Assessment of Renal Tubular Function

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  • I start this talk with a picture of a healthy glomerulus simply because most of the assessment I am going to talk about is to do with the GFR I:\\powerpointGFRtalk020904
  • This is one of the earliest publications on the clinical utility of cystatin C. it is again David Newman’s work and you can see that there is an earlier and greater proportional rise in cystatin C as GFR falls compared to serum creatinine.
  • Just to emphasise the advantages of cystatin C in this group, this figure shows cystatin C and creatinine plotted relative to their respective upper limits of normal. The vertical lines denote the lower limit of normal and the cut-off for mild renal failure. Note the earlier and more pronounced rise in cystatin C.
  • The newborn kidney is immature and one would therefore expect that serum markers of GFR should be increased. However, this is not true of serum creatinine, reflecting the lower muscle mass in babies. By contrast cystatin C is appropriately increased at birth and falls as kidney function completes its development. The usefulness of creatinine in this setting is often further limited by the coexistence of jaundice.
  • Abstract Background: Glomerular filtration rate (GFR) is routinely assessed by measuring the concentrations of endogenous serum markers such as blood urea nitrogen and serum creatinine (SCr). Although widely used, these endogenous markers are not ideal and do not perform optimally in certain clinical settings. The purpose of this review is to critically review the potential utility of cystatin C (CysC), especially in patient populations in which CysC may have an advantage over routinely used endogenous markers of GFR. Approach: In a narrative approach, we extensively review publications, primarily from the last 5 years, that address the development of methods to measure CysC, reference intervals, and the diagnostic accuracy of CysC to assess GFR. Between June 2000 and September 2001 Medline was searched using "cystatin c" as a textword, and articles that examined >75 individuals (except for renal transplant studies) and/or used accepted "gold standards" for assessing GFR were selected for inclusion. A total of 17 studies are reviewed that provide reference interval data for several populations. A total of 24 studies make conclusions about the utility of CysC vs SCr and/or creatinine clearance, with 20 providing data on the sensitivity and specificity of CysC for detecting impaired GFR. These publications are organized into subgroups that deal with specific patient populations or clinical situations. Content: This review focuses on two areas: (a) the evolution of immunoassays used to determine the concentration of CysC in serum, their analytic sensitivity, and reference intervals; and (b) the diagnostic performance of CysC against other renal markers in the general population and in specific subpopulations of patients. Summary: Studies of reference intervals for CysC overwhelmingly demonstrated that CysC values in blood are independent of age and sex. Of the 24 studies that examined clinical utility, 15 concluded that CysC is superior to SCr, whereas 9 concluded that CysC is equivalent but provides no advantage. Summary ROC plot analysis of 20 studies that provide sensitivity and specificity data strongly suggests that CysC will be superior to SCr for detecting impaired GFR. Taken together, it is clear that CysC performs at least as well as SCr in the population at large and that it is likely to be superior to SCr in specific patient populations.
  • There have been many similar studies subsequently and last year a meta-analysis of these studies was published. 46 studies were included In the top panel you can see the correlation coefficients for the reciprocal against GFR and that for Cys C was superior. Similarly, in the lower panel, the AUC values for the reciprocals again showed superior value for Cys C. Abstract Serum cystatin C (Cys C) has been proposed as a simple, accurate, and rapid endogenous marker of glomerular filtration rate (GFR) in research and clinical practice. However, there are conflicting reports regarding the superiority of Cys C over serum creatinine (Cr), with a few studies suggesting no significant difference. METHODS: We performed a meta-analysis of available data from various studies to compare the accuracy of Cys C and Cr in relation to a reference standard of GFR. A bibliographic search showed 46 articles until December 31, 2001. We also retrieved data from eight other studies presented and published in abstract form. RESULTS: The overall correlation coefficient for the reciprocal of serum Cys C (r = 0.816; 95% confidence interval [CI], 0.804 to 0.826) was superior to that of the reciprocal of serum Cr (r = 0.742; 95% CI, 0.726 to 0.758; P < 0.001). Similarly, receiver operating characteristic (ROC)-plot area under the curve (AUC) values for 1/Cys C had greater identity with the reference test for GFR (mean ROC-plot AUC for Cys C, 0.926; 95% CI, 0.892 to 0.960) than ROC-plot AUC values for 1/Cr (mean ROC-plot AUC for serum Cr, 0.837; 95% CI, 0.796 to 0.878; P < 0.001). Immunonephelometric methods of Cys C assay produced significantly greater correlations than other assay methods (r = 0.846 versus r = 0.784; P < 0.001). CONCLUSION: In this meta-analysis using currently available data, serum Cys C is clearly superior to serum Cr as a marker of GFR measured by correlation or mean ROC-plot AUC.
