Research Foundation, India e Department of Community Medicine, Coimbatore Medical College, India f Post-Doctoral Fellow, DST Unit of Nanoscience, IIT Madras, India g Chairman, Kovai Medical Center & Hospital, KMCH Research Foundation, India b
article info
abstract
Article history:
Aim: Our aim was to study the potential role of Cystatin C as a marker for renal function and
Received 18 February 2016
cardiovascular disease in a rural South Indian population.
Accepted 29 August 2016
Methods: Local Ethics Committee approval and informed consent was obtained from all
Available online xxx
participants. Inclusion criterion was that participants had to be ≥20 and ≤85 years of age. All participants were administered a detailed questionnaire, and had their anthropometric
Keywords:
measurements taken including height, weight, and waist circumference. Blood was drawn
Cystatin C
for determining random glucose, glycosylated hemoglobin, nonfasting lipid profile, Cystatin
Creatinine
C, uric acid, and hemoglobin. Urine samples were collected for urine protein creatinine ratio.
Cardiovascular disease
All participants had their carotid intima thickness determined by high-resolution B-mode
Renal impairment
carotid ultrasound.
Marker
Results: A total of 865 participants were screened in this study. Cystatin C value of >1 mg/l and creatinine of >1.1 mg/dl in females and 1.3 mg/dl in males were defined as ‘‘elevated levels.’’ Nearly a third of this population had elevated Cystatin C levels. Higher levels of Cystatin C, even within the normal creatinine range, was associated with cardiovascular risk markers like age, blood pressures, uric acid, and carotid intima thickness. Conclusions: Cystatin C in a rural South Indian population correlates well with creatinine and cardiovascular markers. Long-term follow-up of this cohort stratified on the basis of Cystatin C will be extremely helpful in augmenting the utility of Cystatin C as an atherosclerotic risk marker in an Indian population. # 2016 Indraprastha Medical Corporation Ltd. All rights reserved.
* Corresponding author. Tel.: +91 8526421150; fax: +91 422 2627782. E-mail address: [email protected] (K. Swaminathan). http://dx.doi.org/10.1016/j.apme.2016.08.003 0976-0016/# 2016 Indraprastha Medical Corporation Ltd. All rights reserved.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5
2
apollo medicine xxx (2016) xxx–xxx
1.
Background
Cystatin C is a member of the family of cysteine proteinase inhibitors, which are produced by all nucleated cells at a constant rate.1–3 The history of Cystatin C began more than 50 years ago with a multitude of clinical data over the past decade on its utility as a marker for kidney (dys) function and atherosclerotic cardiovascular disease. Owing to its free filtration in the glomerulus, complete reabsorption, and lack of tubular secretion, the plasma concentration of Cystatin C is thought to depend mainly on the glomerular filtration rate. Several studies have also shown that Cystatin C has a stronger association with cardiovascular outcomes than serum creatinine.4–9 Despite such exciting prospects, Cystatin C still widely remains a research tool rather than an important investigation in clinical practice. The reasons include lack of standardization of available kits, lack of familiarity with the test that has not proven to affect clinical decision-making in a significant manner, and costs of Cystatin C measurement as opposed to creatinine measurements.10 There have been very few Indian studies evaluating the utility of Cystatin C as a marker for renal function and cardiovascular disease.11,12 Our aim, therefore, was to evaluate the utility of Cystatin C in a larger rural South Indian population and follow-up this cohort long-term to assess the implications of doing this test.
2.
Study design
Nallampatti is a typical rural farming village in South India with a population of around 3500. This village was selected due to the ease of logistics and contacts with the local village heads. All participants >20 years of age were invited to participate in this study through pamphlets and word of mouth. The study was conducted over a 4-week period from March 15th to April 15th, 2015. Ethical Committee approval and informed consent were obtained. A detailed questionnaire was administered followed by anthropometry, blood pressure recordings, and blood investigations including random glucose, glycosylated hemoglobin, nonfasting lipids, Cystatin C, hemoglobin, uric acid, urine protein creatinine ratio (PCR), and carotid intima thickness using carotid ultrasound. Cystatin C results were classified into the following three groups: Group A: normal Cystatin C and normal creatinine, Group B: elevated Cystatin C and normal creatinine, and Group C: elevated Cystatin C and elevated creatinine. Cystatin C levels were analyzed using the nephlometric method (BN Prospec Instrument, Seimens). All results were tabulated on Microsoft Excel and transposed to SPSS 20 for further analysis. Discrete values were analyzed for mean and standard deviation. Categorical variables were analyzed using Pearson's chi-square test. Linear regression was also performed at certain instances to measure the strength of association between the testing variables. Student t-test was employed to calculate the absolute difference in mean between Groups A and B. p < 0.05 was considered statistically significant. The
participants were grouped to have abnormal creatinine level based on National Institute of Health's guidelines, in which cutoff value of creatinine for men is fixed at 1.3 mg/dl and that of women is fixed at 1.1 mg/dl. People were considered to have abnormal Cystatin C if their value is more than 1 mg/dl.
3.
