Streszczenie

Wprowadzenie: Wyniki opublikowanych badań dowodzą, że oznaczanie cystatyny C w surowicy może stano­wić lepszy marker czynności wydalniczej nerek niż stężenie kreatyniny w surowicy. Jednakże znaczenie tego markera w diagnostyce upośledzonej czynności nerek u noworodków nie zostało jednoznacznie wyjaśnione. Celem badania jest analiza stężeń cystatyny C w surowicy u nowo­rodków leczonych z powodu sepsy.
Materiał/Metody: Do badań włączono 32 noworodki (wiek płodowy 34-40 tygodni) przyjęte do Oddziału Intensywnej Terapii i Patologii Noworodka w ciągu pierwszych 14 dni życia. Stężenie cystatyny C oznaczono metodą ELISA w 3 kolejnych dobach leczenia z powodu zakażenia. Wśród analizowanych no­worodków u 9 rozpoznano sepsę, u 14 – ciężką sepsę, a u 9 – wstrząs septyczny.
Wyniki/Dyskusja: W chwili włączenia do obserwacji średnie stężenie cystatyny C wynosiło 1,35 mg/L (95% CI 1,20-1,49). Nieoczekiwanie najniższe wartości cystatyny C obserwowano u pacjentów, u których w kolejnych dniach rozwinął się wstrząs septyczny – 1,13 mg/L (95% CI 0,91-1,34). Wyższe war­tości stwierdzono u noworodków z sepsą 1,51 mg/L (95% CI 1,18-1,84) i ciężką sepsą 1,38 mg/L (95% CI 1,13-1,63). Nie wykazano korelacji ze stężeniem kreatyniny w surowicy oraz wiekiem płodowym. Stwierdzono natomiast istotną korelację pomiędzy wiekiem chronologicznym a stę­żeniem cystatyny C (R=-0,439, p=0,01). W kolejnych dniach obserwacji wykazano obniżanie się stężeń cystatyny C u chorych z sepsą (1,34 mg/L; 95% CI 0,89-1,78) i ciężką sepsą (1,24 mg/L; 95% CI 1,00-1,48), podczas, gdy u pacjentów we wstrząsie septycznym obserwowano wzrost wartości, choć niewielki i nieistotny statystycznie (1,25 mg/L; 95% CI 0,97-1,53). Zależność po­między wiekiem a stężeniem cystatyny C zanikała w kolejnych dniach obserwacji. Nawet u pa­cjentów, którzy zmarli w przebiegu wstrząsu septycznego, obserwowane zmiany w stężeniu cy­statyny C były niewielkie i nie przekraczały zmian w stężeniu kreatyniny.
Wnioski: Cystatyna C nie jest przydatnym parametrem uszkodzenia nerek u noworodków z sepsą.

Słowa kluczowe:cystatyna C • kreatynina • noworodki • czynność nerek • sepsa

Summary

Introduction: Several studies have claimed that the estimation of serum cystatin C could be a better marker of kidney excretory function than serum creatinine. However, its role in the diagnosis of reduced kidney function was not unquestionably confirmed. The aim of this study was to analyze the con­centrations of serum cystatin C in neonates with sepsis.
Material/Methods: Thirty-two neonates (gestational age from 34 to 40 weeks) admitted to the NICU during the first 14 days of life were enrolled. Serum cystatin C concentrations were estimated by ELISA during three successive days in neonates treated for infection. The study group consisted of 9 newborns with sepsis, 14 with severe sepsis and 9 with septic shock.
Results/Discussion: At the beginning of the observational period the mean serum concentration of cystatin C in the study group was 1.35 mg/L (95% CI 1.20-1.49). Surprisingly, the lowest concentration of cy­statin was observed in patients with septic shock (1.23 mg/L; 95%CI 0.92-1.54) within the ob­servation period. Higher concentrations were found in neonates with sepsis (1.47 mg/L; 95%CI 1.04-1.90) and severe sepsis (1.50; 1.12-1.87). There was no correlation between serum cystatin C concentration and serum creatinine or gestational age. A significant correlation was discovered between chronological age and cystatin C (R=-0.439, p=0.01). There was a tendency for cystatin C to decline during the second observational day in patients with sepsis (to 1.53 mg/L; 95%CI: 1.19-1.86) and severe sepsis (to 1.32 mg/L; 95%CI: 1.07-1.57), while a slight insignificant increase in patient with septic shock (to 1.28 mg/L; 95%CI: 0.88-1.68) was revealed. The interrelation between age and cystatin C concentration disappeared in the following days of stay in the NICU. Even in patients who died in the course of septic shock the observed changes in cystatin C levels were small and did not exceed those of serum creatinine.
Conclusions: Cystatin C is not a useful marker of kidney function in neonates with sepsis.