  • .
  • Filtered albumin binds to megalin and cubulin. Cubulin has no transmembrane domain and so can only initialise internalization following binding to megalin. Complex taken up in endososme and transported to lysososmes where broken down by cathepsisn B and D and cysteine proteases into 500 to 15,000 Da fragments. Most of these recycled back in circulation but 35% lost in urine. Overall represents approx 10% of daily albumin turnover in the body. Amn=amnionless ( 50 kDa chaperone for cubulin) Megalin deficient mice and patients with cubulin and amnionless mutations have been described who are proteinuric. Impairment of the tubular reabsorptive mechanism or of processing of filtered albumin may be an early event in diabetic nephropathy, pre-dating changes in glomerular permeability.
  • Professor Keen and co-workers at Guy’s first developed assays for low concentrations of albumin in urine in 1963 and 20 years later the same group coined the term microalbuminuria (A misleading term which I shall return to). In the mid-’80’s studies demonstrated what had been suspected for some time, that patients with microalbuminuria were more likely to go in to develop diabetic nephropathy. In the 1990’s several studies (including the DCCT study of type 1 diabetics in the USA and the UKPDS study of type 2 diabetics in the UK) have demonstrated that good glycaemic control and anti-hypertensive medication can slow the progression of diabetic nephropathy in patients with microalbuminuria and t he decrease the number of normoalbuminuric patients who become microalbuminuric. More recently, there is a surge of interest in microalbuminuria as a marker of cardiovadscular risk although its’ clinical role in this setting is unclear..
  • Day to day variation in albumin excretion is about 40% so several tests must be done before the diagnosis is established - we have concurred with the definition that it should be present in two or more samples out of three but our guidelines also suggest that physicians may then proceed to a confirmatory overnight collection in patients identified as having high AER’s. The best possible metabolic control of diabetes should be achieved before investigating patients for microalbuminuria - poor control is commonly associated with an increase in albumin excretion but often this is transient and totally reversible. The co-existence of any acute illness may result in an increase in albumin excretion - the marked fall in serum albumin levels which are seen after surgery, for example, are due to an increased permeability of the vasculature to albumin and exactly the same process occurs in the glomerular capillary bed. Menstrual contamination and haematuria must be avoided and any blood stained samples should be disregarded. A thumb nail calculation will show you that the presence of 1 mL of menstrual blood in 20 mL of urine could increase the albumin concentration by 1000 mg/L.
  • The reference ranges will obviously vary depending on which sample you are using but these cut-offs for random, overnight and 24 h collections are thought to be roughly equivalent. Note that these are not normal ranges - there is a grey area above normal but below a level defined as microalbuminutria. Also note that the albumin/creatinine ratios are different in men and women - this is due to the lower creatinine excretion in women rather than a difference in true albumin excretion rate.
  • Rates of progression very variable between individuals and between types 1 and 2. Type 2 more likely to have overt nephropathy at diagnosis, or to progress to it quickly after diagnosis, but their subsequent progression to ESRD is lower than that of type 1 (20% cf 75%) 80% of type 1 with microalbuminuria will progress to overt nephropathy. Once overt nephropathy established, 75% of type will progress to ESRD by 20 y.
