HIPPOCRATES ( B.C.)

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2 HIPPOCRATES ( B.C.) APHORISMS SEVENTH SECTION XXXIV When bubbles form in the urine, it is a sign that the kidneys are affected, and that the disease will be protracted

3 Thomas Brian cautioned in 1655 against erroneus use of urine for diagnostic purposes in his discourse The pisse-prophet or, certain pisse-pot lectures Wherein are newly discovered the old fallacies, deceit and jugling of the pisse-pot science, used by all those (wether quacks ) who pretend knowledge of diseases, by the urine, in giving judgement of the same.

4 Frederik Dekkers, , professor of medicine in Leyden, described in 1695 in De medicamentis purgantibus heat and acid precipitation to demonstrate proteinuria

5 The discoverers of the first specific urine protein (1845) Henry Bence Jones William MacIntyre Thomas Watson: Dear Dr. Jones. The tube contains urine of very high specific gravity. but as it cools, assumes the consistence and apperance which you see. Heat reliquifies it. What is it?

6 Presently, we have a catastrophic nomenclature for describing normal and abnormal urine protein patterns: It is not only wrong, but also underpins stupidity and ignorance.

7 Plasma-derived proteins in normal urine Albumin IgG free protein HC (α₁-microglobulin) free κ-chains free λ-chains α₂-macroglobulin orosmucoid RBP β₂-microglobulin cystatin C α ₁-antirypsin lysozyme transferrin myoglobin

8 Levels of plasma-derived proteins in normal urine Albumin 1 30 mg/l IgG free protein HC (α₁-microglobulin) free κ-chains free λ-chains α₂-macroglobulin orosmucoid < 5 mg/l 2 10 mg/l 1 6 mg/l mg/l < 10 mg/l < 7 ml/l

9 Kidney-derived proteins in normal urine Tamm-Horsfall glycoprotein = uromodulin (2 200 mg/l) N-acetyl-,D-glucosaminidase (NAG) fructose-1,6-bisphosphatase fibronectin core protein-glycosaminoglycans laminin collagens Na-D-glucose cotransporter

10 The concept Proteinuria Addition of the upper reference values for the plasma- and kidney-derived specific proteins in urine from healthy subjects produces a value for total protein of about 270 mg/l. Is it wise to call 270 mg protein/l No proteinuria, which we must as long as we prefer to use the term proteinuria to indicate a pathological condition?

11 The concept Proteinuria The urine concentration of several kidneyderived specific proteins e.g. Tamm- Horsfall glycoprotein, NAG, core proteinglycosaminoglycans, is below the lower reference value in certain pathological conditions. Such observations cannot be described within the conceptual frame of proteinuriano proteinuria

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13 The concept of Proteinura Alternative terminology Normal urine protein pattern (Normoproteinuria) Abnormal urine protein pattern (+ specifications)

14 Different ways of measuring urinary protein excretion

15 Presently, there is no shortage of suggested Markers of Renal Diseases

16 Protein Markers of Renal Disease Protein Plasma/Urine Damage site Albumin Plasma Glomerulus (leakage) Albumin Urine Glomerulus (leakage) 2 -microglobulin Plasma Glomerulus (GFR) 2 -microglobulin Urine Proximal tubulus -trace protein Plasma Glomerulus (GFR) -trace protein Urine Proximal tubulus core protein- Urine Glomerulus (disintegration) glycosaminoglycans Calbindin Urine Distal tubulus Clusterin Urine Tubulus Cystatin C Plasma Glomerulus (GFR) Cystatin C Urine Proximal tubulus

17 Protein Markers of Renal Disease (Continued) Protein Plasma/Urine Damage site Cystein-rich protein Urine Proximal tubulus EGF Epidermal Growth Factor Urine Distal tubulus Factor D (complement system) Plasma Glomerulus (GFR) Free k- and l-chains Plasma Glomerulus (GFR) Free k- and l-chains Urine Proximal tubulus Free protein HC Plasma Glomerulus (GFR) a 1 -microglobulin Free protein HC Urine Proximal tubulus a-gst a-glutathione-s-transferase Urine Proximal tubulus p-gst p-glutathione-s-transferase Urine Distal tubulus Immunoglobulin G Urine Glomerulus (leakage) Interleukin-18 Urine Multiple

18 Protein Markers of Renal Disease (Continued) Protein Plasma/Urine Damage site KIM-1,Kidney injury Urine Proximal tubulus molecule-1 L-FABPLiver-type fatty acid Urine Proximal tubulus binding protein NAG N-acetyl- -glucosaminidase Urine Proximal tubulus NGAL Neutrophil gelatinase- Plasma Multiple associated lipocalin NGAL Urine Proximal tubulus Podocin Urine Glomerulus (disintegration) RBP Retinol binding protein Plasma Glomerulus (GFR) RBP Urine Proximal tubulus VEGF Vascular endothelial Urine Multiple Growth factor

