Cystatin C, a measure of renal function, as prognostic risk marker in acute heart failure : Studies on the cardiorenal syndrome

Acute heart failure (AHF) is a complex syndrome associated with exceptionally high mortality. Still, characteristics and prognostic factors of contemporary AHF patients have been inadequately studied. Kidney function has emerged as a very powerful prognostic risk factor in cardiovascular disease. This is believed to be the consequence of an interaction between the heart and kidneys, also termed the cardiorenal syndrome, the mechanisms of which are not fully understood. Renal insufficiency is common in heart failure and of particular interest for predicting outcome in AHF. Cystatin C (CysC) is a marker of glomerular filtration rate with properties making it a prospective alternative to the currently used measure creatinine for assessment of renal function. The aim of this thesis is to characterize a representative cohort of patients hospitalized for AHF and to identify risk factors for poor outcome in AHF. In particular, the role of CysC as a marker of renal function is evaluated, including examination of the value of CysC as a predictor of mortality in AHF. The FINN-AKVA (Finnish Acute Heart Failure) study is a national prospective multicenter study conducted to investigate the clinical presentation, aetiology and treatment of, as well as concomitant diseases and outcome in, AHF. Patients hospitalized for AHF were enrolled in the FINN-AKVA study, and mortality was followed for 12 months. The mean age of patients with AHF is 75 years and they frequently have both cardiovascular and non-cardiovascular co-morbidities. The mortality after hospitalization for AHF is high, rising to 27% by 12 months. The present study shows that renal dysfunction is very common in AHF. CysC detects impaired renal function in forty percent of patients. Renal function, measured by CysC, is one of the strongest predictors of mortality independently of other prognostic risk markers, such as age, gender, co-morbidities and systolic blood pressure on admission. Moreover, in patients with normal creatinine values, elevated CysC is associated with a marked increase in mortality. Acute kidney injury, defined as an increase in CysC within 48 hours of hospital admission, occurs in a significant proportion of patients and is associated with increased short- and mid-term mortality. The results suggest that CysC can be used for risk stratification in AHF. Markers of inflammation are elevated both in heart failure and in chronic kidney disease, and inflammation is one of the mechanisms thought to mediate heart-kidney interactions in the cardiorenal syndrome. Inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) correlate very differently to markers of cardiac stress and renal function. In particular, TNF-α showed a robust correlation to CysC, but was not associated with levels of NT-proBNP, a marker of hemodynamic cardiac stress. Compared to CysC, the inflammatory markers were not strongly related to mortality in AHF. In conclusion, patients with AHF are elderly with multiple co-morbidities, and renal dysfunction is very common. CysC demonstrates good diagnostic properties both in identifying impaired renal function and acute kidney injury in patients with AHF. CysC, as a measure of renal function, is also a powerful prognostic marker in AHF. CysC shows promise as a marker for assessment of kidney function and risk stratification in patients hospitalized for AHF.

Regulation of TGF-β1-Driven Differentiation of Human Lung Fibroblasts: Emerging Roles of Cathepsin B and Cystatin C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF) and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover addition of Cat B generated 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation, but had no effect on p38 MAPK and JNK phosphorylation indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. While mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of IPF and CCD-19Lu fibroblasts. In addition cystatin C participated in the control of extracellular Cats, since its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Active site labeling of cysteine cathepsins by a straightforward diazomethylketone probe derived from the N-terminus of human cystatin C

We designed a straightforward biotinylated probe using the N-terminal substrate-like region of the inhibitory site of human cystatin C as a scaffold, linked to the thiol-specific reagent diazomethylketone group as a covalent warhead (i.e. Biot-(PEG)2-Ahx-LeuValGly-DMK). The irreversible activity-based probe bound readily to cysteine cathepsins B, L, S and K. Moreover affinity labeling is sensitive since active cathepsins were detected in the nM range using an ExtrAvidin®-peroxidase conjugate for disclosure. Biot-(PEG)2-Ahx-LeuValGly-DMK allowed a slightly more pronounced labeling for cathepsin S with a compelling second-order rate constant for association (kass = 2,320,000 M−1 s−1). Labeling of the active site is dose-dependent as observed using 6-cyclohexylamine-4-piperazinyl-1,3,5-triazine-2-carbonitrile, as competitive inhibitor of cathepsins. Finally we showed that Biot-(PEG)2-Ahx-LeuValGly-DMK may be a simple and convenient tool to label secreted and intracellular active cathepsins using a myelomonocytic cell line (THP-1 cells) as model.

