Mechanisms of intervention to reduce proteinuria & Biomarkers: beyond proteinuria

1. Mechanisms of intervention to reduce proteinuria&Biomarkers: beyond proteinuria Jeffrey Kopp, MD Kidney Disease Section NIDDK, NIH

2. Possible mechanisms of proteinuria reduction • Reduction in glomerular capillary hydrostatic pressure • Restoring glomerular filtration barrier - Cytoprotection: podocyte, endothelium - Restoration of glomerular basement membrane pore size distribution • Restoring proximal tubule protein reabsorption: cytoprotection

3. Hydrostatic mechanisms Reducing efferent arteriolar tone - ACEI, ARB Treating systemic hypertension - all agents

4. Podocyte injury Mitochondrial dysfunction • Loss of filtration slits and slit diaphragms • - Mutations • Transcription • - ER processing • Signaling • Actin cytoskeleton Detachment, loss of adhesion Apoptosis Loss of anionic charge: podocalyxin (glucose) Replenishment failure (?) Dysregulation (collapsing glomerulopathy) IC, C5b-9

5. Protecting and restoring podocyte phenotype Preventing IC deposition • Glucocorticoids • Transcription • Actin stabilization • Ransom KI 2005 • Anti-apoptotic • Wada JASN 2005 • Transport from ER • Fuji KI 2006 Mizoribine - Transport from ER via energetics Nakajo JASN 2007 • Retinoids • reverse FPE • nephrin, podocin • Vaughan KI 2005 Cyclosporine

6. Glomerular basement membrane • Collagen IV • Mutations • - Isoform shift •  synthesis by glucose, Ang 2 • degradation • Loss of heparan sulfate (?) and HSPG agrin: • production, •  degradation • Glucose, Ang2 Jefferson, KI 2008

7. Endothelium Haraldsson, Physiol Rev 2008

8. Injury to endothelial cell and endothelial surface layer Hyperglycemia, AGE Free fatty acids ROS, oxidative stress, mitochondrial dysfunction Proinflammatory cytokines (TNF) Adiponectin VEGF antagonism Haraldsson, Physiol Rev 2008 Rask- Madsen, Nature Clin Pract 2007

9. Pima diabetics: Macroproteinuria but not microproteinuria is associated with shunt Macro Micro • Shunt magnitude correlates with FPE Lemley, JASN 2000

10. Proximal tubule albumin reabsorption Birn, KI 2006

11. Impaired albumin reabsorption by proximal tubule in PAN nephrosis 0 40 s 14 min CON PAN Russo, KI 2007

12. Gene therapy reduces tubulointerstitial injury in rat overload proteinuria model MCP-1 antagonist (7ND) IB Shimizu, JASN 2003 Takase, KI 2005

13. Does macroalbuminuria cause tubulointerstitial damage? Pro • Overload albuminuria models • Exposure to albumin (or cytokines of FA on albumin) induces RANTES, MCP-1, IL8, fractalkine, TNF-, ET, TGF-; alters integrins, may induce apoptosis • Other proteins: iron carriers, complement, Ig, growth factors • Gene therapy to PTC (MCP-1 reduction, IB) protects Con • Minimal change nephropathy: proteinuria for years without progression • Role of selectivity

14. B B S I Biomarkers • Biomarkers: measures that predict clinical outcome NIH biomarker working group: “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses” • Clinical end point: a variable that reflects how a patient feels or functions or how long a patient survives • Surrogate end point: a biomarker that can substitute for an observed clinically meaningful end point • Intermediate end point: a characteristic that is intermediate in the causal pathway between an intervention and the clinical endpoint Clinical end point Treatment Stevens, CJASN 2006

15. Biomarkers in drug development and use • Pre-clinical/animal Clinical studies: identify pathways Animal studies: screening for leads, rank candidates • Clinical studies Identify pathways Early detection Differential diagnosis, identify subpopulations Prognosis • Surrogate end point for trials Assess drug effect, dose-ranging, more efficient trial design • Clinical therapy: drug dosing Hewitt, JASN 2004

16. Biomarkers and CKD • Increased interest, increased funding • Needed: more systematic searches, validation in prospective observational studies (CRIC, CKID) and interventional trials

17. Biomarkers can address different issues across the course of disease Hewitt, JASN 2004

18. Biomarker discovery approaches SELDI-TOF MALDI-TOF 2D gel

19. Two biomarkers are better than one Hewitt, JASN 2004

20. Cystatin C • Cystatin C: 13.3 kDa, product of all nucleated cells, freely filtered and readily reabsorbed • May have advantages over serum creatinine (MDRD eGFR) in monitoring GFR over time: vs iothalamate r=0.77, 0.31) (Perkins JASN 2005)

21. Podocyturia • Evidence that podocyte depletion characterizes most progressive CKD • Direct counting of urinary podocytes is impractical • Enumeration with FACS has proven difficult • Podocyte proteins: total, exosomes Kuusniemi, KI

22. Podocyturia correlates more closely than proteinuria with disease activity in animal models PAN Thy-1 5/6 Nx Yu JASN 2005

23. Diabetic nephropathy: Nephrinuria • Increased urine nephrin in diabetes, but unrelated to proteinuria Men Women Pätäri, Diabetes 2003

24. Lupus nephritis: urinary cytokines Li Autoimmunity Rev 2006

25. Treatment reduces urinary TGF- in diabetic nephropathy ACEI + ARB Ruboxistaurin Gilbert Diabetes Care 2007 Song NDT 2006

26. Normal FSGS WT-1 WT-1 Female 6.1 8.4 10.9 15.0 g/day proteinuria Normal FSGS Male 2.2 2.8 4.0 6.4 8.4 20 g/day proteinuria Urinary exosomes • Derived from podocytes, RTEC, and lower tract cells • Sample various cellular compartments, including nucleus Zhou KI in press

27. Conclusions • Diverse mechanisms of proteinuria and of proteinuria reduction • Non-albumin protein biomarkers are not yet validated surrogates, demonstrated to lie within the causal pathway to CKD across multiple diseases and multiple interventions Strength of association Glomerular microalbuminuria diabetic vs metabolic Glomerular hypertension Endothelial injury Glomerular macroalbuminuria GBM abnormalities CKD progression Podocyte injury Tubular microalbuminuria Proximal tubule dysfunction Tubular macroproteinnuria