Key words:cystatin C • creatinine • newborns • kidney function • sepsis

Abbreviations:

ABG – arterial blood gases; A&E – accident and emergency; AKI – acute kidney injury; CBC – complete blood count; CI – confidence interval; CRP – C-reactive protein; GA – gestational age; GFR – glomerular filtration rate; NICU – neonatal intensive care unit; SIRS – systemic inflammatory response syndrome.

Introduction

Sepsis is one of the most severe disorders in newborns and young infants, responsible for almost one and a half mil­lion deaths yearly worldwide. Sepsis is typically divided to mild sepsis, severe sepsis and septic shock. The latter two may lead to multiorgan dysfunction syndrome, and if the inflammatory process is not stopped and reversed it may eventually cause multiorgan failure and death. Clinical si­gns and symptoms are not specific in newborns, which makes the disease even more difficult to diagnose. Early introduction of antibiotic therapy is essential in the mana­gement of generalized infection.

Symptoms suggesting kidney dysfunction occur relatively late, but may quickly be followed by acute kidney insufficien­cy. Acute kidney injury (AKI) is claimed to be recognized in about 50% of septic adult patients. The epidemiological data in newborns are missing, but clinical observations confirm the above-mentioned numbers. Both systemic inflammation and AKI are serious risk factors for unfavorable outcome. There are not many research data on kidney function in the short-term post-septic period or in long-term follow-up.

As soon as the diagnosis of AKI is made, the treatment in­cluding adequate hydration, use of diuretics for proper vo­lume control, and diet can be introduced. Currently, serial measurements of serum creatinine and monitoring of diu­resis are the only methods for assessment of kidney func­tion. Serial serum creatinine measurements in the early postnatal period eliminate the impact of muscle mass va­riability or gender, but not that of diet and the declining effect of impaired creatinine elimination during pregnancy [5,7]. Initially raised serum creatinine values in newborns gradually decline during the first months, reaching adult values by the end of the first year [7]. Additionally, inter­ference of bilirubin, hemoglobin and ketone bodies with creatinine measurements was observed [7].

Extensive research on new, more sensitive and specific mar­kers has been conducted in the last twenty years. As a result, cystatin C was proposed for evaluation of kidney function.

Cystatin C (CystC), a 120-amino acid protein, is a cyste­ine protease inhibitor produced by all nucleated cells at a constant rate [1,13]. CystC is filtered in the glomeru­lus, reabsorbed and degraded by epithelium of proximal tubes, and passed into the urine [19,21,22]. The role of CystC in the diagnosis of reduced kidney function has not been confirmed in neonates. It is known that CystC does not cross the placenta and should then reflect only the in­fant’s own renal function [7]. On the other hand, marke­dly higher concentrations of CystC were revealed shor­tly after delivery, caused by failure of placental removal. At birth, CystC concentration is higher than in adults and decreases within 2 months [7]. The reference values pro­posed by Finney [7] were defined as follows: mean value 1.37 (mg/L) with range 0.81-2.32 [7]. Armangil [2] me­anwhile reported 1.25-2.84 mg/L (mean 1.8±0.3) on the first day of life followed by a significant decrease on the third day (mean: 1.65±0.3 mg/L).