Serum cystatin C is a sensitive early marker for changes in the glomerular filtration rate in patients undergoing laparoscopic surgery

OBJECTIVE: Pneumoperitoneum during laparoscopy results in transient oliguria and decreased glomerular filtration and renal blood flow. The presence of oliguria and elevated serum creatinine is suggestive of acute renal injury. Serum cystatin C has been described as a new marker for the detection of this type of injury. In this study, our aim was to compare the glomerular filtration rate estimated using cystatin C levels with the rate estimated using serum creatinine in patients with normal renal function who were undergoing laparoscopic surgery. METHODS: In total, 41 patients undergoing laparoscopic cholecystectomy or hiatoplasty were recruited for the study. Blood samples were collected at three time intervals: first, before intubation (T1); second, 30 minutes after the establishment of pneumoperitoneum (T2); and third, 30 minutes after deflation of the pneumoperitoneum (T3). These blood samples were then analyzed for serum cystatin C, creatinine, and vasopressin. The Larsson formula was used to calculate the glomerular filtration rate based on the serum cystatin C levels, and the Cockcroft-Gault formula was used to calculate the glomerular filtration rate according to the serum creatinine levels. RESULTS: Serum cystatin C levels increased during the study (T1 = T2creatinine levels decreased (T1 = T2>T3; p<0.05). The calculated eGlomerular filtration rate-Larsson decreased, whereas the eGlomerular filtration rate-Cockcroft-Gault increased. There was no correlation between cystatin C and serum creatinine. Additionally, Pearson's analysis showed a better correlation between serum cystatin C and the eGlomerular filtration rate than between serum creatinine and the eGlomerular filtration rate. CONCLUSION: This study demonstrates that serum cystatin C is a more sensitive indicator of changes in the glomerular filtration rate than serum creatinine is in patients with normal renal function who are undergoing laparoscopic procedures.

Fetuin-A and cystatin C are endogenous inhibitors of human meprin metalloproteases

Meprin and , zinc metalloproteinases, play significant roles in inflammation, including inflammatory bowel disease (IBD), possibly by activating cytokines, like interleukin 1 , interleukin 18, or tumor growth factor . Although a number of potential activators for meprins are known, no endogenous inhibitors have been identified. In this work, we analyzed the inhibitory potential of human plasma and identified bovine fetuin-A as an endogenous meprin inhibitor with a K(i) (inhibition constant) of 4.2 × 10(-5) M for meprin and a K(i) of 1.1 × 10(-6) M meprin . This correlated with data obtained for a fetuin-A homologue from carp (nephrosin inhibitor) that revealed a potent meprin and inhibition (residual activities of 27 and 22%, respectively) at a carp fetuin concentration of 1.5 × 10(-6) M. Human fetuin-A is a negative acute phase protein involved in inflammatory diseases, thus being a potential physiological regulator of meprin activity. We report kinetic studies of fetuin-A with the proteolytic enzymes astacin, LAST, LAST_MAM, trypsin, and chymotrypsin, indeed demonstrating that fetuin-A is a broad-range protease inhibitor. Fetuin-A inhibition of meprin activity was 40 times weaker than that of meprin activity. Therefore, we tested cystatin C, a protein structurally closely related to fetuin-A. Indeed, cystatin C was an inhibitor for human meprin (K(i) = 8.5 × 10(-6) M) but, interestingly, not for meprin . Thus, the identification of fetuin-A and cystatin C as endogenous proteolytic regulators of meprin activity broadens our understanding of the proteolytic network in plasma.

Cystatin C is independently associated with total and cardiovascular mortality in individuals undergoing coronary angiography. The Ludwigshafen Risk and Cardiovascular Health (LURIC) study.

AIMS Cystatin C is a well established marker of kidney function. There is evidence that cystatin C concentrations are also associated with mortality. The present analysis prospectively evaluated the associations of cystatin C with all-cause and cardiovascular (CV) mortality in a well-characterized cohort of persons undergoing angiography, but without overt renal insufficiency. METHODS Cystatin C was available in 2998 persons (mean age: 62.7 ± 10.5 years; 30.3% women). Of those 2346 suffered from coronary artery disease (CAD) and 652 (controls) did not. Creatinine (mean ± SD: 83.1 ± 47.8 vs. 74.1 ± 24.7 μmol/L, p = 0.036) but not Cystatin C (mean ± SD: 1.02 ± 0.44 vs. 0.92 ± 0.26 mg/L, p = 0.065) was significantly higher in patients with CAD. After a median follow-up of 9.9 years, in total 898 (30%) deaths occurred, 554 (18.5%) due to CV disease and 326 (10.9%) due to non-CV causes. Multivariable-adjusted Cox analysis (adjusting for eGFR and established cardiovascular risk factors, lipid lowering therapy, angiographic coronary artery disease, and C-reactive protein) revealed that patients in the highest cystatin C quartile were at an increased risk for all-cause (hazard ratio (HR) 1.93, 95% CI 1.50-2.48) and CV mortality (HR 2.05 95% CI 1.48-2.84) compared to those in the lowest quartile. The addition of cystatin C to a model consisting of established cardiovascular risk factors increased the area under the receiver-operating characteristic curve for CV and all-cause mortality, but the difference was statistically not significant. However, reclassification analysis revealed significant improvement by addition of cystatin C for CV and all-cause mortality (p < 0.001), respectively. CONCLUSION The concentration of cystatin C is strongly associated with long-term all-cause and cardiovascular mortality in patients referred to coronary angiography, irrespective of creatinine-based renal function.

The cystatin C/creatinine ratio, a marker of glomerular filtration quality: associated factors, reference intervals, and prediction of morbidity and mortality in healthy seniors.