Outcomes
A total of 865 participants participated in this study. Cystatin C levels were elevated in 28.4% of the study population. Mean Cystatin C level was 0.95 mg/l (SD 0.29, range 0.23–3.4 mg/l). We observed an exponential increase in Cystatin C levels with increasing age groups. The percentage of participants with elevated Cystatin C in the age groups 20–40, 41–60, and >60 years were 7.7%, 31.8%, and 54.2%, respectively (Pearson's chi-square p value <0.001). Linear regression was performed to analyze the strength of association between Cystatin C and various covariates. For every unit increase in age, the Cystatin C increases by 0.009 units (95% CI 0.007–0.010, p < 0.001). Women had lower Cystatin C levels, i.e. 0.131 units, compared to men (95% CI, 0.093–0.17, p < 0.001). Participants were classified into three groups, as detailed in the methods section. Baseline characteristics of the three groups are outlined in Table 1. We compared the means between Group A (normal Cystatin C and normal creatinine) and Group B (elevated Cystatin C and normal creatinine). Participants in Group B were older, and had significantly higher systolic and diastolic blood pressures, creatinine level, uric acid level, urine PCR, and carotid intima media thickness. HDL-C levels were significantly lower in Group B compared to Group A (Table 2). There was a positive correlation between Cystatin C and various cardiovascular risk markers including age, blood pressures, creatinine, uric acid, and carotid intima media
Table 1 – Baseline characteristics of three cystatin groups. Variable Total (n) Mean age (years) Weight (kg) SBP (mmHg) DBP (mmHg) Cystatin C (mg/l) Creatinine (mg/dl) Uric acid (mg/dl) HbA1c (%) Total cholesterol (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Urine PCR CIMT right (cm) CIMT left (cm)
Group A
Group B
Group C
632 44.9 (13.1) 58.7 (12.6) 129.4 (21.4) 81.3 (11.6) 0.83 (0.10) 0.70 (0.11) 4.5 (1.3) 5.9 (1.0) 184.4 (37.6) 109 (30.3) 45 (9.3) 0.06 (0.09) 0.06 (0.01) 0.06 (0.01)
216 56.2 (11.1) 60.2 (13.2) 135.4 (20.7) 84.9 (12.4) 1.15 (0.16) 0.80 (0.13) 5.19 (1.2) 6.1 (1.1) 188 (42.3) 112 (34.6) 41.5 (9.9) 0.11 (0.1) 0.07 (0.02) 0.07 (0.02)
17 64.5 (8.08) 50.6 (7.9) 141.6 (21.3) 84.2 (13.8) 2.36 (0.5) 1.67 (0.3) 7.52 (1.3) 6.1 (0.5) 189 (38.9) 110.3 (26.7) 46.9 (26.7) 0.37 (0.3) 0.08 (0.01) 0.08 (0.01)
Group A: normal Cystatin C and normal creatinine. Group B: elevated Cystatin C and normal creatinine. Group C: elevated Cystatin C and elevated creatinine.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5 apollo medicine xxx (2016) xxx–xxx
Table 2 – Comparison of means between Group A and Group B. Variable Age (years) Body weight (kg) SBP (mmHg) DBP (mmHg) Cystatin C (mg/l) Creatinine (mg/dl) Uric acid (mg/dl) HbA1c (%) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Urine PCR CIMT right (cm) CIMT left (cm)
Group A
Group B
p value*
44.9 58.7 129.4 81.3 0.83 0.70 4.5 5.9 184.4 45 109 0.06 0.06 0.06
56.2 60.2 135.4 84.9 1.15 0.80 5.19 6.1 188 41.5 112 0.11 0.07 0.07
<0.001 0.08 <0.001 <0.001 <0.001 <0.001 <0.001 0.05 0.20 <0.001 0.14 0.003 <0.001 <0.001
* Student t-test used to compare means between two groups, 2tailed, equal variances not assumed.
thickness. Correlations with glycosylated hemoglobin and non-HDL-C were of borderline significance (Table 3).
4.
