Prognostic Value of Isolated High Serum Cystatin C Levels Without Glomerular Filtration Rate Reduction

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INTRODUCTION
Chronic Kidney Disease (CKD) affects 10% of the world's populatio and ranks in the top ten noncommunicable diseases contributing to disease and disability.Its incidence is increasing worldwide, and mortality owing to CKD rose between 2005 and 2017 from 0.9 million to 1.2 million deaths annually.
Serum creatinine and creatinine clearance were rst used to evaluate renal function by the Danish physiologists, Rehberg and Holten in the mid-1920s.Serum creatinine is the only renal plasma biomarker currently used in daily clinical practice to estimate GFR.However, a proper interpretation of the serum creatinine result remains sometimes problematic.Creatinine clearance is a relatively easy method to estimate GFR but it has some important limitation of this measurement (i.e.creatinine tubular secretion which is variable from one subject to another).Creatinine clearance systematically overestimates measured GFR and this overestimation is higher at low GFR levels.Nowadays, the creatinine based equations, especially the Modi cation of Diet in Renal Disease study (MDRD) equation and the CKD-EPI one, are used all over the world to estimate GFR.
Cystatin C (CysC) is an interesting new marker for the estimation of GFR.It does offer several advantages over creatinine or other similar molecular weight proteins.CysC is produced by all nucleated cells in the human body and because the protein is coded by a housekeeping gene, (i.e. a gene expressed both constitutively and in an unregulated manner), CysC is considered to be constantly produced.After being ltered without restriction by the glomeruli because of its low molecular mass and absence of protein binding, CysC is entirely reabsorbed by the proximal tubules, where it is almost entirely catabolized.
Current KDIGO Guidelines for CKD suggests measuring CysC in adults with GFR 45-59 ml/min/1.73m2 who do not have markers of kidney damage if con rmation of CKD is required.
The proportion of CKD patients de ned by GFR who progress to end-stage renal disease is extremely small.The KDIGO Guidelines recognized the importance of albuminuria, and the underlying diagnosis as well as GFR.A table was designed to categorized risk iIn populations with CKD using group eGFR and albuminuria severity, although the evidence was not graded 5 .We have tried to evaluate the value of isolated high plasma CysC levels in patients with normal plasma creatinine level and estimated GFR higher than 60 ml/min as renal risk marker.

DESIGN AND METHODS
A group of 608 patients were studied: 401 males and 207 females; mean age was 53.3 ± 12.8 years; 34.6% have diabetes mellitus.Serum cystatin C was measured using a BNII nephelometer (Dade Behring Inc., Deer eld, IL, USA) that used a particle-enhanced immunonephelometric assay (N Latex Cystatin-C).
The assay range is 0.195-7.330mg/L, with the reference range for young healthy individuals reported as 0.53-0.95mg/L.Microalbuminuria was measured in 24 h urine collection and 18.5% showed increased urine albumin excretion (≥ 30 mg/day).GFR was estimated from serum creatinine using the CKD-EPI equation for every sex.Only Caucasian patients were included in the study, so that race was not included in calculation.Albuminuria was analyzed in 24h urine collection.
Patients were classi ed according to K/DIGO stages of chronic renal disease: 13.6% were in stage IV or V, 30.9% were in stage III, and the remaining patients had GFR higher than 60 mL/min (39.1%).The cut-off point for the highest quartile of serum cystatin C distribution was 1.03 mg/L.GFR was estimated from cystatin C using the CKD-EPI formulation for cystatin C. Microalbuminuria was de ned as an urinary albumin excretion ≥ 30 and < 300 mg/day; macroalbuminuria was diagnosed when albuminuria was equal or higher than 300 mg/day.Patients were split into three groups: CONTROL (normal cystatin C levels and GFR > 60 ml/min, n = 193), HIGH CYSTATIN (HCy), (cystatin C higher than 1.03 mg/l but GFR > 60 ml/min,, n = 40 ) and CKD (Chronic Kidney Disease, cystatin C above 1.03 mg/g and GFR < 60 ml/min, n = 160).´Table 1 show the characteristics of each group.
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The ethical approval was supplied by the University of Extremadura Ethics Committee (#21/2014).Informed consent was obtained from all individual participants included in the study.