Studies performed in adults showed a strong correlation be­tween CystC and GFR [3,9,10,18], and these findings were confirmed in children and newborns [4,11]. The lack of in­fluence of muscle mass (approx. 1 of body weight) or in­flammatory status on CystC value is considered to be an advantage [1,10,18], and allows this marker to be used in GFR evaluation [7]. Body weight, gender, diet, hydration and nutritional status have no influence on CystC concen­tration, but thyroid function may have some impact [5].

The aim of this study is to analyze the concentrations of cystatin C in neonates suffering from sepsis, intensively treated at the NICU, and to compare cystatin C values with creatinine concentration.

Material and Methods

Thirty-two neonates in the first 14 days of life, diagnosed with sepsis based on clinical symptoms and/or clinical history with risk factors of infection, confirmed later by positive microbiological blood culture in 24 cases, were enrolled. Sepsis (N=9) was defined as SIRS and eviden­ce of infection (clinical symptoms or positive blood cul­ture) and severe sepsis (N=14) was identified when the course of sepsis was complicated by dysfunction of two or more organs or systems. Septic shock (N=9) was reco­gnized when the decrease in blood pressure occurred to­gether with symptoms of compromised peripheral blood perfusion. All studied patients were outborns and were admitted to a single, third level university children’s ho­spital from home (A&E department) or transferred to the unit from other hospitals by a neonatal ambulance trans­port service. Clinical evaluation of the study group is pre­sented in Table 1. Clinical condition at the moment of in­clusion was objectified using SNAP (Score for Neonatal Acute Physiology) and the mean value for the study gro­up was 9.2 (7.3-11.0).

Table 1. Clinical evaluation of the study group (N=32) – mean values (95%CI)

Inclusion criteria consisted of gestational age equal to or higher than 34 weeks of gestation and signs and symptoms of neonatal sepsis. Malformations of urinary system and confirmed severe perinatal asphyxia were the exclusion criteria. The study group consisted of 20 males and 12 fe­males. The average gestational age was 37 weeks (range: 34-40) and the average birth weight was 2920 g (range: 1720-4470g). Mean Apgar score after 1 minute was 7 po­ints (range: 1-10), and 8 after 5 minutes (range: 5-10). One patient was given 5 points in the Apgar score after 5 mi­nutes and 1 point after the first minute, but further clinical observation and biochemical tests did not confirm perina­tal asphyxia. The reasons for hospital admission included hyperbilirubinemia, failure to thrive, symptoms of respi­ratory distress and suspected infection. The mean chrono­logical age on admission was 8 days of life (range: 1-14), and the sepsis was diagnosed within 5 days of hospitali­zation on average (range: 1-13).

Acute kidney injury (AKI) was scored according to the RIFLE classification based on serum creatinine (at least 1.5-fold increase from baseline values) and urinary output changes after admission to the NICU [15].

The study protocol was approved by the Institutional Bioethics Committee. The protocol did not allow for any changes from the basic algorithm of diagnosis and treat­ment accepted in the unit only for the purpose of this stu­dy. All included patients were cared for and treated in the standard way, and the preservation of some samples of se­rum for further biochemical analysis was the only exception.

All study participants were screened for microbiological colo­nization on admission and basic septic screening was perfor­med, including CBC with blood smear, CRP, glucose, lactate, electrolytes and blood culture, as well as basic biochemical evaluation including creatinine, total protein, serum albumin and bilirubin. Urinalysis and urine culture were also perfor­med. Serum samples were collected after the routine tests were completed, and frozen to -70°C in polypropylene tubes.

The observational period started when the patient presen­ted evident clinical symptoms of infection and antibio­tic therapy was introduced. The standard protocol inclu­ded biochemical screening (glucose, lactate, creatinine, aspartate aminotransferase [AST], alanine aminotrans­ferase [ALT]), CBC and ABG every second day or more often, according to clinical requirements. Cystatin C me­asurements were performed by ELISA according to the manufacturer’s instructions (BioVendor, Modrice, Czech Republic). Creatinine was measured in serum samples by the Jaffe method (Olympus AU 640).