  • These issues are best illustrated by examination of data from the United Kingdom External Quality Assessment Scheme (UKNEQAS). For example, the results shown below represent data collected from 219, predominantly UK, laboratories participating in the urinary total protein scheme. The mean concentration is 0.28 g/L (280 mg/L), approximately equivalent to 1+ using a reagent strip device. A variety of methods are in use and between-laboratory agreement is poor, with a coefficient of variation of 12.4%. Clearly a patient submitting a sample to a local laboratory could get a result anywhere between 0.20 and 0.40 g/L. The UKNEQAS also provide a scheme for reagent strip device quality assessment. Although several types of device are in use, the predominant method group (87 of 135 participants) is the manufacturer Bayer plc. In relation to the above sample, 56.3% of Bayer users classified the above sample as 1+, 29.9% as ‘trace’, 8.0% as negative and 5.7% as 2+ or greater.
  • PAGE showed that major protein in urine was albumin Large differences between RIA (actually non-RIA) and HPLC, particularly at lower albunin concs. Denisty of PAGE bands correlates more closely with HPLC results.
  • Reducing conditions breaks the S-S bonds and results in aseries of protein fragmenst Suggests that the primary structure has been cut at multiple sites ? By lysosomal processing but that the fragments are held together in urine by S-S bonds. The scissions have destroyed antibody recognition sites so that immunoassays no longer identify the molecule as albumin.
  • Assessment of Renal Tubular Function

    1. 1. Kidney Function Testing - 2 Dr Edmund Lamb ACB National Training Course, September 2007 U cr x V P cr x T
    2. 2. Overview <ul><li>Part one </li></ul><ul><li>Classification of CKD </li></ul><ul><li>GFR </li></ul><ul><li>Creatinine and eGFR </li></ul><ul><li>Part two </li></ul><ul><li>Cystatin C </li></ul><ul><li>Proteinuria/albuminuria </li></ul>
    3. 3. Cystatin C <ul><li>13 kD basic protein </li></ul><ul><li>120 AAs, single pp. chain </li></ul><ul><li>cysteine-protease inhibitor </li></ul><ul><li>produced at constant rate by all nucleated cells </li></ul><ul><li>freely filtered at glomerulus </li></ul><ul><li>reabsorbed/catabolised in proximal tubule </li></ul><ul><li>serum concentration mainly determined by GFR </li></ul><ul><li>proposed as improved GFR marker </li></ul><ul><li>? especially useful in moderate CKD </li></ul>
    4. 4. CKD: sensitivity to nephron loss Cystatin C Creatinine EDTA GFR (ml/min/1.73 m 2 ) Proportional increase in analyte ** ** * * Newman et al KI 1995 206 nephrology out-patients with SCr <300 umol/L <30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 101-110 111-120 >120 0 1 2 3 4
    5. 5. Sensitivity in older people O’Riordan et al 2003 53 patients, mean age 80 y
    6. 6. Newman, Ann Clin Biochem 2002 Cystatin C reflects GFR in children
    7. 7. Other settings <ul><li>CKD (numerous studies) </li></ul><ul><li>Paediatrics (numerous studies) </li></ul><ul><li>Renal Tx monitoring (Le Bricon et al Clin Chem 1999) </li></ul><ul><li>Chemotherrapy monitoring (Stabuc et al Clin Chem 2000) </li></ul><ul><li>Pre-eclampsia (Strevens et al BJOG 2003) </li></ul><ul><li>Type 2 diabetes (Mussap et al KI 2002) </li></ul><ul><li>Spinal cord injury (Jenkins et al Ann Clin Biochem 2003) </li></ul><ul><li>Renovascular disease (Olivieri et al Clin Chem 2002) </li></ul><ul><li>Myeloma (Lamb et al 2004) </li></ul><ul><li>Rheumatoid arthritis on NSAIDs (Mangge et al CCA 2000) </li></ul><ul><li>All demonstrate benefits c.f. creatinine </li></ul>