Discussion
Our study is the largest Indian study to assess the performance of Cystatin C in a rural population. Cystatin C is elevated in nearly a third of our study population and seems to correlate well with creatinine and cardiovascular risk markers. The results of our study are consistent with similar studies from Caucasian population linking higher levels of Cystatin C with cardiovascular risk factors. Among Framingham Heart study offsprings, higher Cystatin C levels were associated with cardiovascular risk factors even in the absence of chronic kidney disease (CKD).13 In a crosssectional study of nearly 5000 participants ≥20 years without CKD, the prevalence of cardiovascular risk factors was higher in individuals with higher Cystatin C concentrations, and Cystatin C was independently associated with cardiovascular disease.14 In our study, individuals with elevated Cystatin C levels with normal creatinine were significantly older, had significantly higher systolic and
Table 3 – Pearson's correlation test. Cystatin C
Pearson's correlation
p value (2-tailed)
Age SBP DBP Creatinine Uric acid HbA1c Non-HDL-C CIMT right CIMT left
0.394 0.149 0.115 0.758 0.415 0.067 0.07 0.281 0.251
<0.001 <0.001 0.001 <0.001 <0.001 0.05 0.05 <0.001 <0.001
3
diastolic blood pressures, serum creatinine, uric acid, glycosylated hemoglobin, and carotid intima thickness, and lower HDL cholesterol levels (Table 2). Higher Cystatin C levels seem to be independently associated not only with hypertension but also with the development of hypertension in the future. In the MultiEthnic Study of Atherosclerosis, it was established that for every 0.2 mg/l increase in Cystatin C levels, there was a significant 15% greater incidence of hypertension.15 In this study, during a median follow-up of 3.1 years, nearly 20% of the participants developed hypertension. This was found to be associated with elevated Cystatin C levels after multivariable adjustment for important clinical factors. In our study, there was a significant difference in both systolic and diastolic blood pressures (6 mm and 3 mmHg, respectively) in participants with higher Cystatin C levels in the absence of renal impairment as defined by creatinine, compared to those with normal Cystatin C levels. Participants in our study with higher Cystatin C levels in the absence of renal impairment (Group B) had significantly higher urine PCR with lower high-density lipoprotein (HDL cholesterol) levels but similar low-density lipoprotein (LDL cholesterol) levels compared to those with normal Cystatin C levels (Table 2). Similar findings were noted in a large study of 5633 participants where greater urine albumin creatinine ratio was associated with lower HDL-C and higher triglycerides but not with LDL-C calculated using conventional methods.16 However, using nuclear magnetic resonance techniques, it was found that each two-fold increase in urine ACR was associated with a significant increase in small LDL-C particle concentration that was not observed directly with a standard lipid panel. Therefore, it may be reasonable to speculate that individuals with higher Cystatin C levels with increased urine PCR, lower HDL-C, but apparently normal LDL-C may still be at a higher cardiovascular risk due to atherogenic lipoprotein abnormalities that may not be directly observed with a standard lipid panel. Carotid intima thickness has been widely used as a marker for early detection of cardiovascular disease. The association between Cystatin C and carotid intima thickness is not very clear. A recently published Chinese study on 927 participants showed that Cystatin C was more strongly associated with carotid thickening and plaque than any other measure of kidney function.17 On the contrary, there have been other studies showing no association between Cystatin C and carotid intima thickness.18,19 In our study, there was a significant increase in carotid intima thickness with increasing Cystatin C levels even in those with normal creatinine levels. The relationship between Cystatin C and diabetes is intriguing. Cystatin C is secreted by adipose tissue, suggesting a possible role of this marker in incident diabetes.20 Cystatin C has shown to predict incident type 2 diabetes and progression from normal to pre-diabetes.21,22 Within normal creatinine levels, participants with elevated Cystatin C levels in our study had higher glycosylated hemoglobin, which was of borderline clinical significance. In those with documented diabetes in our study, the Cystatin C levels were significantly higher compared to those without diabetes (1.04 vs 0.93 mg/ dl, p < 0.001).
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5
4
apollo medicine xxx (2016) xxx–xxx
The question is whether Cystatin C is a marker of renal injury alone or whether there is a larger role in inflammation and atherogenesis? High Cystatin C levels have been found with elevated CRP levels and could be directly linked to inflammation and atherosclerosis.23,24 Inflammatory cytokines linked with atherosclerosis stimulate the production of lysosomal cathepsins. Cystatin C is a cathepsin inhibitor, and elevated levels of Cystatin C may potentially reflect an attempt by the body to counterbalance a damaging effect of elastolytic activity.25 Increased Cystatin C levels, therefore, is emerging as a marker for both kidney and cardiovascular disease. Well-designed larger studies are the need of the hour to explore this marker in our population. The mean Body Mass Index (BMI) was 23.2. The prevalence of generalized obesity as defined by a BMI cutoff of ≥25 was 31.6% in this rural farming population.
5.
Conclusions
In conclusion, we demonstrated elevations in Cystatin C in nearly 30% of a rural South Indian population. Cystatin C levels correlate well with creatinine and cardiovascular risk markers. Long-term follow-up of this cohort stratified by Cystatin C levels will be helpful in augmenting the utility of this test as a renal and atherosclerotic risk marker.
Funding The financial support for this study was provided by an unrestricted educational and research grant through corporate social responsibility funding by Kovai Medical Center and Hospital, Coimbatore, India.
Conflicts of interest The authors have none to declare.
Acknowledgements We acknowledge all the members of the KMCH Research Foundation, volunteers and villagers from Nallampatti, and Mr. Velmurugan from Madurai Kamaraj University. We also thank Dr. Mani, Microbiological Labs, Coimbatore for analyzing the blood investigations.
references
1. Newman DJ. Cystatin C. Ann Clin Biochem. 2002;39(March (Pt 2)):89–104. 2. Abrahamson M, Olafsson I, Palsdottir A, et al. Structure and expression of the human Cystatin C gene. Biochem J. 1990;268 (June (2)):287–294. 3. Mussap M, Plebani M. Biochemistry and clinical role of human Cystatin C. Crit Rev Clin Lab Sci. 2004;41(5–6): 467–550.