The ratio of cystatin C (cysC) to creatinine (crea) is regarded as a marker of glomerular filtration quality associated with cardiovascular morbidities. We sought to determine reference intervals for serum cysC-crea ratio in seniors. Furthermore, we sought to determine whether other low-molecular weight molecules exhibit a similar behavior in individuals with altered glomerular filtration quality. Finally, we investigated associations with adverse outcomes. A total of 1382 subjectively healthy Swiss volunteers aged 60 years or older were enrolled in the study. Reference intervals were calculated according to Clinical & Laboratory Standards Institute (CLSI) guideline EP28-A3c. After a baseline exam, a 4-year follow-up survey recorded information about overall morbidity and mortality. The cysC-crea ratio (mean 0.0124 ± 0.0026 mg/μmol) was significantly higher in women and increased progressively with age. Other associated factors were hemoglobin A1c, mean arterial pressure, and C-reactive protein (P < 0.05 for all). Participants exhibiting shrunken pore syndrome had significantly higher ratios of 3.5-66.5 kDa molecules (brain natriuretic peptide, parathyroid hormone, β2-microglobulin, cystatin C, retinol-binding protein, thyroid-stimulating hormone, α1-acid glycoprotein, lipase, amylase, prealbumin, and albumin) and creatinine. There was no such difference in the ratios of very low-molecular weight molecules (urea, uric acid) to creatinine or in the ratios of molecules larger than 66.5 kDa (transferrin, haptoglobin) to creatinine. The cysC-crea ratio was significantly predictive of mortality and subjective overall morbidity at follow-up in logistic regression models adjusting for several factors. The cysC-crea ratio exhibits age- and sex-specific reference intervals in seniors. In conclusion, the cysC-crea ratio may indicate the relative retention of biologically active low-molecular weight compounds and can independently predict the risk for overall mortality and morbidity in the elderly.

Elevated cystatin C & heart failure prognosis a systematic review

Renal insufficiency is one of the most common co-morbidities present in heart failure (HF) patients. It has significant impact on mortality and adverse outcomes. Cystatin C has been shown as a promising marker of renal function. A systematic review of all the published studies evaluating the prognostic role of cystatin C in both acute and chronic HF was undertaken. A comprehensive literature search was conducted involving various terms of 'cystatin C' and 'heart failure' in Pubmed medline and Embase libraries using Scopus database. A total of twelve observational studies were selected in this review for detailed assessment. Six studies were performed in acute HF patients and six were performed in chronic HF patients. Cystatin C was used as a continuous variable, as quartiles/tertiles or as a categorical variable in these studies. Different mortality endpoints were reported in these studies. All twelve studies demonstrated a significant association of cystatin C with mortality. This association was found to be independent of other baseline risk factors that are known to impact HF outcomes. In both acute and chronic HF, cystatin C was not only a strong predictor of outcomes but also a better prognostic marker than creatinine and estimated glomerular filtration rate (eGFR). A combination of cystatin C with other biomarkers such as N terminal pro B- type natriuretic peptide (NT-proBNP) or creatinine also improved the risk stratification. The plausible mechanisms are renal dysfunction, inflammation or a direct effect of cystatin C on ventricular remodeling. Either alone or in combination, cystatin C is a better, accurate and a reliable biomarker for HF prognosis. ^

Cystatin C and cardiovascular disease : A mendelian randomization study

BACKGROUND: Epidemiological studies show that high circulating cystatin C is associated with risk of cardiovascular disease (CVD), independent of creatinine-based renal function measurements. It is unclear whether this relationship is causal, arises from residual confounding, and/or is a consequence of reverse causation. OBJECTIVES: The aim of this study was to use Mendelian randomization to investigate whether cystatin C is causally related to CVD in the general population. METHODS We incorporated participant data from 16 prospective cohorts (n ¼ 76,481) with 37,126 measures of cystatin C and added genetic data from 43 studies (n ¼ 252,216) with 63,292 CVD events. We used the common variant rs911119 in CST3 as an instrumental variable to investigate the causal role of cystatin C in CVD, including coronary heart disease, ischemic stroke, and heart failure. RESULTS: Cystatin C concentrations were associated with CVD risk after adjusting for age, sex, and traditional risk factors (relative risk: 1.82 per doubling of cystatin C; 95% confidence interval [CI]: 1.56 to 2.13; p ¼ 2.12 1014). The minor allele of rs911119 was associated with decreased serum cystatin C (6.13% per allele; 95% CI: 5.75 to 6.50; p ¼ 5.95 10211), explaining 2.8% of the observed variation in cystatin C. Mendelian randomization analysis did not provide evidence for a causal role of cystatin C, with a causal relative risk for CVD of 1.00 per doubling cystatin C (95% CI: 0.82 to 1.22; p ¼ 0.994), which was statistically different from the observational estimate (p ¼ 1.6 105 ). A causal effect of cystatin C was not detected for any individual component of CVD. CONCLUSIONS: Mendelian randomization analyses did not support a causal role of cystatin C in the etiology of CVD. As such, therapeutics targeted at lowering circulating cystatin C are unlikely to be effective in preventing CVD.