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PubMed1999

Cystatin C as a marker for glomerular filtration rate in pediatric patients.

Cystatin C is a non-glycated 13-kilodalton basic protein produced by all nucleated cells. The low molecular mass and the basic nature of cystatin C, in combination with its stable production rate, suggest that the glomerular filtration rate (GFR) is the major determinant of cystatin C concentration in the peripheral circulation. Recently published studies have shown that cystatin C correlates more strongly than creatinine with GFR measured using the 51Cr-EDTA clearance. The aim of this study was to evaluate serum cystatin C as a marker for GFR in children. GFR was determined on medical indications using the 51Cr-EDTA technique in pediatric patients (2-16 years) in our renal unit. Simultaneously their cystatin C and creatinine concentrations were also measured. Of our 52 patients, 19 had a reduced renal function (<GFR 89 ml/min per 1.73 m2) based on the 51Cr-EDTA clearance. The correlation of cystatin C with the isotopic measurement of GFR tended to be stronger (r=0.89, P=0.073) than that of creatinine (r=0.80). Receiver operating characteristic analysis showed that the diagnostic accuracy of cystatin C was better (P=0.037) than that of creatinine in discriminating between subjects with normal renal function and those with reduced GFR. This study demonstrates that serum cystatin C has an increased diagnostic accuracy for reduced GFR when compared with serum creatinine. Hence, cystatin C seems to be an attractive alternative for the estimation of GFR in children.

Adolescent↗
PubMed2004

Family 2 cystatins inhibit osteoclast-mediated bone resorption in calvarial bone explants.

Osteoclastic bone resorption depends on the activity of various proteolytic enzymes, in particular those belonging to the group of cysteine proteinases. Biochemical studies have shown that cystatins, naturally occurring inhibitors of these enzymes, inhibit bone matrix degradation. Since the mechanism by which cystatins exert this inhibitory effect is not completely resolved yet, we studied the effect of cystatins on bone resorption microscopically and by Ca-release measurements. Calvarial bone explants were cultured in the presence or absence of family 2 cystatins and processed for light and electron microscopic analysis, and the culture media were analyzed for calcium release. Both egg white cystatin and human cystatin C decreased calcium release into the medium significantly. Microscopic analyses of the bone explants demonstrated that in the presence of either inhibitor, a high percentage of osteoclasts was associated with demineralized non-degraded bone matrix. Following a 24-h incubation in the presence of cystatin C, 41% of the cells were adjacent to areas of demineralized non-degraded bone matrix, whereas in controls, this was only 6%. If bone explants were cultured with both PTH and cystatin C, 60% of the osteoclasts were associated with demineralized non-degraded bone matrix, compared to 27% for bones treated with PTH only (P < 0.01). Our study provides evidence that cystatins, the naturally occurring inhibitors of cysteine proteinases, reversibly inhibit bone matrix degradation in the resorption lacunae adjacent to osteoclasts. These findings suggest the involvement of cystatins in the modulation of osteoclastic bone degradation.

Animals↗
PubMed2005

Expression of cystatin C prevents oxidative stress-induced death in PC12 cells.

Cystatin C, an inhibitor of cysteine proteinases, is suggested to be involved in oxidative stress-induced apoptosis of cultured CNS neurons and various neuronal diseases in vivo; however, little is known about its mechanism of action. To address the role cystatin C plays in oxidative stress-induced neuronal cell death, we established PC12 cell lines that stably expressed rat cystatin C. These cystatin C-expressing PC12 cells showed remarkable resistance to high (50%) oxygen atmosphere. This resistance correlate with expression levels of cystatin C, demonstrating that cystatin C has a protective effect on high oxygen-induced cell death. In contrast, in a normal (20%) oxygen atmosphere neither control nor cystatin C-expressing PC12 cells showed a significant change in the number of living cells, indicating that cystatin C does not play an important role in the regulation of cellular proliferation. Furthermore, the cystatin C-expressing cell line also resisted other oxidative stresses, including glutamate- and 13-L-hydroperoxylinoleic acid (LOOH)-induced cell death. These results demonstrate that cystatin C has protective effects against various oxidative stresses that induce cell death.