4. Shlipak MG, Sarnak MJ, Katz R, et al. Cystatin C and the risk of death and cardiovascular events among elderly persons. N Engl J Med. 2005;352:2049–2060. 5. Sarnak MJ, Katz R, Stehman-Breen CO, et al. Cystatin C concentration as a risk factor for heart failure in older adults. Ann Intern Med. 2005;142:497–505. 6. Shlipak MG, Katz R, Sarnak MJ, et al. Cystatin C and prognosis for cardiovascular and kidney outcomes in elderly persons without chronic kidney disease. Ann Intern Med. 2006;145:237–246. 7. Ix JH, Shlipak MG, Chertow GM, Whooley MA. Association of Cystatin C with mortality, cardiovascular events, and incident heart failure among persons with coronary heart disease: data from the Heart and Soul Study. Circulation. 2007;115:173–179. 8. Hoke M, Pernicka E, Niessner A, et al. Renal function and long-term mortality in patients with asymptomatic carotid atherosclerosis. Thromb Haemost. 2012;107:150–157. 9. O'Hare AM, Newman AB, Katz R, et al. Cystatin C and incident peripheral arterial disease events in the elderly: results from the Cardiovascular Health Study. Arch Intern Med. 2005;165:2666–2670. 10. Chew JS, Saleem M, Florkowski CM, George PM. Cystatin C – a paradigm of evidence based laboratory medicine. Clin Biochem Rev. 2008;29(May (2)):47–62. 11. Manocha A, Gupta F, Jain R, et al. The potential of Cystatin C and small dense LDL as biomarkers of coronary artery disease risk in a young Indian population. Mol Cell Biochem. 2014;389(April (1–2)):59–68. 12. Surendar J, Anuradha S, Ashley B, et al. Cystatin C and Cystatin glomerular filtration rate as markers of early renal disease in Asian Indian participants with glucose intolerance (CURES-32). Metab Syndr Relat Disord. 2009;7 (October (5)):419–425. 13. Parikh NI, Hwang SJ, Yang Q, et al. Clinical correlates and heritability of Cystatin C (from the Framingham Offspring Study). Am J Cardiol. 2008;102(November (9)):1194–1198. 14. Muntner P, Mann D, Winston J, Bansilal S, Farkouh ME. Serum Cystatin C and increased coronary heart disease prevalence in US adults without chronic kidney disease. Am J Cardiol. 2008;102(July (1)):54–57. 15. Kestenbaum B, Rudser KD, de Boer IH, et al. Differences in kidney function and incident hypertension: the multi-ethnic study of atherosclerosis. Ann Intern Med. 2008;148(April (7)):501–508. 16. De Boer IH, Astor BC, Kramer H, et al. Mild elevations of urine albumin excretion are associated with atherogenic lipoprotein abnormalities in the Multi-Ethnic Study of Atherosclerosis (MESA). Atherosclerosis. 2008;197(March (1)):407–414. 17. Zhu Y, Zhang HP, Wang YC, et al. Serum Cystatin C level is associated with carotid intima-media thickening and plaque. Scand J Clin Lab Investig. 2015;75(May (3)):265–272. 18. Monteiro Junior FD, Ferreira PA, Nunes JA, et al. Correlation between serum Cystatin C and markers of subclinical atherosclerosis in hypertensive patients. Arq Bras Cardiol. 2012;99(October (4)):899–906. 19. Nand N, Jain R, Seth S, Sen J, Sharma M. A new marker of carotid atherosclerosis in middle aged adults: Cystatin C or microalbuminuria. Indian Heart J. 2010;62(July–August (4)):320–323. 20. Naour N, Fellahi S, Renucci JF, et al. Potential contribution of adipose tissue to elevated serum Cystatin C in human obesity. Obesity (Silver Spring). 2009;17(December (12)):2121– 2126. 21. Sahakyan K, Lee KE, Shankar A, Klein R. Serum Cystatin C and the incidence of type 2 diabetes mellitus. Diabetologia. 2011;54(June (6)):1335–1340.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5 apollo medicine xxx (2016) xxx–xxx
22. Donahue RP, Stranges S, Rejman K, et al. Elevated Cystatin C concentration and progression to pre-diabetes: the Western New York study. Diabetes Care. 2007; 30(July (7)):1724–1729. 23. Shlipak MG, Katz R, Cushman M, et al. Cystatin-C and inflammatory markers in the ambulatory elderly. Am J Med. 2005;118(December (12)):1416.
24. Keller C, Katz R, Cushman M, Fried LF, Shlipak M. Association of kidney function with inflammatory and procoagulant markers in a diverse cohort: a cross-sectional analysis from the Multi-Ethnic Study of Atherosclerosis (MESA). BMC Nephrol. 2008;9:9. 25. Taglieri N, Koenig W, Kaski JC. Cystatin C and cardiovascular risk. Clin Chem. 2009;55(November (11)):1932–1943.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/apme
Research Article
Cystatin levels in a rural South Indian population Krishnan Swaminathan a,*, Ganesh Veerasekar b, Mangalakumar Veerasamy c, Sundaresan Mohanraj d, Kuppusamy Sujatha e, Ganesan Velmurugan f, Nalla G. Palaniswami g, on behalf of KMCH Research Foundation, Coimbatore, India a
Endocrinology, KMCH Research Foundation, India Clinical Epidemiologist, KMCH Research Foundation, India c Consultant Nephrologist, Kovai Medical Center & Hospital, India d Research Coordinator, KMCH Research Foundation, India e Department of Community Medicine, Coimbatore Medical College, India f Post-Doctoral Fellow, DST Unit of Nanoscience, IIT Madras, India g Chairman, Kovai Medical Center & Hospital, KMCH Research Foundation, India b
article info
abstract
Article history:
Aim: Our aim was to study the potential role of Cystatin C as a marker for renal function and
Received 18 February 2016
cardiovascular disease in a rural South Indian population.