    8. 8. ROC Meta-Analysis <ul><li>ROC curve analysis of relative diagnostic accuracy </li></ul><ul><li>20 studies included </li></ul><ul><li>AUC Cystatin C = 0.95 </li></ul><ul><li>AUC Creatinine = 0.91 </li></ul><ul><li>P=0.003 </li></ul>Laterza et al 2002
    9. 9. 1/CysC 1/Creat META-ANALYSIS Serum Cystatin C Is Superior to Serum Creatinine as a Marker of Kidney Function Also, PENIA studies (r=0.846) better than PETIA studies (r=0.784) Dharnidharka et al 2002 1/CysC 1/Creat p<0.001 p<0.001 p<0.001
    10. 10. Measurement of cystatin C <ul><li>Measured by immunoassay </li></ul><ul><li>No international standard. </li></ul><ul><li>Generally free from spectral interferences (haemolysis, icterus, lipaemia) ? Effects of rheumatoid factor </li></ul><ul><li>Precision as good as creatinine </li></ul><ul><li>Cost £2-£3 </li></ul>
    11. 11. Possible caveats <ul><li>Malignant progression </li></ul><ul><li>Thyroid disease </li></ul><ul><li>Biological variation </li></ul>
    12. 12. Malignant progression Suggested up-regulation of cystatin C in tumour progression BUT, didn’t present renal function data (other than “creatinines equivalent”)! Kos et al 1998
    13. 13. Malignant progression <ul><li>Expression of cystatin C has been observed in human lung and colon cancer cell lines </li></ul><ul><li>Cathepsins (which cystatin C inhibit) implicated in a variety of models of malignant progression </li></ul><ul><li>But, to date: </li></ul><ul><li>Multiple myeloma - no evidence of effect (Lamb et al 2004) </li></ul><ul><li>Multiple myeloma – no evidence of effect (Finney et al 2001) </li></ul><ul><li>Proliferative haematological disorders - no evidence of effect (Mojiminiyi et al 2002) </li></ul>
    14. 14. Thyroid function and cystatin C Discrepancy between GFR assessed by creatinine and cystatin C (BUT, no gold standard GFR used) Jayagopal et al 2003
    15. 15. Thyroid function “ Cystatin C should not be used without knowledge of thyroid status” (BUT, no gold standard GFR used) den Hollander et al 2003
    16. 16. Biological variability and cystatin C <ul><li>Healthy volunteers </li></ul><ul><li>Cystatin C better as a screening test than creatinine </li></ul><ul><li>Creatinine better for following changes in an individual patient </li></ul><ul><li>Children with CKD </li></ul><ul><li>Total variability (analytical + biological) </li></ul><ul><li>Cystatin C 12%, creatinine 13% (p=0.0012) </li></ul>Keevil et al 1998 Sambasivan et al 2005
    17. 17. Monitoring function over time <ul><li>20 Pima Indians with type 2 diabetes </li></ul><ul><li>All hyperfiltering </li></ul><ul><li>Iothalamate GFR over 4 years </li></ul><ul><li>Cystatin C </li></ul><ul><li>MDRD </li></ul><ul><li>C&G </li></ul>Perkins et al 2005
    18. 18. Monitoring function over time Perkins et al 2005 -4.4 127 MDRD -4.5 166 C&G -3.8 148 100/creatinine -6.9 163 100/cystatin C -8.1 156 Iothalamate GFR Annual % change Baseline Measure
    19. 19. Cystatin C can predict GFR <ul><li>536 adults and children </li></ul><ul><li>Iohexol gold standard </li></ul><ul><li>GFR = 84.69 x cystatin C -1.680 [ x 0.948 if female] </li></ul><ul><li>R 2 = 0.868, median bias 1.9%, within 30% =82% </li></ul><ul><li>Estimation superior/equivalent to MDRD </li></ul><ul><li>R 2 = 0.846, median bias 0.02%, within 30% =79% </li></ul><ul><li>(Also better than Counahan-Barrat, Schwartz) </li></ul>Grubb et al 2005