Animals↗
PubMed2004

Binding of cystatin C to Alzheimer's amyloid beta inhibits in vitro amyloid fibril formation.

The colocalization of cystatin C, an inhibitor of cysteine proteases, with amyloid beta (Abeta) in parenchymal and vascular amyloid deposits in brains of Alzheimer's disease (AD) patients may reflect cystatin C involvement in amyloidogenesis. We therefore sought to determine the association of cystatin C with Abeta. Immunofluorescence analysis of transfected cultured cells demonstrated colocalization of cystatin C and beta amyloid precursor protein (betaAPP) intracellularly and on the cell surface. Western blot analysis of immunoprecipitated cell lysate or medium proteins revealed binding of cystatin C to full-length betaAPP and to secreted betaAPP (sbetaAPP). Deletion mutants of betaAPP localized the cystatin C binding site within betaAPP to the Abeta region. Cystatin C association with betaAPP resulted in increased sbetaAPP but did not affect levels of secreted Abeta. Analysis of the association of cystatin C and Abeta demonstrated a specific, saturable and high affinity binding between cystatin C and both Abeta(1-42) and Abeta(1-40). Notably, cystatin C association with Abeta results in a concentration-dependent inhibition of Abeta fibril formation.

Alzheimer Disease↗
PubMed2006

The upregulation of cystatin C in oral submucous fibrosis.

Cystatin C is a 13kDa non-glycosylated basic protein belonging to cystatin family. It is consistently and dramatically upregulated in a variety of fibrotic diseases. The aim of this study was to compare cystatin C expression in normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induction of cystatin C expression. Twenty-five OSF specimens and six of normal buccal mucosa were examined by immunohistochemistry. The activity of cystatin C from fibroblasts cultured from OSF and normal buccal mucosa were evaluated by using reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Furthermore, the effect of arecoline, the major areca nut alkaloid, was explored. Cystatin C expression was significantly higher in OSF specimens (p<0.05) and expressed mainly by fibroblasts, endothelial cells, and inflammatory cells. OSF demonstrated significantly higher cystatin C expression than normal buccal mucosa fibroblasts both in mRNA and protein levels (p<0.05). In addition, arecoline was also found to elevate cystatin C mRNA and protein expression in a dose-dependent manner (p<0.05). Taken together, the data demonstrate that cystatin C expression is significantly upregulated in OSF from areca quid chewers and arecoline may be responsible for the enhanced cystatin C expression in vivo.

Arecoline↗
PubMed2004

Cystatin C in cerebrospinal fluid is not a diagnostic test for pain in humans.

A recent subtractive cDNA cloning study in rats demonstrated an unexpected increase in expression of the proteinase inhibitor, cystatin C in the spinal cord during acute peripheral inflammation, suggesting this protein may be involved in the pathogenesis of persistent pain. A subsequent study of 10 women suggested that prolonged labor pain resulted in increased cystatin C concentrations in cerebrospinal fluid, and that this could be used as a biomarker for pain. To confirm and extend these observations, we measured cystatin C concentrations in cerebrospinal fluid in 131 subjects: 30 normal volunteers without pain, 25 women at elective cesarean section without pain, 60 women in labor with severe pain, and 16 patients with chronic neuropathic pain and tactile allodynia. The median cystatin C concentration in normal volunteers, 2.2 microg/ml, was similar to that previously reported by multiple investigators, and cystatin C concentrations were increased in women in labor (3.9 microg/ml). However, contrary to the previous report, cystatin C concentrations in laboring women with pain did not differ from those of pregnant women without pain (3.7 microg/ml). There was no relationship between duration of painful labor and cystatin C concentration. Patients with neuropathic pain had similar cystatin C concentrations (2.4 microg/ml) to controls. Logistic regression analysis indicated that cystatin C concentrations could not be used to reliably predict the presence of pain in either acute or chronic settings. These data suggest that cystatin C concentration in cerebrospinal fluid is an unreliable diagnostic marker for pain in humans.

Adult↗
PubMedDate not supplied

Examination of the potential structure of human salivary cystatins based on computer modelling.