Intra and inter-assay coefficients of variability were 5.0-9.6% and 4.8-6.2% for high and low concentrations, respectively.

Statistical analysis

STATISTICA 8.0 (StatSoft Polska, Kraków, Poland) so­ftware was used for statistical analysis. All presented data were expressed as means and 95% confidence intervals. Normality of distribution was tested with the Kolmogorov-Smirnov test. Mann-Whitney U pair-wise comparison for independent variables and Wilcoxon pair-wise comparison for dependent variables were used as appropriate. Chi² test and Chi² test with Yates’ correction were used to compare distributions between groups. Correlation coefficients were calculated according to Spearman. The p values <0.05 were considered as statistically significant.

Results

At the time of enrolment the mean serum concentration of CystC was 1.35 mg/L (95% CI 1.20-1.49). The lowest concentration was 1.23 mg/L (95% CI 0.92-1.54) and was observed in patients who developed septic shock during the following days. Surprisingly, slightly higher concentra­tions were found in neonates with sepsis (1.47 mg/L; 95% CI 1.04-1.90) and severe sepsis (1.50; 1.12-1.87).

Analysis of demographic factors revealed lack of a corre­lation between CystC and gestational age, 5′ minute Apgar score, birth weight and gender. A significant correlation was found between chronological age and CystC levels (R=-0.439, p=0.01), but the interrelation disappeared on the following days of hospitalization. There was also no correlation with serum creatinine (Figure 1).

Figure 1. Correlation between serum cystatin C and creatinine levels

Comparison of creatinine and CystC values at successive time points (at the beginning of the observational period, and after 24 and 48 hours) in the three subgroups of pa­tients (groups A, B and C) showed some statistically si­gnificant differences of creatinine concentrations betwe­en the subgroups. Such results were not found in regard to CystC (Table 2, 3).

Table 2. Values of creatinine and cystatin C in three subgroups of newborns at successive time points. Mean value, 95%CI and statistical significance of differences between the subgroups A, B and C (Mann-Whitney U test) are presented

Table 3. Serum cystatin C concentration in neonates with and without acute kidney injury (AKI) during the three successive days of observation (Mann-Whitney U test), values presented as mean value and 95%CI

During the 3-day observation period a tendency for CystC level to decline in patients with sepsis (1.53 mg/L, 95%CI: 1.19-1.86) and severe sepsis (1.32 mg/L; 95%CI: 1.07-1.57) was found. In patients with septic shock CystC levels in­significantly increased (1.28 mg/L; 95%CI: 0.88-1.68) – Figure 2. Even in patients who died in the course of sep­tic shock the observed changes in CystC were small and did not exceed those of serum creatinine.

Figure 2. Serum cystatin C levels in patients with sepsis, severe sepsis and septic shock on successive days of observation

Discussion

The results of the study revealed that assessment of CystC is not better, and in fact seems to be even worse than that of serum creatinine, in newborns treated for sepsis in the detection of sepsis-related AKI.

The study group was quite homogeneous and consisted only of newborns admitted to the hospital on their first days of life, not older than 15 days. On admission their cli­nical condition was diverse, ranging from very stable to unstable with symptoms of cardio-respiratory dysfunction.

Regardless of the severity of clinical condition the initial serum creatinine levels were elevated, which is considered physiological in newborns in their first 2-3 days of life. It has been shown that serum creatinine in the first three days of life revealed the kidney status of the mother, as creati­nine is eliminated through the placenta along a gradient [2]. Analysis of serum creatinine concentrations separate­ly in all three groups of patients revealed higher values in newborns suffering from severe sepsis and septic shock, indicating AKI in some patients, even taking into account limitations such as maternal kidney status, chronological age and muscle mass.