    20. 20. Summary – cystatin C <ul><li>Cystatin C detects CKD earlier than creatinine </li></ul><ul><li>It more sensitively predicts earlier complications of CKD </li></ul><ul><li>We need better markers of GFR </li></ul><ul><li>If we are really serious about early detection (and management) of CKD, then cystatin C may find a place </li></ul><ul><li>Possible roles – Tx monitoring, paediatric nephrology, pregnancy </li></ul>
    21. 21. Proteinuria – the cardinal sign of kidney disease
    22. 22. History of proteinuria <ul><li>Hippocrates (400 bc) noted association between bubbles on surface of urine and kidney disease </li></ul><ul><li>Richard Bright (1827), Guy’s Hospital, London discovered that oedema and proteinuria linked with renal disease – Bright’s disease (albuminous nephritis). </li></ul><ul><li>Detected protein by boiling urine until white precipitate appeared </li></ul>
    23. 23. Proteinuria is the strongest predictor of progressive disease GISEN study, KI 1998 Progression to ERF per tertile of protein excretion
    24. 24. … Independently of hypertension GISEN study, KI 1998
    25. 25. ‘ Clinical’ proteinuria <ul><li>Normal protein excretion <150 mg/day (of which albumin about 30 mg, THG predominates) </li></ul><ul><li>Proteinuria typically considered present when ‘1+’ on disptick </li></ul><ul><li>Equivalent to approx 300 mg/L or 500 mg/day (0.5 g/day) </li></ul>
    26. 26. “ Proteinuria can be assessed from a single urine sample (preferably an EMU)…24 h urine collections are therefore unnecessary for this”
    27. 27. Protein:creatinine ratio and 24 h protein excretion are closely related Ruggenenti et al (1998) study of 177 non-diabetic patients with nephropathy Ginsberg (NEJM 1983) – proposed 24 h urine collections could be replaced by PCR ratios
    28. 28. Ruggenenti et al 1998 … also showed that the ratio (r=-0.40) is a better predictor of progression than 24 h excretion (r=-0.27)
    29. 29. Protein:Creatinine Ratios <ul><li>“ The protein:creatinine ratio on a random urine specimen provides evidence to rule-out the presence of significant proteinuria as defined by a 24 h urine excretion measurement” </li></ul>Systematic review. Price et al, Clin Chem September 2005
    30. 30. Protein:Creatinine Ratios <ul><li>Assumptions: normal volume 1.5 L/24 h, normal creatinine excretion 10 mmol/24 h </li></ul><ul><li>‘ Normal’ protein excretion often considered <150 mg/24 h </li></ul><ul><li>‘ 1+’ on a dipstick = 300 mg/L or 450 mg/24 h </li></ul><ul><li>Therefore, ‘normal’ becomes <15 mg/mmol and ‘1+’ becomes 45 mg/mmol </li></ul>
    31. 31. Protein:Creatinine Ratios (2) <ul><li>correct for urinary dilution/concentration </li></ul><ul><li>easier </li></ul><ul><li>cheaper </li></ul><ul><li>more acceptable to the patient </li></ul><ul><li>closely predict 24 h excretion </li></ul><ul><li>consistent with guidelines (K-DOQI, PARADE, NSF) </li></ul><ul><li>more accurately predict progression </li></ul><ul><li>… but require re-education in interpretation </li></ul>
    32. 32. Classification of proteinuria <ul><li>Glomerular </li></ul><ul><li>Tubular </li></ul><ul><li>Overflow </li></ul><ul><li>Quantitatively and clinically, glomerular proteinuria is most significant </li></ul>
    33. 33. Glomerular proteinuria <ul><li>The glomerulus is a filter, retaining proteins of Mr > approximately 65 kDa (e.g. albumin) </li></ul><ul><li>Therefore the appearance of high Mr proteins in urine implies glomerular damage </li></ul><ul><li>May be selective (mainly albumin) or unselective (larger proteins e.g. IgG) – classification rarely used </li></ul><ul><li>In most conditions, albumin is quantitatively the most significant protein </li></ul>