The cystatin family of proteins exists in both excreted and intracellular forms, and appears to be involved in protective and regulatory roles, inhibiting a variety of bacterial, viral and intracellular proteases. The amino acid sequences of several human forms of cystatin are known, but currently only the structure of chicken cystatin (approx. 40% homologous to the human forms) has been experimentally determined. The objective of this study was to use the X-ray coordinates of chicken cystatin to construct computer models of the structures of three human salivary forms (SN, S and SA). These structures were energy-minimized and subjected to dynamic simulations. The resultant structures were compared to determine conformational differences. Global root mean square deviations between equivalent atoms ranged from 1.4 A to 3.9 A. The closest structural similarity to chicken cystatin involved cystatin SN, which also showed the highest (68%) functional sequence homology. Local secondary structure was examined in more detail. In comparisons of alpha-carbon position the third beta-strand (77% functional sequence conservation) and its preceding loop (60% conserved) showed the highest structural conservation in S, while beta-strand 4 showed the highest structural conservation in SN and SA. Throughout their structures, SN and SA were more structurally similar to chicken cystatin than to salivary cystatin S. There are two regions of conserved, negatively charged residues in the salivary cystatins, which appear to be spaced so that they are capable of interaction with hydroxypatite. It is concluded that not only does structural modelling by analogy provide detailed models of salivary cystatins that can be tested by future experimentation, but also that examination of the models has revealed potential sites of interaction with hydroxyapatite.

Amino Acid Sequence↗
PubMed1998

Production of rat salivary cystatin S variant polypeptides in Escherichia coli.

Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.

Alternative Splicing↗
PubMed1999

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.

Animals↗
PubMed2000

Rainbow trout (Oncorhynchus mykiss) cystatin C: expression in Escherichia coli and properties of the recombinant protease inhibitor.

Cystatins are a superfamily of low Ki cysteine proteinase inhibitors found in both plants and animals. Cystatin C, a secreted molecule of this family, is of interest from biochemical and evolutionary points of view, and also has biotechnological applications. Recently we cloned and sequenced the cDNA for rainbow trout (Oncorhynchus mykiss) cystatin C [Li et al., 1998. Molecular cloning, sequence analysis and expression distribution of rainbow trout (Oncorhynchus mykiss) cystatin C. Comp. Biochem. Phys. B 121, 135-143]. To explore the relationship between protein structure and function of trout cystatin C, we established a bacterial system for expression of the protein. Trout cystatin C expressed in the cytoplasm of bacterial cells did not have detectable protease inhibitory activity. Activity was regained by Ni-NTA chromatography under denaturing conditions followed by dialysis-based refolding. Titration of purified cystatin C preparations with papain indicated that approximately 20% of the total protein had been converted to the active form after one refolding cycle. Expression levels were 3-5 mg/l. The protease-inhibitory properties of recombinant trout cystatin C were similar to those of human and chicken cystatin C derived from biological sources and recombinant cystatin C derived from rat and mouse genes. The Ki for papain was 1.2 x 10(-15) M, exhibiting the high affinity binding unique to this family of protease inhibitors.

Animals↗
PubMed1990

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.

Adrenergic beta-Agonists↗
PubMed1993

Importance of the evolutionarily conserved glycine residue in the N-terminal region of human cystatin C (Gly-11) for cysteine endopeptidase inhibition.