Statistics
Results are expressed as mean ± 1 standard deviation.All statistical tests were two-sided.P values lower than 0.05 were considered as signi cant.For comparisons between groups, Anova test and Bonferroni post-hoc analysis was used for continuous variables and Chi-square test for categorical variables.Since Kolmogorov-Smirnov Z test found that albuminuria did not follow a normal distribution, Kruskall-Wallis test was used to compare values.These parameters have been expressed as median (IR, interquartilic range).The statistical analysis was developed with the package SPSS 21.0.
Kaplan-Meier survival analysis was used to calculate survival before death, reaching stage V KDIGO CKD or renal replacement therapy.Associations between the three subgroups of patients and risk of death or reaching renal replacement therapy were assessed using Cox proportional hazards survivorship model.
Hazard ratios (HR) and corresponding 95% con dence intervals (CI) were calculated, and a p value < 0.05 was considered to be statistically signi cant.All variables achieving a signi cance level of p < 0.1 in univariate analysis were considered for inclusion in the construction of the Cox model.The data were analyzed using the IBM® statistical program SPSS ® Statistics V.21 (IBM Corporation, Armonk, NY, USA).
Kaplan-Meier survival for general mortality at 5 years was 93.9% for CONTROL group, 78.8% for CKD group and 82.3% in the HCy group (p < 0,001, Log Rank test for the difference between CONTROL group and HCy one) (Fig. 1).Regarding survival before starting renal replacement therapy or reaching stage V KDIGO, the Kaplan-Meier method shows a survival at 5 years 99.0% for CONTROL group, 73.0% for CKD group and 94.3% in the HCy group (p < 0,001 Log Rank for the CKD group, there is no difference between HCy and Control ones, p = 0.08) (Fig. 2).
In the global sample, after adjusting for possible confounding factors, Cox analysis showed a signi cant relationship of high cystatin C levels with mortality (p < 0.001), only age and diabetes mellitus showed a signi cant relationship (see Table 2).Contrariwise, after adjusting for possible confounding factors, survival before reaching renal replacement therapy was signi cantly associated with cystatin C levels (p = 0.008), GFR measured by CKD-EPI equation and urinary albumin excretion (see signi cances in Table 3).