Surprisingly, CystC values were all in the normal range. There are few authors reporting on CystC concentrations, measured by nephelometric or turbidimetric methods, on the first days of life: Novo – 1.70±0.26 mg/L on the first and 1.51±0.19 mg/L on the third day of life [17]; Finney [7] – 0.81-2.32 mg/L during the first three months; Harmoinen [11] – 1.36-2.23 mg/L in the first week of life; and Treiber [20] 1.28-2.66 mg/L on the third day. The range 0.81-2.6 mg/L detected in our patients corresponds with the abo­ve data of clinically stable newborns, but measured with a different method (ELISA).

In contrast to a suggestion of Nejat et al. [16], who claimed that CystC was an effective and earlier surrogate marker of decreased renal function than plasma creatinine in a gene­ral ICU population, we could not confirm that on the basis of our results. Recently, Martensson et al. reported [14] a similar increase in serum CystC and creatinine levels in pa­tients with AKI and sepsis. Thus the assessment of CystC as a functional marker of AKI seems to be useless at least in septic newborns. Some authors suggest that CystC is a better marker only for detection of chronic kidney injury, and that only chronic damage causes a steady and signifi­cant increase in CystC concentration [6,8,12,22].

The relatively low values found in the most severely ill pa­tients are rather surprising. Regarding the normal range of CystC in newborns, patients with septic shock presented values in the lower range or even below. Cystatin C is fo­und in the lysosomes of every cell, and actively participate in protein metabolism inside the cell. The marker is known for its resistance to the influence of inflammation. It seems that severe inflammatory status may disturb cell metabo­lism, causing a decrease in concentrations of some sub­stances. Unfortunately, the literature on the topic is spar­se, and fails to provide a good explanation.

Recently Matterson et al. reported that sepsis per se does not affect circulating CystC level, and thus is a potential­ly valuable marker of kidney function, including in septic patients. Moreover, this group did not show a correlation between CRP and cystatin C values [14].

The main limitation of the study is the lack of glomeru­lar filtration rate measurement by insulin clearance tech­nique or similar. Moreover, we did not check the thyroid status, although all newborns were screened for congenital hypothyroidism. Finally, we did not enroll a group of heal­thy neonates as the control group. Thus we cannot compa­re the cystatin C values observed in septic neonates with those in healthy subjects.

We conclude that cystatin C is not a useful marker of kid­ney function in neonates with sepsis.

REFERENCES

[1] Abrahamson M., Olafsson I., Palsdottir A., Ulvsbäck M., Lundwall A., Jensson O., Grubb A.: Structure and expression of the human cystatin C gene. Biochem. J., 1990; 268: 287-294
[PubMed]  [Full Text PDF]  

[2] Armangil D., Yurdakök M., Canpolat F.E., Korkmaz A., Yigit S., Tekinalp G.: Determination of reference values for plasma cystatin C and comparison with creatinine in premature infants. Pediatr. Nephrol., 2008; 23: 2081-2083
[PubMed]  

[3] Bökenkamp A., Domanetzki M., Zinck R., Schumann G., Byrd D., Brodehl J.: Cystatin C – a new marker of glomerular filtration rate in children independent of age and height. Pediatrics, 1998; 101: 875-881
[PubMed]  

[4] Cataldi L., Mussap M., Bertelli L., Ruzzante N., Fanos V., Plebani M.: Cystatin C in healthy women at term pregnancy and in their infant newborns: relationship between maternal and neonatal serum levels and reference values. Am. J. Perinatol., 1999; 16: 287-295
[PubMed]  

[5] Corrao A.M., Lisi G., Di Pasqua G., Guizzardi M., Marino N., Ballone E., Chiesa P.L.: Serum cystatin C as a reliable marker of changes in glomerular filtration rate in children with urinary tract malformations. J. Urol., 2006; 175: 303-309
[PubMed]  