Human cystatin C variants in which the evolutionarily conserved Gly-11 residue has been replaced by residues with positively charged (Arg), negatively charged (Glu), bulky hydrophobic (Trp), or small (Ser or Ala) side-chains have been produced by site-directed mutagenesis and expression in Escherichia coli. The five variants were isolated and structurally verified. Their inhibitory properties were compared with those of wild-type recombinant cystatin C by determination of the equilibrium constants for dissociation (Ki) of their complexes with the cysteine endopeptidases papain and human cathepsin B and with the cysteine exopeptidase dipeptidyl peptidase I. The Ser-11 and Ala-11 cystatin C variants displayed Ki values for the two endopeptidases that were approx. 20-fold higher than those of wild-type cystatin C, while the corresponding values for the Trp-11. Arg-11 and Glu-11 variants were increased by a factor of about 2000. In contrast, the Ki values for the interactions of all five variants with the exopeptidase differed from that of wild-type cystatin C by a factor of less than 10. Wild-type cystatin C and the Ser-11, Ala-11 and Glu-11 variants were incubated with neutrophil elastase, which in all cases resulted in the rapid hydrolysis of a single peptide bond, between amino acid residues 10 and 11. The Ki values for the interactions with papain of these three N-terminal-decapeptide-lacking cystatin C variants were 20-50 nM, just one order of magnitude higher than the value for N-terminally truncated wild-type cystatin C, which in turn was similar to the corresponding values for the full-length Glu-11, Arg-11 and Trp-11 variants. These data indicate that the crucial feature of the conserved Gly residue in position 11 of wild-type cystatin C is that this residue, devoid of a side-chain, will allow the N-terminal segment of cystatin C to adopt a conformation suitable for interaction with the substrate-binding pockets of cysteine endopeptidases, resulting in high-affinity binding and efficient inhibition. The functional properties of the remaining part of the proteinase contact area, which is built from more C-terminal inhibitor segments, are not significantly affected even when amino acids with bulky or charged side-chains replace the Gly-11 residue of the N-terminal segment.

Amino Acid Sequence↗
PubMed2001

Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.

Amino Acid Substitution↗
PubMed1995

Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases.

The structural basis for the biological specificity of human cystatin C has been investigated. Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor. Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression. A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S. Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed. Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity. In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains. The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes. The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B. The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.

Amino Acid Sequence↗
PubMed2000

Evaluation of serum cystatin C and chromogranin A as markers of nephropathy in patients with type 2 diabetes mellitus.

Nephropathy is a significant cause of morbidity and mortality in patients with diabetes mellitus (DM). The condition is characterized by persistent albuminuria and years of progressive renal structural changes associated with decline in the glomerular filtration rate (GFR). This study evaluates whether serum concentrations of the endogenous markers of GFR, cystatin C and chromogranin A could be used as indicators of nephropathy in 77 patients with Type 2 DM. On the basis of early morning urine microalbumin:creatinine ratio, patients were divided into patients without diabetic nephropathy (DN) who were normoalbuminuric (n = 27) and patients with DN who were microalbuminuric (n = 8) or macroalbuminuric (n = 42). Patients with reduced GFR or elevated serum cystatin C did not show the expected increase in serum chromogranin A. Twenty-six percent of the patients with normoalbuminuria and 6% of those with DN had serum chromogranin A below the detection limit of the assay (< 2 U/L). In patients with DN, serum chromogranin A showed significant correlation with serum cystatin C, but not with serum creatinine and creatinine clearance. Serum cystatin C and creatinine showed poor correlation with duration of DM and HbA1c. Serum cystatin C and creatinine were significantly higher in patients with DN than in normoalbuminuric patients. Serum cystatin C showed significant correlation with serum creatinine (rs = 0.45, p = 0.002), but not with creatinine clearance (rs = 0.23, p = 0.17) in patients with DN. Four of nine patients with creatinine clearance between 50 and 80 mL/min had increased (> or = 1.4 mg/L) serum cystatin C compared with only two patients with increased serum creatinine concentration. Twenty of 50 (40%) patients with DN had elevated serum cystatin C compared with 6 of 50 (12%) with elevated serum creatinine. If microalbuminuria is regarded as the "gold-standard" test, serum cystatin C has a sensitivity of 40% and specificity of 100% for the detection of DN. However, further studies are required to confirm the usefulness of serum cystatin C estimation as a screening test and as an early indicator and predictor of the development of DN.

Biomarkers↗
PubMed2004

Calculation of glomerular filtration rate expressed in mL/min from plasma cystatin C values in mg/L.