DISCUSSION
We have found that in a group of patients with high plasma cystatin C levels and GFR below 60 ml/min mortality was higher than in those patients with normal cystatin C levels and GFR > 60 ml/min.Contrariwise, the risk of progression of chronic kidney disease was not higher in the HCy group.Those patients with high cystatin levels and reduced GFR both mortality and CKD have the highest risk of mortality and progression to stage V KDIGO of CKD or renal replacement therapy.
The renal system carries several physiologic roles but GFR is considered the best surrogate of overall kidney function and, for this reason, its assessment has become an important tool in clinical practice.GFR cannot be measured directly, but instead can be estimated by the clearance of ltration markers.Regardless, the clinical assessment of GFR can aid the clinician in estimating the degree of renal dysfunction and/or progression of established kidney disease.Various creatinine-based equations have been developed in an attempt to improve the estimation of GFR from serum creatinine.Current KDIGO Guidelines recommend CKD-EPI Eq. 7 and that is the one that we have selected.In this regard, the current de nition for chronic kidney disease is a GFR < 60 ml/min for more than three months and this is the threshold that was selected to split the study group.
Patients with CKD exhibit a pronounced risk for cardiovascular events: 50% of all patients with CKD stage 4 to 5 have CVD,2 and cardiovascular mortality accounts for ≈ 40-50% of all deaths in patients with advanced CKD (stage 4) as well as end-stage kidney disease (stage 5), compared with 26% in controls with normal kidney function.The proportions of deaths from heart failure and valvular disease speci cally increased with declining eGFR along with the proportions of deaths from infectious and other causes, whereas the proportion of deaths from cancer decreased.In the same way, lower eGFR is associated with an increased mortality risk caused by infection.The cut point used was, as in our study, a GFR < 60 ml/min but mortality it is specially increased when GFR get down of 45 ml/min.Therefore, it could explain why mortality is higher in the CKD group but cannot give light about the relationship of higher plasma cystatin levels with a GFR above 60 ml/min and mortality.
Cystatin C is an interesting marker for the estimation of GFR.It does offer several advantages over creatinine or other similar molecular weight proteins.It is produced by all nucleated cells in the human body and is considered to be constantly produced.After being ltered without restriction by the glomeruli because of its low molecular mass and absence of protein binding, cystatin C is entirely reabsorbed by the proximal tubules, where it is almost entirely catabolized.Current KDIGO Guidelines for CKD suggests measuring cystatin C in adults with GFR 45-59 ml/min/1.73m2 who do not have markers of kidney damage if con rmation of CKD is required.Cystatin C is also a correlate of cardiovascular risk.These associations persisted with the additional exclusion of persons with CKD or with microalbuminuria. 23  Nevertheless, these two abilities of cystatin C seem not to be correlated in our study.In fact, mortality was independently associated with cystatin C levels but this relationship was not found with eGFR or albuminuria.These ndings can be interpreted in various ways.First of all, persons with high cystatin C levels but without CKD may have preclinical CKD and an associated elevated risk factor burden that is similar to persons with CKD.Because of measurement error involved in the quanti cation of eGFR, it is also possible that persons with high cystatin C but GFR that is not in the CKD range are more accurately classi ed as renal patients by using cystatin C levels.Nevertheless, this hypothesis seems unlikely since GFR calculated from cystatin C or creatinine rendered very close results.Moreover, cystatin C -without disminished GFR-is not associated to CKD progression.Alternatively, high cystatin C in persons without CKD may be the result of extra-renal sources of cystatin C variability and so that they were not related to kidney function.There is some evidence that serum cystatin C level is heritable and high serum cystatin C levels were associated with most major CVD risk factors. 23  Increased urinary albumin excretion in diabetic patients has been found to be a predictor of progression of diabetic nephropathy and also as a powerful independent risk factor for cardiovascular morbidity and mortality ,, .In nondiabetic hypertensive patients urinary albumin excretion has been shown to predict cardiovascular events, and a continuous relation between urinary albumin excretion and cardiovascular, as well as general, mortality has been demonstrated in a general population study ,,,, .Therefore, searching for microalbuminuria is currently recommended, because of the evidence that it may be a sensitive marker of target organ damage, not only in diabetes but also in hypertension , .Therefore, current KDIGO Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease state to assess GFR and albuminuria at least annually in people with CKD.Moreover, GFR and albuminuria should be assessed more often for individuals at higher risk of progression, and/ or where measurement will impact therapeutic decisions 5 .Although a signi cant proportion of subjects in the CONTROL and HCy had increased urinary albumin excretion, it was not associated to mortality risk, but it was signi cantly related to CKD progression.

Strengths and limitations
The limitation of this study is the small size of the sample with high cystatin C values and GFR below 60 ml/min.Nevertheless, this kind of patients are uncommon in the clinical practice and, therefore, the information resulting for our results become more important.As a matter of fact, few data have been reported on this issue.Shlipak et al 14 used data from de Cardiovascular Health Study where, at baseline, 78% of participants have estimated GFR > or = 60 mL/min.Cystatin C concentrations had strong associations with death, cardiovascular death, and major cardiovascular events among these  Comparative life survival using Kaplan-Meier method.The differences among the three groups are signi cant.

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