Accepted 29 August 2016
Methods: Local Ethics Committee approval and informed consent was obtained from all
Available online xxx
participants. Inclusion criterion was that participants had to be ≥20 and ≤85 years of age. All participants were administered a detailed questionnaire, and had their anthropometric
Keywords:
measurements taken including height, weight, and waist circumference. Blood was drawn
Cystatin C
for determining random glucose, glycosylated hemoglobin, nonfasting lipid profile, Cystatin
Creatinine
C, uric acid, and hemoglobin. Urine samples were collected for urine protein creatinine ratio.
Cardiovascular disease
All participants had their carotid intima thickness determined by high-resolution B-mode
Renal impairment
carotid ultrasound.
Marker
Results: A total of 865 participants were screened in this study. Cystatin C value of >1 mg/l and creatinine of >1.1 mg/dl in females and 1.3 mg/dl in males were defined as ‘‘elevated levels.’’ Nearly a third of this population had elevated Cystatin C levels. Higher levels of Cystatin C, even within the normal creatinine range, was associated with cardiovascular risk markers like age, blood pressures, uric acid, and carotid intima thickness. Conclusions: Cystatin C in a rural South Indian population correlates well with creatinine and cardiovascular markers. Long-term follow-up of this cohort stratified on the basis of Cystatin C will be extremely helpful in augmenting the utility of Cystatin C as an atherosclerotic risk marker in an Indian population. # 2016 Indraprastha Medical Corporation Ltd. All rights reserved.
* Corresponding author. Tel.: +91 8526421150; fax: +91 422 2627782. E-mail address: [email protected] (K. Swaminathan). http://dx.doi.org/10.1016/j.apme.2016.08.003 0976-0016/# 2016 Indraprastha Medical Corporation Ltd. All rights reserved.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5
2
apollo medicine xxx (2016) xxx–xxx
1.
Background
Cystatin C is a member of the family of cysteine proteinase inhibitors, which are produced by all nucleated cells at a constant rate.1–3 The history of Cystatin C began more than 50 years ago with a multitude of clinical data over the past decade on its utility as a marker for kidney (dys) function and atherosclerotic cardiovascular disease. Owing to its free filtration in the glomerulus, complete reabsorption, and lack of tubular secretion, the plasma concentration of Cystatin C is thought to depend mainly on the glomerular filtration rate. Several studies have also shown that Cystatin C has a stronger association with cardiovascular outcomes than serum creatinine.4–9 Despite such exciting prospects, Cystatin C still widely remains a research tool rather than an important investigation in clinical practice. The reasons include lack of standardization of available kits, lack of familiarity with the test that has not proven to affect clinical decision-making in a significant manner, and costs of Cystatin C measurement as opposed to creatinine measurements.10 There have been very few Indian studies evaluating the utility of Cystatin C as a marker for renal function and cardiovascular disease.11,12 Our aim, therefore, was to evaluate the utility of Cystatin C in a larger rural South Indian population and follow-up this cohort long-term to assess the implications of doing this test.
2.
Study design
Nallampatti is a typical rural farming village in South India with a population of around 3500. This village was selected due to the ease of logistics and contacts with the local village heads. All participants >20 years of age were invited to participate in this study through pamphlets and word of mouth. The study was conducted over a 4-week period from March 15th to April 15th, 2015. Ethical Committee approval and informed consent were obtained. A detailed questionnaire was administered followed by anthropometry, blood pressure recordings, and blood investigations including random glucose, glycosylated hemoglobin, nonfasting lipids, Cystatin C, hemoglobin, uric acid, urine protein creatinine ratio (PCR), and carotid intima thickness using carotid ultrasound. Cystatin C results were classified into the following three groups: Group A: normal Cystatin C and normal creatinine, Group B: elevated Cystatin C and normal creatinine, and Group C: elevated Cystatin C and elevated creatinine. Cystatin C levels were analyzed using the nephlometric method (BN Prospec Instrument, Seimens). All results were tabulated on Microsoft Excel and transposed to SPSS 20 for further analysis. Discrete values were analyzed for mean and standard deviation. Categorical variables were analyzed using Pearson's chi-square test. Linear regression was also performed at certain instances to measure the strength of association between the testing variables. Student t-test was employed to calculate the absolute difference in mean between Groups A and B. p < 0.05 was considered statistically significant. The
participants were grouped to have abnormal creatinine level based on National Institute of Health's guidelines, in which cutoff value of creatinine for men is fixed at 1.3 mg/dl and that of women is fixed at 1.1 mg/dl. People were considered to have abnormal Cystatin C if their value is more than 1 mg/dl.