19 Markers of Renal Diseases We need markers for GFR Glomerular damage Tubular/interstitial damage Assessment of Risk for Acute Renal Failure

20 Markers for Glomerular Damage Urine albumin: Selective and unselective glomerular damage and systemic endothelial dysfunction (mg/mmol creatinine) Urine IgG: Unselective (i.e. worse) glomerular damage (mg/mmol creatinine) Urine IgM: Unselective (i.e. worse) glomerular damage (mg/mmol creatinine) Urine Core protein-glycosaminoglycans: Glomerular disintegration e.g. amyloidosis (mg/mmol creatinine) Urine Podocin: Glomerular disintegration

21 Size selectivity proteins IgG mw 150 kda, P mg/l, U< 8mg/L α₂-macroglobulin mw 720 kda, P 2000 mg/l U? IgM mw 900 kda, P 1200 mg/l, U? SI =

22 Charge selectivity proteins: IgG2: uncharged IgG4: negatively charged mw 150 kda P- conc. IgG1>IgG2>IgG3>IgG4 IgG2/4 ratio =

23 Markers for Tubular/interstitial damage Urine free protein HC (free a 1 -microglobulin): Both reversible and non-reversible damage Urine 2 -microglobulin: Both reversible and nonreversible damage Urine free k- and l-chains: Both reversible and nonreversible damage (and, if abnormal ratio, immunodyscracies like myelomatosis and MGUS)

24 Protein HC, alias a 1 -microglobulin, is a Human Complex-forming protein, Heterogenous in Charge Plasma contains approximately equal molar amounts of free protein HC (28 kda) and HC-IgA-complexes (180 kda) and lower amounts of several other protein HC-complexes (HCalbumin, HC-fibrinogen etc). Urine contains virtually only free protein HC. In contrast to 2 -microglobulin, protein HC is stable in native urine, in native urine stored for one week at room temperature or for one month in the refrigerator and its high urine concentrations allows the use of rapid, cheap assays methods.

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27 Markers for Assessment of Risk for Acute Renal Failure Most accepted/used: Plasma and urine Neutrophil Gelatinase Associated Lipocalin (NGAL) Promising, awaiting confirmation: Urine Kidney Injury Molecule-1 (KIM-1) Urine free protein HC (a 1 -microglobulin) Plasma cystatin C

28 Markers for GFR Plasma cystatin C Plasma creatinine Plasma -trace protein Plasma factor D (complement system) Plasma 2 -microglobulin Plasma free protein HC (Free a 1 -microglobulin) Plasma RBP (Retinol Binding Protein alias a 2 -microglobulin)

29 Advantages of cystatin C as a GFR-marker Demonstrates the early, potentially reversible, decrease of GFR in the creatinine-blind area Independent of diet No tubular secretion Low influence by muscle mass, gender and race (African American) Independent of age for children and adults above 1 year Demonstrates the decrease of GFR in old persons Can be used to measure the renal reserve Mirrors the diurnal GFR variation Elucidates filtration quality and life expectancy

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32 GFR-markers for patients with muscle atrophy Non-parametric ROC plots for serum cystatin C (solid line) AUC = and serum creatinine (dotted line) AUC = AUC = 0.50 equals the diagnostic efficiency of tossing a coin

33 Disadvantages of cystatin C High doses of corticosteroids increase significantly the P-Cystatin C level (but not low doses e.g. ointments, inhalation) Hyperthyreosis moderately increases the P-Cystatin C level (and decreases the P-Creatinine level due to increased tubular secretion) The cost of analysing P-Cystatin C is higher than that of analysing P-Creatinine (prices in Lund 2013: 1.5 versus 3.5 $)

34 Suggestion for optimal diagnostic use of egfr Run both cystatin C and creatinine and calculate egfr (cystatin C) and egfr (creatinine) at the initial patient contact. If they agree: GFR is correct, no invasive clearance determination is required. Use mean of egfr (cystatin C) and egfr (creatinine)! If they do not agree: Try to find a biomedical explanation (low muscle mass, high dose of glucocorticoid). If an obvious explanation is found no invasive clearance determination is required. The non-affected egfr is used. If the two egfrs do not agree and no obvious explanation for the difference can be found: An invasive clearance determination is justified (iohexol clearance). Site: egfr.se

35 Verified Markers of Renal Diseases GFR-decrease: Plasma cystatin C Glomerular damage: Urine albumin and IgG Tubular/interstitial damage: Urine free protein HC (a 1 -microglobulin) Assessment of Risk for Acute Renal Failure: Plasma and urine NGAL