    34. 34. Total protein versus albumin <ul><li>Proteinuria is predominantly albuminuria, but there is not a linear relationship between the two: </li></ul><ul><li>150 mg/L TP contains 30 mg/L albumin (20%) </li></ul><ul><li>300 mg/L TP contains 150 mg/L albumin (50%) </li></ul><ul><li>1000 mg/L TP contains 700 mg/L albumin (70%) </li></ul><ul><li>Relationship more variable at low protein concentrations </li></ul><ul><li>TP stick tests and laboratory methods particularly sensitive to albumin </li></ul>
    35. 35. Albuminuria
    36. 36. Renal handling of albumin <ul><li>r=3.6 nm, filtration fraction <0.01 (cf dextran of same r=0.1) </li></ul><ul><li>pI 4.7 – highly anionic – repulsed by glomerular polyanion </li></ul><ul><li>37,000 g/day pass through glomerular capillaries, </li></ul><ul><li>1.3 g/day pass into urinary space (0.004%) </li></ul><ul><li>Where is the barrier to filtration? </li></ul>
    37. 37. The filtration barrier The glomerular basement membrane is a size- and charge-selective filter Foot processes are the final barrier to filtration
    38. 38. (A) Healthy (B) MCN MCN associated with flattening (effacement) of the foot processes in scanning EM Mathieson, Clin Sci 2004;107:533-8
    39. 40. Renal handling (2) – post glomerular <ul><li>1.3 g pass into urinary space </li></ul><ul><li>(0.004% of handled) </li></ul><ul><li>Approx 10-30 mg/day passed in urine </li></ul><ul><li>(<1% of filtered) </li></ul><ul><li>What happens to the remainder? </li></ul>
    40. 41. Albumin 66,000 Da Cub Meg TUBULAR LUMEN Cub Meg Meg Endosome Lysosome 500-15,000 Da fragments 35% 65% Lost in urine (only intact albumin measured) Returns to circulation 1.3 g/day 10 mg/day Amn
    41. 42. Urinary albumin/‘microalbuminuria’ <ul><li>1963 - Keen & Chlouverakis @ Guy’s developed immunoassay for low concentrations of urine albumin </li></ul><ul><li>Such immunoassays can detect increased albumin in urine before clinical proteinuria is detectable </li></ul><ul><li>1982 - Viberti et al @ Guy’s coined term “microalbuminuria” </li></ul><ul><li>“ An increase in the urinary excretion of albumin above the reference range for healthy non-diabetic subjects but at a level not detectable by crude clinical tests (protein stix tests)” </li></ul><ul><li>‘ Microalbuminuria’ is common in diabetes mellitus and predicts progression to ESRD </li></ul>
    42. 43. NOTE!!! <ul><li>‘ microalbuminuria’ is not about a small form of albumnin </li></ul><ul><li>‘ microalbuminuria’ is about increased, not decreased, amounts of albumin in the urine </li></ul>
    43. 44. Microalbuminuria and progression <ul><li>mid-1980's - studies showed that microalbuminuria predicted development of clinical nephropathy in diabetic patients and that good control slowed progression (e.g. Kroc study) but studies were small/too short </li></ul><ul><li>1990’s </li></ul><ul><li>- good glycaemic control prevents progression to microalbuminuria (DCCT, n>1400) and effect persists (‘metabolic memory’ – EDIC) </li></ul><ul><li>- antihypertensive medication in patients with micro- (& macro-) albuminuria may delay progressive loss of glomerular filtration </li></ul><ul><li>- association with other disease (e.g. CVD) appreciated, both in diabetics and non-diabetics (e.g. PREVEND) </li></ul><ul><li>In diabetes, microalbuminuria has become established as a marker of potentially treatable disease </li></ul>