3.
Outcomes
A total of 865 participants participated in this study. Cystatin C levels were elevated in 28.4% of the study population. Mean Cystatin C level was 0.95 mg/l (SD 0.29, range 0.23–3.4 mg/l). We observed an exponential increase in Cystatin C levels with increasing age groups. The percentage of participants with elevated Cystatin C in the age groups 20–40, 41–60, and >60 years were 7.7%, 31.8%, and 54.2%, respectively (Pearson's chi-square p value <0.001). Linear regression was performed to analyze the strength of association between Cystatin C and various covariates. For every unit increase in age, the Cystatin C increases by 0.009 units (95% CI 0.007–0.010, p < 0.001). Women had lower Cystatin C levels, i.e. 0.131 units, compared to men (95% CI, 0.093–0.17, p < 0.001). Participants were classified into three groups, as detailed in the methods section. Baseline characteristics of the three groups are outlined in Table 1. We compared the means between Group A (normal Cystatin C and normal creatinine) and Group B (elevated Cystatin C and normal creatinine). Participants in Group B were older, and had significantly higher systolic and diastolic blood pressures, creatinine level, uric acid level, urine PCR, and carotid intima media thickness. HDL-C levels were significantly lower in Group B compared to Group A (Table 2). There was a positive correlation between Cystatin C and various cardiovascular risk markers including age, blood pressures, creatinine, uric acid, and carotid intima media
Table 1 – Baseline characteristics of three cystatin groups. Variable Total (n) Mean age (years) Weight (kg) SBP (mmHg) DBP (mmHg) Cystatin C (mg/l) Creatinine (mg/dl) Uric acid (mg/dl) HbA1c (%) Total cholesterol (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Urine PCR CIMT right (cm) CIMT left (cm)
Group A
Group B
Group C
632 44.9 (13.1) 58.7 (12.6) 129.4 (21.4) 81.3 (11.6) 0.83 (0.10) 0.70 (0.11) 4.5 (1.3) 5.9 (1.0) 184.4 (37.6) 109 (30.3) 45 (9.3) 0.06 (0.09) 0.06 (0.01) 0.06 (0.01)
216 56.2 (11.1) 60.2 (13.2) 135.4 (20.7) 84.9 (12.4) 1.15 (0.16) 0.80 (0.13) 5.19 (1.2) 6.1 (1.1) 188 (42.3) 112 (34.6) 41.5 (9.9) 0.11 (0.1) 0.07 (0.02) 0.07 (0.02)
17 64.5 (8.08) 50.6 (7.9) 141.6 (21.3) 84.2 (13.8) 2.36 (0.5) 1.67 (0.3) 7.52 (1.3) 6.1 (0.5) 189 (38.9) 110.3 (26.7) 46.9 (26.7) 0.37 (0.3) 0.08 (0.01) 0.08 (0.01)
Group A: normal Cystatin C and normal creatinine. Group B: elevated Cystatin C and normal creatinine. Group C: elevated Cystatin C and elevated creatinine.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5 apollo medicine xxx (2016) xxx–xxx
Table 2 – Comparison of means between Group A and Group B. Variable Age (years) Body weight (kg) SBP (mmHg) DBP (mmHg) Cystatin C (mg/l) Creatinine (mg/dl) Uric acid (mg/dl) HbA1c (%) Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Urine PCR CIMT right (cm) CIMT left (cm)
Group A
Group B
p value*
44.9 58.7 129.4 81.3 0.83 0.70 4.5 5.9 184.4 45 109 0.06 0.06 0.06
56.2 60.2 135.4 84.9 1.15 0.80 5.19 6.1 188 41.5 112 0.11 0.07 0.07
<0.001 0.08 <0.001 <0.001 <0.001 <0.001 <0.001 0.05 0.20 <0.001 0.14 0.003 <0.001 <0.001
* Student t-test used to compare means between two groups, 2tailed, equal variances not assumed.
thickness. Correlations with glycosylated hemoglobin and non-HDL-C were of borderline significance (Table 3).
4.
