Cystatins as calpain inhibitors: Engineered chicken cystatin- and stefin B-kininogen domain 2 hybrids support a cystatin-like mode of interaction with the catalytic subunit of μ-calpain

Within the cystatin superfamily, only kininogen domain 2 (KD2) is able to inhibit μ- and m-calpain. In an attempt to elucidate the structural requirements of cystatins for calpain inhibition, we constructed recombinant hybrids of human stefin B (an intracellular family 1 cystatin) with KD2 and Delta L110 deletion mutants of chicken cystatin-KD2 hybrids. Substitution of the N-terminal contact region of stefin B by the corresponding KD2 sequence resulted in a calpain inhibitor of K-i = 188 nM. Deletion of L110, which forms a beta -bulge in family 1 and 2 cystatins but is lacking in KD2, improved inhibition of mu -calpain 4- to 8-fold. All engineered cystatins were temporary inhibitors of calpain due to slow substrate-like cleavage of a single peptide bond corresponding to Gly9-Ala10 in chicken cystatin. Biomolecular interaction analysis revealed that, unlike calpastatin, the cystatin-type inhibitors do not bind to the calmodulin-like domain of the small subunit of calpain, and their interaction with the mu -calpain heterodimer is completely prevented by a synthetic peptide comprising subdomain B of calpastatin domain 1. Based on these results we propose that (i) cystatin-type calpain inhibitors interact with the active site of the catalytic domain of calpain in a similar cystatin-like mode as with papain and (ii) the potential for calpain inhibition is due to specific subsites within the papain-binding regions of the general cystatin fold

The Comparison of Creatinine and Cystatin C Value in Preeclampsia Severity and Neonatal Outcome

Objectives: to compare the levels of creatinine and cystatin C with the severity of preeclampsia, and assess neonatal outcomes.Materials and Methods: Creatinine, cystatin C, and neonatal outcomes were assesed in 17 normotensive samples, 17 samples of mild preeclampsia and 17 samples of severe preeclampsia. Analysis of data with statistical tests of ANOVA and t test differences between 2 proportions.Results: The mean levels of creatinine in the normotensive group, mild preeclampsia, severe preeclampsia are 0.56 mg/dL, 0.67 mg/ dL, and 0.75 mg/dL, p=0.138; While on cystatin C are 0.82 mg/L, 1.03 mg/L and 1.32 mg/L, p=0.000. The adverse neonatal out-come wasn't found in the normotensive group. In mild pre-eclampsia obtained 1 preterm birth and 1 intrauterine fetal death (IUFD), whereas in severe preeclampsia obtained 3 babies born preterm, 1 IUFD, and 1 intrauterine growth restriction (IUGR).Conclusion: levels of cystatin C was increased significantly in line with increased severity of preeclampsia, whereas creatinine was not increased significantly. Cystatin C is better than crea-tinine as a marker of renal dysfunction in preeclampsia patients. There was an increase in adverse neonatal outcomes in the group of preeclampsia

Estimated glomerular filtration rate correlates poorly with four-hour creatinine clearance in critically ill patients with acute kidney injury.

Introduction. RIFLE and AKIN provide a standardised classification of acute kidney injury (AKI), but their categorical rather than continuous nature restricts their use to a research tool. A more accurate real-time description of renal function in AKI is needed, and some published data suggest that equations based on serum creatinine that estimate glomerular filtration rate (eGFR) can provide this. In addition, incorporating serum cystatin C concentration into estimates of GFR may improve their accuracy, but no eGFR equations are validated in critically ill patients with AKI. Aim. This study tests whether creatinine or cystatin-C-based eGFR equations, used in patients with CKD, offer an accurate representation of 4-hour creatinine clearance (4CrCl) in critically ill patients with AKI. Methods. Fifty-one critically ill patients with AKI were recruited. Thirty-seven met inclusion criteria, and the performance of eGFR equations was compared to 4CrCl. Results. eGFR equations were better than creatinine alone at predicting 4CrCl. Adding cystatin C to estimates did not improve the bias or add accuracy. The MDRD 7 eGFR had the best combination of correlation, bias, percentage error and accuracy. None were near acceptable standards quoted in patients with chronic kidney disease (CKD). Conclusions. eGFR equations are not sufficiently accurate for use in critically ill patients with AKI. Incorporating serum cystatin C does not improve estimates. eGFR should not be used to describe renal function in patients with AKI. Standards of accuracy for validating eGFR need to be set