    44. 45. Confounding factors <ul><li>Biological variation (day-to-day CV 45%) </li></ul><ul><li>Metabolic control </li></ul><ul><li>Intercurrent illness (e..g. sepsis, post-myocardial infarction, surgery, SIRS) </li></ul><ul><li>Haematuria/menstrual contamination </li></ul><ul><li>Non-diabetic renal disease </li></ul><ul><li>Uncontrolled hypertension </li></ul><ul><li>Strenuous exercise </li></ul><ul><li>Urinary tract infection </li></ul><ul><li>Microalbuminuria should be present in at least two out of three urine samples preferably collected within a 6(1) month period in the absence of ketonuria or infection </li></ul>
    45. 46. Microalbuminuric ranges <ul><li>Overnight AER 20-200 ug/min [seen as ‘gold standard’ method] </li></ul><ul><li>equivalent to: </li></ul><ul><li>30-300 mg/24 h [or 20-200 mg/L (NICE)] </li></ul><ul><li>which if you excrete 10 mmol creatinine/day is equivalent to: </li></ul><ul><li>3.0 – 30 mg/mmol creatinine [or 30-300 ug/mg in US] </li></ul><ul><li>or: </li></ul><ul><li>Males  2.5 mg/mmol, Females  3.5 mg/mmol </li></ul>
    46. 47. Natural history of diabetic renal disease 10-15 y 10-20 y
    47. 48. PREVEND (1) <ul><li>40,000/85,000 residents of Groningen recruited in 1997 </li></ul><ul><li>Urine albumin measured </li></ul><ul><li>Followed for 3 y </li></ul><ul><li>516 deaths </li></ul><ul><li>Mortality and cause of mortality recorded </li></ul>Hillege et al, JIM 2001, Circulation 2002
    48. 49. PREVEND (2) <ul><li>Microalbuminuria present in 7.2% of population </li></ul><ul><li>Independently associated with hypertension, diabetes, CV disease </li></ul><ul><li>After excluding diabetics and hypertensives, microalbuminuria still present in 6.6% of population. </li></ul>
    49. 50. PREVEND (3) <ul><li>Increasing albuminuria associated with increasing CV and, to a lesser extent, non-CV mortality (esp. cancer) </li></ul><ul><li>Albuminuria is a strong predictor of all cause mortality in general popn. </li></ul><ul><li>Risk begins at levels not considered microalbuminuric </li></ul><ul><li>(Similar data from Framingham Offspring Study on non-diabetic, non-HT subjects [Arnlov et al, Circulation 2005]) </li></ul>
    50. 51. PREVEND (4) <ul><li>Microalbuminuria common and associated with CV risk factors and death </li></ul><ul><li>Advocates population screening approach to microalbuminuria detection, but especially hypertensives </li></ul><ul><li>Non-diabetics with microalbuminuria should be on ACEI/ARBs </li></ul><ul><li>Threshold for pathological albuminuria should be revised </li></ul>
    51. 52. Measurement issues
    52. 53. Standardised against albumin Immunoassay, approx £0.40 Albumin Results differ between dyes, often standardised against albumin, mainly sensitive to albumin Mainly colorimetric (e.g. pyrogallol red, coomassie blue, benzethonium chloride), approx £0.10 Lab total protein Semi-quantitative, inconsistency between manufacturers, mainly measure albumin Colorimetric, approx £0.10 Stick tests
    53. 54. Urinary Total Protein UKNEQAS Bayer stix tests: 8% negative, 30% trace, 56% 1+, 6% 2+
    54. 55. Urinary albumin UKNEQAS
    55. 56. Non-immunoreactive albumin (1) <ul><li>HPLC (total albumin) assays suggest large amounts of albumin present in diabetic urine not measured by immunoassays – termed ‘non-immunoreactive’ albumin </li></ul><ul><li>No difference between non-diabetic urines </li></ul>Comper et al AJKD 2003, Osick and Comper, Clin Chem 2005
