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Proteinuria and albuminuria

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  • Albumin is a single chain plasma protein, consisting of three homologius globular domains, that are stabilised by 17 internal disulfide bridges. Is also contains a single reactive thiol, Cys 34, which is involved in dimerization of albumin molecules by forming disulfide bridges between two albumin molecules.
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    • 1. Ron T. Gansevoort Coordinator PREVEND Study Department of Nephrology University Medical Center Groningen The Netherlands Assessing albuminuria Methodological considerations with clinical impact
    • 2. Introduction Albuminuria is a urinary biomarker that has been shown to be a predictor of renal and CV events. As such albuminuria has a place in clinical practice: kDOQI stages 1 and 2 are defined by presence of micro-albuminuria. There is strong lobby for standardisation of measurement serum creatinine (Cleveland Clinic / IDMS traceable) to obtain the most reliable GFR estimate. Untill recently little attention has been paid to standardisation of albuminuria ( exception Miller et al, Clin Chem 2009;55:24-38 )
    • 3. Micro-albuminuria - Definition and classification - Spot urines (first morning void, or random) 17 - 170 25 - 250 < 17 < 25 Alb/creat ratio (mg/gram) > 200 > 300 > 200 Macro-albuminuria 20 – 200 30 – 300 M F 20 – 200 Micro-albuminuria < 20 < 30 M F < 20 Normal Albumin Excretion (  g/min) Albumin Excretion (mg/24h) Albumin Concentration (mg/l) Overnight (timed) 24h urine > 170 > 250 M F
    • 4. Assessment of albuminuria Questions to address
      • What assay to use? (answer: immunochemistry polyclonal)
      • What urine sample to use?
        • 24hr urine collection, first morning void or a spot sample?
      • Which albuminuria measure to use: urinary albumin concentration, albumin/creatinine ratio, or 24hr albumin excretion?
      • Does it matter whether we use fresh urine samples or stored samples?
      • If we are going to use frozen urine samples, what is important?
        • pre-storage handling, storage temperature, sample handling
    • 5. Monomer Bakker , Gansevoort et al, Curr Hypert Rep 2009;11:111-7 Loss of immunoreactivity ?? Assessment of albuminuria Which assay to use? Dimerization Polymerization Fragmentation Type of assay Monoclonal AB Polyclonal AB Colorimetric test strips Immunochemistry based Size exclusion (HPLC)
    • 6. 24-hour FMV Spot (morning) Albumin concentration (mg/L) 0 20 40 60 80 Median 24-hour [IQ-range] 7.6 [4.8-12.7] Median Overnight [IQ-range] 7.2 [4.5-12.0] Median Spot (morning) [IQ-range] 11.9 [7.8-25.8] P = < 0.01 P = < 0.01 P = 0.43 N=250 Witte et al, JASN 2009;20:436-43 What urine samples to use ? Median urinary albumin concentration PREVEND
    • 7. Urinary Albumin Concentration 24-hour FMV Spot (morning) Intra-subject coefficient of Variation (%) 0 20 40 60 80 100 P = < 0.01 P = < 0.01 P = 0.08 What urine samples to use ? Coefficient of variation N=250 Witte et al, JASN 09;20:436-43 PREVEND
    • 8. 24-hour FMV Spot (morning) Albumine:creatinine ratio (mg/mmol) 0 2 4 6 8 10 Median 24-hour [IQ-range] 1.00 [0.65-1.54] Median Overnight [IQ-range] 0.67 [0.50-1.17] Median Spot (morning) [IQ-range] 1.21 [0.68-2.37] P = 0.023 P = < 0.001 P < 0.001 Witte et al, JASN 09;20:436-43 What urine samples to use ? Median albumin:creatinine ratio N=250 PREVEND
    • 9. What urine samples to use ? Coefficient of variation Albumin:creatinine ratio 24-hour FMV Spot (morning) 0 20 40 60 80 100 P = < 0.01 P = < 0.01 P = 0.58 N=250 Intra-subject coefficient of Variation (%) Witte et al, JASN 09;20:436-43 PREVEND
    • 10. 0 10 20 30 Overall Male Female Prevalence of microalbuminuria (%) # * # * # * # * # * # * 24hr UAE UAC ACR 0 10 20 30 Overall Male Female Prevalence of microalbuminuria (%) First Morning Void # * # * # * # * # * # * Spot Morning Urine Sample Witte et al, JASN 09;20:436-43 UAC ACR What urine samples to use ? Prevalence of microalbuminuria PREVEND
    • 11. Which albuminuria measure to use? Predicting CV outcome * p < 0.05 vs UAC, # p < 0.05 vs UAE AUC ROC curve N=3432 Random sample of the general population Lambers-Heerspink et al, Am J Epidemiol 08;168:897-905 PREVEND 24 hr urine First morning void UAE (mg/24hr) UAC (mmol/L ACR (mg/mmol) Overall 0.65 0.62 0.66* Subgroups Male 0.64 0.62 0.68* Female 0.66 0.59 # 0.66* <47 yr 0.58 0.52 0.52 >47yr 0.65 0.64 0.64
    • 12. Lambers Heerspink et al, submitted N=701 RENAAL: DM2 nephropathy Which albuminuria measure to use? Predicting renal outcome 100 80 60 40 20 0 0 20 40 60 80 100 Sensitivity (%) Specificity (%) UAE 24hr; AUC = 0.78 UPE 24hr; AUC = 0.78 p<0.001 ACR FMV; AUC = 0.82
    • 13. First morning void -60 -40 -20 0 20 40 60 80 35 45 55 65 75 Age (years) Difference (%) UAC UCrC ACR 24-hour urine collection -60 -40 -20 0 20 40 60 80 35 45 55 65 75 Age (years) Difference (%) UAE UCrE ACR Lambers-Heerspink, Gansevoort et al, Am J Epidemiol 2008;168:897-905 Which albuminuria measure to use? ACR “incorporates” the influence of age PREVEND
    • 14. Frozen storage (-20 C) of urine samples Influence of duration of storage and sample handling 3 to 5 5 to 8 8 to 12 12 to 18 18 to 24 months * Not significantly different from zero Hand-inversion Brinkman et al, Clin Chem 2005;51:2181-3 PREVEND * * Percentage change in UAC, % Vortex mixing No sample handling -80 -60 -40 -20 0 20 40
    • 15. Predictive value of albuminuria Does it matter when urine has been stored frozen ? Brinkman et al, Clin Chem 2007;53:153-4 P<0.01 Predictive value of UAE for CV endpoints PREVEND
    • 16. Frozen storage of urine samples Does urinary pH matter ? - 100 - 75 - 50 - 25 0 25 50 75 Change in urinary albumin concentration (%) -20 ° C - 20  C pH8 -80  C -80  C pH8 Storage condition - - - - - Lambers Heerspink et al, Diabetic Med 2009;26:556-9 PREVEND
    • 17. Screening for albuminuria The past (1892) Gansevoort and Ritz, Nephrol Dial Transplant 2008
    • 18. Conclusions
      • When assessing the clinical impact of urinary biomarkers it is
      • essential to take into consideration methodological issues
      • Which assay was used? Polyclonal? Intra- and interassay CV?
      • What urine samples were used? Preferably 24hr collections or first morning voids
      • In case first morning void samples are used, normalise for creatine concentration
      • Fresh or frozen? Preferably use fresh urine samples.
      • If frozen, what were storage conditions and how was sample handling? Frozen at -80 0 Celsius, pH adjustment (or protease inhibitors?), vortexing?

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