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CLASSIC AND NEW APPROACHES FOR THE ASSESSMENT OF KIDNEY FUNCTION Добрин А. Свинаров


SYNOPSIS ™DISCOVERY OF NEW BIOMARKERS ™PROTEINURIA & UROPROTEIN BIOMARKERS ™ACUTE KIDNEY INJURY (AKI): FAST, SLOW & INTERMEDIATE BIOMARKERS ™FUTURE TRENTS: MOLECULAR MEDICINE

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DISCOVERY OF NEW BIOMARKERS ™SUROGATES (LEVEL C), PATHOPHYSIOLOGY (LEVEL B) ™BIOMARKERS DIFINING HEALTH & DISEASE ™EVENT & DISEASE BIOMARKERS ™PREVENTION & RISK ASSESSMENT ™MEASUREMENT OF THERAPEUTIC INTERVENTION ™PHARMACOKINETIC ™PHARMACODYNAMIC ™PHARMACOGENETIC D.Svinarov©2009


DISCOVERY OF NEW BIOMARKERS Human genome genome analysis analysis Human Gen expression expression activity activity assessment assessment Gen Analysis of of protein protein synthesis synthesis && signaling signaling Analysis Metabolite profiling profiling in in cells cells && body body fluids fluids Metabolite Clinical validation validation of of candidate candidate biomarkers biomarkers Clinical D.SvinarovŠ2009


Uroprotein biomarkers ™ Albumin ™ GF marker, indicates loss of selectivity by charge ™ IgG ™ GF marker, indicates loss of selectivity by dimention ™ α1-Microglobulin ™ Marker of tubular reabsorption ™ α2-Macroglobulin ™ Marker of post-renal proteinuria

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PD W Hofmann, Munich


Tubular mechanism of protein reabsorption a1-microglobulin

Проксимална тубулна клетка

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PD W Hofmann, Munich


Glomerulus

Albumin IgG

Îą1-Microglobulin

Basal membrane

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PD W Hofmann, Munich


Biological variability of uroprotein biomarkers Interindividual (%) Total protein

/L 36

g /Creat. 33

IgG

29

25

Albumin

29

22

α1-Microglobulin

26

20

N-acetyl-ß-gucosaminidase

16

13

Creatinine

20

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PD W Hofmann, Munich


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PD W Hofmann, Munich


Interpretation of Uroprotein Biomarker Findings D.SvinarovŠ2009


α1-mircroglobuin mg/g creatinine

g

mg/g creatinine

>

0m 0 0 2

rea C /g

Albumin D.Svinarov©2009

IgG

a1-microglobulin

Albumin (mg/g Crea)

PD W Hofmann, Munich


α1-Mikroglobulin mg/g Kreatinin

Referenzkollektiv primäre Glomerulopathien sekundäre Glomerulopathien (Nephrosklerosen) interstitielle Nephropathien 1000

100

10

1 1 D.Svinarov©2009

10

100

1000

10000

Albumin mg/g Kreatinin

100000

PD W Hofmann, Munich


Îą1-microglobulin (mg/g creatinine)

1000

Glomerulonephritis Tubular Nephropathy Diabetes Type 1 Diabetes Type 2

3

100

2

upper reference limit

10

1 test strip negative

1 1

10

100

test strip positive 1000

10000

100000

albumin (mg/g /creatinine) D.SvinarovŠ2009

PD W Hofmann, Munich


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PD W Hofmann, Munich


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AKI: A Common, Serious Problem ™5% of hospital discharges, up to 50% of patients in ICUs ™The incidence is increasing at an alarming rate ™Mortality rate remains dismally high –about 50% in dialyzed ICU patients ™25% of ICU dialysis survivors progress to end stage renal disease within 3 years ™The diagnosis of AKI is frequently delayed ™•Potentially effective preventive and therapeutic measures are frequently delayed due to lack of early diagnostic markers 2006 D.Svinarov©2009

Devarajan P & R Christenson, AACC, Online,


Diagnosis of AKI is Often Delayed ™Elevation in serum creatinine is the current gold standard, but this is problematic ™Normal serum creatinine varies widely with age, gender, diet, muscle mass, muscle metabolism, medications, and hydration status ™In AKI, serum creatinine can take several days to reach a new steady state ™Up to 50% of kidney function may be lost before serum creatinine even begins to rise Devarajan P & R Christenson, AACC, Online, 2006 D.Svinarov©2009


AKI: Urgent Need for Early Diagnosis ™Early forms of AKI are often reversible ™There is a direct relationship between duration of renal failure and mortality ™Early diagnosis will enable timely institution of measures for treatment of renal injuries and prevention of progression -animal and human studies reveal a narrow “window of opportunity” Devarajan P & R Christenson, AACC, Online, 2006 D.Svinarov©2009