    56. 57. Non-immunoreactive albumin (2) <ul><li>Albumin has similar Mr to normal albumin </li></ul><ul><li>May represent </li></ul><ul><li>(a) albumin which has undergone minimal tubular processing (‘scissions’ with # held together by S-S bonds) or </li></ul><ul><li>(b) filtered forms of albumin not recognised by immunoassay (e.g. FA binding induces conformational change) </li></ul><ul><li>These processes may be affected by diabetes </li></ul>2. Purified NIA from urine 3. Purified NIA under reducing conditions 1. Diabetic urine
    57. 58. Non-immunoreactive albumin (3) <ul><li>Retrospective analysis of 15 y of stored urine samples from diabetics with progressive (n=41) or non-progressive (n=50) kidney disease </li></ul><ul><li>HPLC predicted onset of diabetic nephropathy 2-4 y earlier than immunoassay </li></ul><ul><li>But: </li></ul><ul><li>Only this group publishing in this area </li></ul><ul><li>Assay being marketed by the authors </li></ul><ul><li>When is albumin not albumin? </li></ul>Comper et al KI 2004
    58. 59. ACR to replace PCR? Nephrology literature based on PCR Consistent with International practice (KDIGO/KDOQI) No IRP Single protein (know what you are measuring and how it is calibrated) More expensive? Consistency with diabetic nephropathy literature Improved precision More sensitive than nephrologists want? More sensitive – essential to identify all CKD stage 1 and 2 patients KDIGO define kidney damage = ACR >30 mg/g (approx 3.5 mg/mmol) Disadvantages Advantages
    59. 60. Functional ‘tubular’ proteinuria <ul><li>Small Mr proteins filtered at glomerulus and reabsorbed in proximal tubule </li></ul><ul><li>Appearance of proteins in urine implies tubular damage </li></ul><ul><li>E.g. urinary alpha-1 microglobulin, beta-2 microglobulin, retinol binding protein </li></ul>
    60. 61. Tubular proteinuria due to cell damage <ul><li>Certain proteins present at high concentration in tubular cells </li></ul><ul><li>E.g. Tamm Horsfall glycoprotein and N-acetyl-B-D-glucosaminidase (NAG) </li></ul><ul><li>Appearance of these proteins in urine implies tubular damage </li></ul><ul><li>Useful in drug toxicity studies or occupational monitoring (e.g. heavy metal workers) </li></ul>
    61. 62. Overflow proteinuria <ul><li>Excess formation of a low molecular weight protein that is freely filtered </li></ul><ul><li>E.g. BJP, myoglobin </li></ul><ul><li>Such proteins may be directly toxic to the tubular cells </li></ul>
    62. 63. Summary <ul><li>Kidney disease is common </li></ul><ul><li>Perhaps more than any other disease state, its assessment relies on quantitative laboratory tests </li></ul><ul><li>… and renal units rely on the laboratory </li></ul><ul><li>There are a range of tests available to assess the functions of the kidney - the most important of these are GFR and proteinuria </li></ul><ul><li>No current tests are perfect for assessing GFR and there is inconsistency in approach to proteinuria </li></ul>
    63. 64. END
    64. 65. Major filtration barrier now widely believed to be the filtration slits (*) between inter-digitating foot processes ( ↓) Mutations of nephrin and podocin, which co-localise at the slit diaphragm (and several other protein mutations) associated with congenital types of nephrotic syndrome (e.g. Finnish-type congenital NS) Mathieson, Clin Sci 2004;107:533-8

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