36 Diagnosing kidney disease 2014 A To measure relative and absolute glomerular filtration rate (GFR): Iohexolor 51 Cr-EDTA-clearance (Invasive procedures) To estimate relative glomerular filtration rate (GFR): Pt-eGFR (Cystatin C) or Pt-eGFR (Creatinine), unit: ml/min/1.73m 2 body surface area. Best estimation is the mean of Pt-eGFR (Cystatin C) and Pt-eGFR (Creatinine) Internet-tool to do it: To estimate absolute GFR for dosage of drugs excreted by the kidneys; unit: ml/min: Pt-eGFR (Cystatin C) or/and Pt-eGFR (Creatinine) + weight + height using internet calculators e.g. To calculate risk for end-stage renal disease (hemodialysis), myocardial infarction, hospitalization and death at reduced GFR: Pt-eGFR (Cystatin C) Unit: ml/min/1.73m 2 body surface area

37 Diagnosing kidney disease 2014 B To analyse glomerular damage (endothelial damage), selective proteinuria : U-Albumin, spot sample, unit: mg/mmol creatinine To analyse more severe glomerular damage (endothelial damage), unselective proteinuria : U-IgG, (U-IgM) spot sample, unit: mg/mmol creatinine To analyse tubular damage: U-Protein HC (=a 1 -microglobulin), spot sample, unit: mg/mmol creatinine To analyse Bence Jones proteinuria: Ratio U-(Total) Kappa/U-(Total) Lambda-chains, spot sample, or Immunofixation of urine for kappa-, lambda-, IgG-, IgA- and IgM-immunoreactivity or Ratio P-Free Kappa/P-Free Lambdachains

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39 Thank you for your attention and for not caressing your smartphones too much!

40 Extras

41 PNAS 1982

42 THE HUMAN CYSTATIN SUPERFAMILY Family 1 Family 2 Family 3 (intracellular) (extracellular) (intravascular) cystatin A cystatin C LMW-kininogen cystatin B cystatin D HMW-kininogen cystatin E cystatin F cystatin G cystatin S cystatin SA cystatin SN

43 Kyhse-Andersen J, Schmidt C, Nordin G, Andersson B, Nilsson-Ehle P, Lindström V, Grubb A. Serum cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method is a better marker than serum creatinine for glomerular filtration rate Clin Chem 40: , (1994)

44 PubMed search April 2013 for cystatin C AND (renal OR glomerular) generated 1860 hits Google search April 2013 for cystatin C : hits

45 Agarose gel electrophoresis of urine from patients with tubular damage

46 40 Å 30 Å 5 Å

47 Annual numbers of deaths mycardial infarctions strokes hospitalizations U-Albumin (mg/mmol creatinine) P-Cystatin C (mg/l)

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49 Functional glomerular pore size

50 GFR-estimating equations Relative GFR in ml/min/1.73m 2 Creatinine-based for adults: MDRDIDMS-traceable: egfr = 175 x (creatinine/88.4) x age x (if female) x (if African American) The CKD-EPI equation is more complicated Cystatin C-based for adults and children: egfr = 85 x cystatin C -1.68

51 Challenge To reduce/eliminate the large number of cystatin C-based GFR estimating equations Differences in diagnostic performance (bias, P30%) between equations may be due to 1. Differences in cystatin C assays used 2. Different ways of measuring GFR (clearance of iohexol, inulin, 125 I-iothalamate, 51 Cr-EDTA, 99m Tc-DTPA, creatinine clearance etc) 3. Differences between batches of cystatin C-reagents 4. Population differences

52 IFCC-Cystatin C-based GFR-estimating equation for adults and children: egfr = 136 x cystatin C -1 x age Based upon a population of about 4000 Caucasians (Swedish, Dutch), including about 700 children and 800 Asians (Japanese) and Seven commutable cystatin C-assays adjusted to the cystatin C international calibrator ERM-DA471/IFCC (Abbott, Dako, Gentian, Roche, Sentinel, Siemens, Alfresa) and GFR measured by renal inulin clearance and plasma iohexol clearance

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58 Creatinine-based egfr equations MDRD : x [S- creat ( mol/l)/88.4] x age x (if female) x (if African American) or e x ln(creat) x ln(age) (if female) (if African American) Only for adults Schwartz: 0.55 x height (cm) x [P- creat ( mol/l)/88.4] -1 Only for children Counahan Barratt : 0.43 x height (cm) x [P- creat ( mol/l)/88.4] -1 Only for children Lund-Malmö : e x creat x ln(age) x age (if female) For children and adults

59 Age-related GFR-predictions at a constant creatinine level of 80 micromol/l for 3 prediction equations

60 The P30%-value for a GFR-estimating equation is the percentage of all estimations which is between +/- 30% of measured GFR (using an invasive clearance-method). The best cystatin C- and creatinin- (+sex, age and race) based equation have P30%-values of 85-90%. Equations based upon both cystatin C- och creatinine display P30%-values of 90-95%.

61 Present knowledge GFR-estimating equations based upon both cystatin C and creatinine, egfr mean, display the best diagnostic performance (For example, Inker et al. N Engl J Med 2012;367:20-9) (Automatic) comparison of egfr cystatinc and egfr creatinine increases diagnostic performance further and can test the reliability of the estimation (Grubb et al. Scand J Clin Lab Invest. 2012;72:73-7)

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66 BMI: 35