Discussion
Our study is the largest Indian study to assess the performance of Cystatin C in a rural population. Cystatin C is elevated in nearly a third of our study population and seems to correlate well with creatinine and cardiovascular risk markers. The results of our study are consistent with similar studies from Caucasian population linking higher levels of Cystatin C with cardiovascular risk factors. Among Framingham Heart study offsprings, higher Cystatin C levels were associated with cardiovascular risk factors even in the absence of chronic kidney disease (CKD).13 In a crosssectional study of nearly 5000 participants ≥20 years without CKD, the prevalence of cardiovascular risk factors was higher in individuals with higher Cystatin C concentrations, and Cystatin C was independently associated with cardiovascular disease.14 In our study, individuals with elevated Cystatin C levels with normal creatinine were significantly older, had significantly higher systolic and
Table 3 – Pearson's correlation test. Cystatin C
Pearson's correlation
p value (2-tailed)
Age SBP DBP Creatinine Uric acid HbA1c Non-HDL-C CIMT right CIMT left
0.394 0.149 0.115 0.758 0.415 0.067 0.07 0.281 0.251
<0.001 <0.001 0.001 <0.001 <0.001 0.05 0.05 <0.001 <0.001
3
diastolic blood pressures, serum creatinine, uric acid, glycosylated hemoglobin, and carotid intima thickness, and lower HDL cholesterol levels (Table 2). Higher Cystatin C levels seem to be independently associated not only with hypertension but also with the development of hypertension in the future. In the MultiEthnic Study of Atherosclerosis, it was established that for every 0.2 mg/l increase in Cystatin C levels, there was a significant 15% greater incidence of hypertension.15 In this study, during a median follow-up of 3.1 years, nearly 20% of the participants developed hypertension. This was found to be associated with elevated Cystatin C levels after multivariable adjustment for important clinical factors. In our study, there was a significant difference in both systolic and diastolic blood pressures (6 mm and 3 mmHg, respectively) in participants with higher Cystatin C levels in the absence of renal impairment as defined by creatinine, compared to those with normal Cystatin C levels. Participants in our study with higher Cystatin C levels in the absence of renal impairment (Group B) had significantly higher urine PCR with lower high-density lipoprotein (HDL cholesterol) levels but similar low-density lipoprotein (LDL cholesterol) levels compared to those with normal Cystatin C levels (Table 2). Similar findings were noted in a large study of 5633 participants where greater urine albumin creatinine ratio was associated with lower HDL-C and higher triglycerides but not with LDL-C calculated using conventional methods.16 However, using nuclear magnetic resonance techniques, it was found that each two-fold increase in urine ACR was associated with a significant increase in small LDL-C particle concentration that was not observed directly with a standard lipid panel. Therefore, it may be reasonable to speculate that individuals with higher Cystatin C levels with increased urine PCR, lower HDL-C, but apparently normal LDL-C may still be at a higher cardiovascular risk due to atherogenic lipoprotein abnormalities that may not be directly observed with a standard lipid panel. Carotid intima thickness has been widely used as a marker for early detection of cardiovascular disease. The association between Cystatin C and carotid intima thickness is not very clear. A recently published Chinese study on 927 participants showed that Cystatin C was more strongly associated with carotid thickening and plaque than any other measure of kidney function.17 On the contrary, there have been other studies showing no association between Cystatin C and carotid intima thickness.18,19 In our study, there was a significant increase in carotid intima thickness with increasing Cystatin C levels even in those with normal creatinine levels. The relationship between Cystatin C and diabetes is intriguing. Cystatin C is secreted by adipose tissue, suggesting a possible role of this marker in incident diabetes.20 Cystatin C has shown to predict incident type 2 diabetes and progression from normal to pre-diabetes.21,22 Within normal creatinine levels, participants with elevated Cystatin C levels in our study had higher glycosylated hemoglobin, which was of borderline clinical significance. In those with documented diabetes in our study, the Cystatin C levels were significantly higher compared to those without diabetes (1.04 vs 0.93 mg/ dl, p < 0.001).
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5
4
apollo medicine xxx (2016) xxx–xxx
The question is whether Cystatin C is a marker of renal injury alone or whether there is a larger role in inflammation and atherogenesis? High Cystatin C levels have been found with elevated CRP levels and could be directly linked to inflammation and atherosclerosis.23,24 Inflammatory cytokines linked with atherosclerosis stimulate the production of lysosomal cathepsins. Cystatin C is a cathepsin inhibitor, and elevated levels of Cystatin C may potentially reflect an attempt by the body to counterbalance a damaging effect of elastolytic activity.25 Increased Cystatin C levels, therefore, is emerging as a marker for both kidney and cardiovascular disease. Well-designed larger studies are the need of the hour to explore this marker in our population. The mean Body Mass Index (BMI) was 23.2. The prevalence of generalized obesity as defined by a BMI cutoff of ≥25 was 31.6% in this rural farming population.
5.
Conclusions
In conclusion, we demonstrated elevations in Cystatin C in nearly 30% of a rural South Indian population. Cystatin C levels correlate well with creatinine and cardiovascular risk markers. Long-term follow-up of this cohort stratified by Cystatin C levels will be helpful in augmenting the utility of this test as a renal and atherosclerotic risk marker.
Funding The financial support for this study was provided by an unrestricted educational and research grant through corporate social responsibility funding by Kovai Medical Center and Hospital, Coimbatore, India.
Conflicts of interest The authors have none to declare.
Acknowledgements We acknowledge all the members of the KMCH Research Foundation, volunteers and villagers from Nallampatti, and Mr. Velmurugan from Madurai Kamaraj University. We also thank Dr. Mani, Microbiological Labs, Coimbatore for analyzing the blood investigations.
references
1. Newman DJ. Cystatin C. Ann Clin Biochem. 2002;39(March (Pt 2)):89–104. 2. Abrahamson M, Olafsson I, Palsdottir A, et al. Structure and expression of the human Cystatin C gene. Biochem J. 1990;268 (June (2)):287–294. 3. Mussap M, Plebani M. Biochemistry and clinical role of human Cystatin C. Crit Rev Clin Lab Sci. 2004;41(5–6): 467–550.