Renal Hyperfiltration and the Development of Microalbuminuria in Type 1 Diabetes

OBJECTIVE: The purpose of this study was to examine prospectively whether renal hyperfiltration is associated with the development of microalbuminuria in patients with type 1 diabetes, after taking into account known risk factors. RESEARCH DESIGN AND METHODS: The study group comprised 426 participants with normoalbuminuria from the First Joslin Kidney Study, followed for 15 years. Glomerular filtration rate was estimated by serum cystatin C, and hyperfiltration was defined as exceeding the 97.5th percentile of the sex-specific distribution of a similarly aged, nondiabetic population (134 and 149 ml/min per 1.73 m2 for men and women, respectively). The outcome was time to microalbuminuria development (multiple albumin excretion rate >30 μg/min). Hazard ratios (HRs) for microalbuminuria were calculated at 5, 10, and 15 years. RESULTS: Renal hyperfiltration was present in 24% of the study group and did not increase the risk of developing microalbuminuria. The unadjusted HR for microalbuminuria comparing those with and without hyperfiltration at baseline was 0.8 (95% CI 0.4–1.7) during the first 5 years, 1.0 (0.6–1.7) during the first 10 years, and 0.8 (0.5–1.4) during 15 years of follow-up. The model adjusted for baseline known risk factors including A1C, age at diagnosis of diabetes, diabetes duration, and cigarette smoking resulted in similar HRs. In addition, incorporating changes in hyperfiltration status during follow-up had minimal impact on the HRs for microalbuminuria. CONCLUSION;S Renal hyperfiltration does not have an impact on the development of microalbuminuria in type 1 diabetes during 5, 10, or 15 years of follow-up.National Institutes of Health Grant (DK 041526

Clinical review: Biomarkers of acute kidney injury: where are we now?

The recognition that acute kidney injury (AKI) is a significant independent risk factor for morbidity and mortality has resulted in a substantial number of publications over the past 5 years or more. In no small part these have, to a degree, highlighted the inadequacy of conventional markers of renal insufficiency in the acute setting. Much effort has been invested in the identification of early, specific AKI markers in order to aid early diagnosis of AKI and hopefully improve outcome. The search for a 'biomarker' of AKI has seen early promise replaced by a degree of pessimism due to the lack of a clear candidate molecule and variability of results. We outline the major studies described to date as well as discuss potential reasons for the discrepancies observed and suggest that evolution of the field may result in success with ultimately an improvement in patient outcomes

Elevated cystatin-C concentration is associated with progression to prediabetes: the Western New York Study

OBJECTIVE – We conducted a nested case-control investigation to examine if elevated baseline concentrations of cystatin-C predicted progression from normoglycaemia to prediabetes over 6 years of follow-up from the Western New York Health Study. RESEARCH DESIGN AND METHODS – 1,455 participants from the Western New York Health Study, free of type 2 diabetes and known cardiovascular disease at baseline (1996-2001), were reexamined in 2002-2004. An incident case of prediabetes was defined as one with fasting glucose below 100 mg/dl at the baseline examination and ≥ 100 mg/dl and ≤ 125 mg/dl at the follow-up examination. All cases (n=91) were matched 1:3 to control participants based upon sex, race/ethnicity and year of study enrollment. All controls had fasting glucose levels < 100 mg/dl at both baseline and follow-up examinations. Cystatin-C concentrations and the urinary albumin to creatinine ratio were measured from frozen (-196 Cº) baseline blood and urine samples. Serum creatinine concentrations were available from the baseline examination. RESULTS –Multivariate conditional logistic regression analyses adjusted for age, baseline glucose level, HOMA-IR, body mass index, hypertension, eGFR, cigarette smoking, and alcohol use revealed a significantly increased risk of progression to prediabetes among those with elevated baseline concentrations of cystatin-C (Odds Ratio, 95% CI: 3.04, 1.34, 6.89) (upper quintile vs. the remainder). Results of secondary analyses that considered hs-CRP, IL-6, E-selectin, or sICAM did not alter these results. CONCLUSIONS - These results suggest that early renal impairment indexed with cystatin-C imparted a three-fold excess risk of progression to prediabetes in this study population. Recent evidence from randomized clinical trials (1,2) among people with prediabetes have provided convincing evidence that early intervention can significantly delay or prevent the progression to type 2 diabetes. The identification of those with prediabetes is assuming greater importance (3) especially in light of the fact that approximately 35 million adults aged 40-74 years old in the United States have prediabetes defined as impaired fasting glucose (4). Microalbuminuria occurs frequently in nondiabetic subjects and places them at increased risk for cardiovascular disease (5-7). The mechanisms behind this observation are poorly understood, however. Albuminuria may reflect underlying vascular damage (8), hypertension (9, 10) endothelial dysfunction (11, 12) and/or low-grade inflammation (13). A large percentage of type 2 individuals pass through a period of prediabetes (14) and may experience early renal dysfunction e.g., a glomerular filtration rate (GFR) above 60 ml/minute per 1.73m2. Currently used estimating equations are poor at identifying early renal impairment and better indices are of great interest (15, 16). Recently, several studies have suggested that cystatin-C levels may be a more sensitive marker of early renal impairment than either albuminuria or serum creatinine concentration (17-20). Therefore, a better understanding of a putative role for cystatin-C in the etiology of prediabetes could shed light on the renal/heart disease connection (21). Given the reported superiority of cystatin C over conventional measures of renal function, we hypothesized that cystatin-C would predict progression to prediabetes independent of serum creatinine or estimated GFR. We also investigated the role of intervening mechanisms including hypertension, insulin resistance, endothelial dysfunction and inflammation