The Cockcroft Gault formula is often used to calculate the glomerular filtration rate (GFR) from plasma creatinine results. In Sweden this calculation is not usually done in the laboratory, but locally in the wards. These manual calculations could cause erroneous results. In several studies plasma cystatin C has been shown to be superior to plasma creatinine for estimation of GFR. One limitation of using cystatin C as a GFR marker is that there is no conversion formula transforming cystatin C expressed as mg/L to GFR expressed as mL/min. In this study plasma creatinine and cystatin C were compared with iohexol clearance. A stronger correlation (p < 0.0001) was found between cystatin C and iohexol clearance (r2 = 0.91) than between creatinine and iohexol clearance (r2 = 0.84). From the correlation data a formula was calculated to convert cystatin C expressed as mg/L to GFR (mL/min). The formulas y = 77.24x(-1.2623) (Dade Behring cystatin C calibration) or y = 99.43x(-1.5837) (DakoCytomation cystatin C calibration) are used to calculate GFR expressed in mL/min from the cystatin C value in mg/L and both results are reported to the referral doctor. These formulas can provide the clinicians with reliable and readily available GFR data based on single measurements of cystatin C concentrations.

Adolescent↗
PubMed2007

Increased cystatin C expression in the pre-eclamptic placenta.

Trophoblast invasion is regulated by proteinases and their inhibitors. Cystatin C inhibits cysteine proteinases. The serum concentration of cystatin C is increased in late pregnancy and pre-eclampsia. We aimed to investigate whether the expression of cystatin C is increased in the pre-eclamptic placenta and to investigate the expression pattern of cystatin C mRNA and protein in placental tissue. Tissue samples from the central part of the placenta from 13 normal and 22 pre-eclamptic pregnancies were included. We used real-time polymerase chain reaction (RT-PCR) and in situ hybridization for mRNA expression analysis and immunohistochemistry and Western blotting for protein expression analysis. RT-PCR showed a significantly higher expression of cystatin C mRNA in pre-eclampsia than in normal pregnancy, with the highest expression in cases with severe pre-eclampsia. In situ hybridization revealed a distinct pattern of high expression in the extravillous trophoblast cells of the basal plate and low expression in the syncytiotrophoblast covering villi. The cystatin C protein distribution matched the mRNA expression pattern. Western blot analysis revealed an increased protein expression in cases with severe pre-eclampsia and confirmed the presence of cystatin C in amniotic fluid samples. The high expression of cystatin C mRNA in the extravillous trophoblast cells of the basal plate suggests a role for cystatin C in the regulation of proteases in placentation. Placental expression and secretion of cystatin C could contribute to the elevated maternal plasma levels seen in pre-eclampsia.

Adult↗
PubMed2004

Serum cystatin C advantageous compared with serum creatinine in the detection of mild but not severe diabetic nephropathy.

OBJECTIVE: To determine whether serum cystatin C is more accurate than serum creatinine in the detection of diabetic nephropathy, also after adjustment for age. METHODS: Forty-one patients with type 1 and 82 patients with type 2 diabetes were evaluated with serum creatinine, serum cystatin C, and (51)Cr-EDTA clearance (reference method). Cystatin C was measured by a particle-enhanced turbidimetric method and creatinine by an enzymatic method. Statistical estimations were performed both without and with age adjustment created by z-scores for (51)Cr-EDTA clearance, creatinine, and cystatin C. The cut-off levels for glomerular filtration rate (GFR) ((51)Cr-EDTA clearance) were 60 and 80 mL min(-1) 1.73 m(-2), respectively, in absolute values and 80, 90 and 95% CIs, respectively, in age-adjusted values (z-scores). RESULTS: Estimations without age adjustment showed significantly (P = 0.0132) closer correlation for cystatin C (r = 0.817) versus (51)Cr-EDTA clearance as compared with creatinine (r = 0.678). However, when using age-adjusted values, the correlation for cystatin C and creatinine, respectively, versus (51)Cr-EDTA clearance did not differ. When comparing the diagnostic utilities for serum cystatin C versus serum creatinine in manifest renal impairment (GFR < 60 mL min(-1) 1.73 m(-2) or z-scores <-1.28 SD), there were no significant differences between the two markers whether age adjusted or not. However, for diagnosing mild nephropathy (GFR < 80 mL min(-1) 1.73 m(-2) or z-score -0.84 SD), serum cystatin C is significantly more useful. CONCLUSIONS: Serum cystatin C performed better compared with serum creatinine even when measured enzymatically, to detect mild diabetic nephropathy. However, serum creatinine was as efficient as serum cystatin C to detect advanced diabetic nephropathy.

Adult↗