4. Shlipak MG, Sarnak MJ, Katz R, et al. Cystatin C and the risk of death and cardiovascular events among elderly persons. N Engl J Med. 2005;352:2049–2060. 5. Sarnak MJ, Katz R, Stehman-Breen CO, et al. Cystatin C concentration as a risk factor for heart failure in older adults. Ann Intern Med. 2005;142:497–505. 6. Shlipak MG, Katz R, Sarnak MJ, et al. Cystatin C and prognosis for cardiovascular and kidney outcomes in elderly persons without chronic kidney disease. Ann Intern Med. 2006;145:237–246. 7. Ix JH, Shlipak MG, Chertow GM, Whooley MA. Association of Cystatin C with mortality, cardiovascular events, and incident heart failure among persons with coronary heart disease: data from the Heart and Soul Study. Circulation. 2007;115:173–179. 8. Hoke M, Pernicka E, Niessner A, et al. Renal function and long-term mortality in patients with asymptomatic carotid atherosclerosis. Thromb Haemost. 2012;107:150–157. 9. O'Hare AM, Newman AB, Katz R, et al. Cystatin C and incident peripheral arterial disease events in the elderly: results from the Cardiovascular Health Study. Arch Intern Med. 2005;165:2666–2670. 10. Chew JS, Saleem M, Florkowski CM, George PM. Cystatin C – a paradigm of evidence based laboratory medicine. Clin Biochem Rev. 2008;29(May (2)):47–62. 11. Manocha A, Gupta F, Jain R, et al. The potential of Cystatin C and small dense LDL as biomarkers of coronary artery disease risk in a young Indian population. Mol Cell Biochem. 2014;389(April (1–2)):59–68. 12. Surendar J, Anuradha S, Ashley B, et al. Cystatin C and Cystatin glomerular filtration rate as markers of early renal disease in Asian Indian participants with glucose intolerance (CURES-32). Metab Syndr Relat Disord. 2009;7 (October (5)):419–425. 13. Parikh NI, Hwang SJ, Yang Q, et al. Clinical correlates and heritability of Cystatin C (from the Framingham Offspring Study). Am J Cardiol. 2008;102(November (9)):1194–1198. 14. Muntner P, Mann D, Winston J, Bansilal S, Farkouh ME. Serum Cystatin C and increased coronary heart disease prevalence in US adults without chronic kidney disease. Am J Cardiol. 2008;102(July (1)):54–57. 15. Kestenbaum B, Rudser KD, de Boer IH, et al. Differences in kidney function and incident hypertension: the multi-ethnic study of atherosclerosis. Ann Intern Med. 2008;148(April (7)):501–508. 16. De Boer IH, Astor BC, Kramer H, et al. Mild elevations of urine albumin excretion are associated with atherogenic lipoprotein abnormalities in the Multi-Ethnic Study of Atherosclerosis (MESA). Atherosclerosis. 2008;197(March (1)):407–414. 17. Zhu Y, Zhang HP, Wang YC, et al. Serum Cystatin C level is associated with carotid intima-media thickening and plaque. Scand J Clin Lab Investig. 2015;75(May (3)):265–272. 18. Monteiro Junior FD, Ferreira PA, Nunes JA, et al. Correlation between serum Cystatin C and markers of subclinical atherosclerosis in hypertensive patients. Arq Bras Cardiol. 2012;99(October (4)):899–906. 19. Nand N, Jain R, Seth S, Sen J, Sharma M. A new marker of carotid atherosclerosis in middle aged adults: Cystatin C or microalbuminuria. Indian Heart J. 2010;62(July–August (4)):320–323. 20. Naour N, Fellahi S, Renucci JF, et al. Potential contribution of adipose tissue to elevated serum Cystatin C in human obesity. Obesity (Silver Spring). 2009;17(December (12)):2121– 2126. 21. Sahakyan K, Lee KE, Shankar A, Klein R. Serum Cystatin C and the incidence of type 2 diabetes mellitus. Diabetologia. 2011;54(June (6)):1335–1340.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
APME-377; No. of Pages 5 apollo medicine xxx (2016) xxx–xxx
22. Donahue RP, Stranges S, Rejman K, et al. Elevated Cystatin C concentration and progression to pre-diabetes: the Western New York study. Diabetes Care. 2007; 30(July (7)):1724–1729. 23. Shlipak MG, Katz R, Cushman M, et al. Cystatin-C and inflammatory markers in the ambulatory elderly. Am J Med. 2005;118(December (12)):1416.
24. Keller C, Katz R, Cushman M, Fried LF, Shlipak M. Association of kidney function with inflammatory and procoagulant markers in a diverse cohort: a cross-sectional analysis from the Multi-Ethnic Study of Atherosclerosis (MESA). BMC Nephrol. 2008;9:9. 25. Taglieri N, Koenig W, Kaski JC. Cystatin C and cardiovascular risk. Clin Chem. 2009;55(November (11)):1932–1943.
Please cite this article in press as: Swaminathan K, et al. Cystatin levels in a rural South Indian population, Apollo Med. (2016), http://dx.doi. org/10.1016/j.apme.2016.08.003
5