Evaluation of cystatin C for the detection of chronic kidney disease in cats

BackgroundSerum cystatin C (sCysC) and urinary cystatin C (uCysC) are potential biomarkers for early detection of chronic kidney disease (CKD) in cats. An in-depth clinical validation is required. ObjectivesTo evaluate CysC as a marker for CKD in cats and to compare assay performance of the turbidimetric assay (PETIA) with the previously validated nephelometric assay (PENIA). AnimalsNinety cats were included: 49 CKD and 41 healthy cats. MethodsSerum CysC and uCysC concentrations were prospectively evaluated in cats with CKD and healthy cats. Based on plasma exo-iohexol clearance test (PexICT), sCysC was evaluated to distinguish normal, borderline, and low GFR. Sensitivity and specificity to detect PexICT<1.7mL/min/kg were calculated. Serum CysC results of PENIA and PETIA were correlated with GFR. Statistical analysis was performed using general linear modeling. ResultsCats with CKD had significantly higher meanSD sCysC (1.4 +/- 0.5mg/L) (P<.001) and uCysC/urinary creatinine (uCr) (291 +/- 411mg/mol) (P<.001) compared to healthy cats (sCysC 1.0 +/- 0.3 and uCysC/uCr 0.32 +/- 0.97). UCysC was detected in 35/49 CKD cats. R-2 values between GFR and sCysC or sCr were 0.39 and 0.71, respectively (sCysC or sCr=+GFR+epsilon). Sensitivity and specificity were 22 and 100% for sCysC and 83 and 93% for sCr. Serum CysC could not distinguish healthy from CKD cats, nor normal from borderline or low GFR, in contrast with sCr. ConclusionSerum CysC is not a reliable marker of reduced GFR in cats and uCysC could not be detected in all CKD cats

Serum cystatin C and chemerin levels in diabetic retinopathy

Peer reviewedPublisher PD

Use of cystatin C to inform metformin eligibility among adult veterans with diabetes.

AimsRecommendations for metformin use are dependent on eGFR category: eGFR &gt;45 ml/min/1.73 m2 - "first-line agent"; eGFR 30-44 - "use with caution"; eGFR&lt;30 - "do not use". Misclassification of metformin eligibility by creatinine-based MDRD GFR estimates (eGFRcr) may contribute to its misuse. We investigated the impact of cystatin c estimates of GFR (eGFRcys) on metformin eligibility.MethodsIn a consecutive cohort of 550 Veterans with diabetes, metformin use and eligibility were assessed by eGFR category, using eGFRcr and eGFRcys. Discrepancy in eligibility was defined as cases where eGFRcr and eGFRcys categories (&lt;30, 30-44, 45-60, and &gt;60 ml/min/1.73 m2) differed with an absolute difference in eGFR of &gt;5 ml/min/1.73 m2. We modeled predictors of metformin use and eGFR category discrepancy with multivariable relative risk regression and multinomial logistic regression.ResultsSubjects were 95% male, median age 68, and racially diverse (45% White, 22% Black, 11% Asian, 22% unknown). Metformin use decreased with severity of eGFRcr category, from 63% in eGFRcr &gt;60 to 3% in eGFRcr &lt;30. eGFRcys reclassified 20% of Veterans into different eGFR categories. Factors associated with a more severe eGFRcys category compared to eGFRcr were older age (aOR = 2.21 per decade, 1.44-1.82), higher BMI (aOR = 1.04 per kg/m2, 1.01-1.08) and albuminuria &gt;30 mg/g (aOR = 1.81, 1.20-2.73).ConclusionsMetformin use is low among Veterans with CKD. eGFRcys may serve as a confirmatory estimate of kidney function to allow safe use of metformin among patients with CKD, particularly among older individuals and those with albuminuria