[6] Filler G., Bökenkamp A., Hofmann W., Le Bricon T., Martinez-Brú C., Grubb A.: Cystatin C as a marker of GFR – history, indications, and future research. Clin. Biochem., 2005; 38: 1-8
[PubMed]  

[7] Finney H., Newman D.J., Thakkar H., Fell J.M., Price C.P.: Reference ranges for plasma cystatin C and creatinine measurements in premature infants, neonates, and older children. Arch. Dis. Child., 2000; 82: 71-75
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[8] Franco M.C., Nishida S.K., Sesso R.: GFR estimated from cystatin C versus creatinine in children born small for gestational age. Am. J. Kidney Dis., 2008; 51: 925-932
[PubMed]  

[9] Grubb A.: Diagnostic value of analysis of cystatin C and protein HC in biological fluids. Clin. Nephrol., 1992; 389(Suppl. 1): S20-S27
[PubMed]  

[10] Grubb A., Simonsen O., Sturfelt G., Truedsson L., Thysell H.: Serum concentration of cystatin C, factor D and beta 2-microglobulin as a measure of glomerular filtration rate. Acta Med. Scand., 1985; 218: 499-503
[PubMed]  

[11] Harmoinen A., Ylinen E., Ala-Houhala M., Janas M., Kaila M., Kouri T.: Reference intervals for cystatin C in pre- and full-term infants and children. Pediatr. Nephrol., 2000; 15: 105-108
[PubMed]  

[12] Herget-Rosenthal S., Marggraf G., Hüsing J., Göring F., Pietruck F., Janssen O., Philipp T., Kribben A.: Early detection of acute renal failure by serum cystatin C. Kidney Int., 2004; 66: 1115-1122
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[13] Laterza O.F., Price C.P., Scott M.G.: Cystatin C: an improved estimator of glomerular filtration rate? Clin. Chem., 2002; 48: 699-707
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[14] Martensson J., Martling C.R., Oldner A., Bell M.: Impact of sepsis on levels of plasma cystatin C in AKI and non-AKI patients. Nephrol. Dial. Transplant., 2012; 27: 576-581
[PubMed]  

[15] Mehta R.L., Kellum J.A., Shah S.V., Molitoris B.A., Ronco C., Warnock D.G., Levin A.: Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit. Care, 2007; 11: R31
[PubMed]  [Full Text PDF]  

[16] Nejat M., Pickering J.W., Walker R.J., Endre Z.H.: Rapid detection of acute kidney injury by plasma cystatin C in the intensive care unit. Nephrol. Dial. Transplant., 2010; 25: 3283-3289
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[17] Novo A.C., Sadeck Ldos S., Okay T.S., Leone C.R.: Longitudinal study of Cystatin C in healthy term newborns. Clinics, 2011; 66: 217-220
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[18] Simonsen O., Grubb A., Thysell H.: The blood serum concentration of cystatin C (gamma-trace) as a measure of the glomerular filtration rate. Scand. J. Clin. Lab. Invest., 1985; 45: 97-101
[PubMed]  

[19] Tenstad O., Roald A.B., Grubb A., Aukland K.: Renal handling of radiolabelled human cystatin C in the rat. Scand. J. Clin. Lab. Invest., 1996; 56: 409-414
[PubMed]  

[20] Treiber M., Pecovnik-Balon B., Gorenjak M.: Cystatin C versus creatinine as a marker of glomerular filtration rate in the newborn. Wien Klin. Wochenschr., 2006; 118(Suppl. 2): 66-70
[PubMed]  

[21] Warwas M., Piwowar A.: Moczowa cystatyna C jako biomarker uszkodzenia kanalików nerkowych. Postępy Hig. Med. Dośw., 2011; 65: 562-568
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[22] Zappitelli M., Parvex P., Joseph L., Paradis G., Grey V., Lau S., Bell L.: Derivation and validation of cystatin C-based prediction equations for GFR in children. Am. J. Kidney Dis., 2006; 48: 221-230
[PubMed]  

The authors have no potential conflicts of interest to declare.

Pełna treść artykułu