At what level of creatinine does a 65year-old white woman have CKD? A 95 μmol/L B 133 μmol/L C 265 μmol/L Actual eGFRs at these creatinine levels ARE A = 54 mL/min/1.73m2 B = 37 mL/min/1.73m2 C = 17 mL/min/1.73m2 Creatinine is 87 μmol/L when eGFR = 60 mL/min/1.73 m2 (www.mdrd.com) D.Svinarov©2009


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Are there fast biomarker candidates for ACI? Neutrophil Gelatinase-Associated Lipocalin: ™Most up-regulated gene in the kidney after AKI ™Protein product is easily detected in U and B ™Elevated at 2 h (х10 в P, х 15 в U) early marker! ™Sensitive, specific and highly predictive for AKI ! ™Creatinine rise requires 48-72 hrs following AKI (slow); Cystatin С rises after 8 h (intermediate) D.Svinarov©2009


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Newer candidate biomarkers for AKI Urine IL-18 ™Elevated in acute tubular necrosis (ATN) ™Indicates delayed graft function ™Marker for proximal tubular injury in ATN.

Kidney Injury Molecule 1 (KIM-1) ™Type 1 transmembrane protein ™Up-regulated in the proximal tubule cells in AKI ™Ectodomain of KIM-1 is shed in U upon injury D.Svinarov©2009


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FUTURE TRENDS ™From single biomarker to disease signatures ™From diagnosis and treatment to prevention ™“Omics” revolution in clinical medicine

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The ballance Single biomarker – disease signature in clinical medicine

Is there an ideal disease biomarker?

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genomic, proteomic, … “omic” disease signature!


CLINICAL CHEMOME nonenzymatic chemical changes of biomolecules Major reasons: aging and disease • Mechanism : • FREE RADICAL DAMAGE • Common Diseases : • Cancer • Diabetes • Cardiovascular • Neurodegenerative

Major constituents: oxidized nucleotides, AAs, sugars and lipids • Low molecular components of body tissues and fluids • Modify proteins and DNA, forming advanced glyoxidation and lipoxidation products • Examples: glycohemoglobin, isprostanes, malondialdehyde, nitro and ortho-tyrosine, …

Methods for analysis: LC/MS/MS coupled to bioinformatic system • Provide new understanding for health, aging, disease , risk and response to therapy • By revealing the “omics” signatures of disease • Result - nonlinear technologic advance and management in clinical medicine


PROTEOMICS

METABOLOMICS

COMPONENTS OF CLINICAL CHEMOME

METABONOMICS

(Baynes, Clin Chem, 2004)

GENOMICS D.Svinarov©2009

TRANSCRIPTOMICS


“OMICS” THERMINOLOGY Genomics: analysis analysis of of genetic genetic program program Genomics: Transcriptomics: analysis analysis of of gene gene expression expression Transcriptomics: Proteomics: protein protein abandance abandance and and signals signals Proteomics: Metabolomics: intracellular intracellular metabolites metabolites Metabolomics: Metabonomics: metabolite metabolite profiling profiling Metabonomics: “Interactosomics”: concert concert action action of of “omics” “omics” “Interactosomics”: D.Svinarov©2009


PROTEOMIC SIGNATURES OF HEPATOCELLULAR CARCINOMA (HCC) ™Objective: HCC versus CLD at alpha-FP < 500 µg/L ™Methods: 20 HCC vs 38 CLD Patients , MS-TOF-Bioinfomatics with artificial network ™Results: 2 384 plasma proteins up to 200 KD assayed! 250 with specific differences in HCC vs CLD! ™Case clustering: initial HCC vs advanced disease, lymph node invasion, distant metastases

™Specificity 90%; Sensitivity 92% Poon T at all. Clin. Chem., 2003 D.Svinarov©2009


MASS SPECTROMETRY â&#x20AC;&#x201C; CLINICAL CHEMISTRY ANALYSER OF THE NEAR FUTURE High throughput: Mass spectrometry analysis of nucleic acids, proteins, low molecular metabolites provides dramatic advantages

Analysis of thousands of components in a drop of blood in several minutes >> hundreds of samples in a single batch

Absolute specificity: Structural identification of known and unknown components >> direct analysis of PCR products!

Extreme sensitivity: Quantitative assays in the femtomolar range with use of microvolumes of sample

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Clin-Lab Relationship Compliance model

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Concordance model


CONCLUSION ™New analytical techniques coupled to adaptive and vigilant bioinformatic pattern-recognition tools will change how disease is detected and monitored ™Thus a transfer from single biomarkers to disease signatures will open the era of “omics” diagnostics and management in clinical medicine ™The result will be a nonlinear advance in our understanding of health, aging, disease, prevention, risk assessment, individualization of therapy, monitoring of relapse... (Petricoin & Liotta, Clin Chem, 2003) D.Svinarov©2009


Thank you!

D.SvinarovŠ2009


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