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

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

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

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

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

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

Endothelium Haraldsson, Physiol Rev 2008

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

Pima diabetics: Macroproteinuria but not microproteinuria is associated with shunt Lemley, JASN 2000 MacroMicro  Shunt magnitude correlates with FPE

Proximal tubule albumin reabsorption Birn, KI 2006

Impaired albumin reabsorption by proximal tubule in PAN nephrosis CON Russo, KI 2007 PAN 040 s14 min

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

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

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 Stevens, CJASN 2006 Treatment Clinical end point B BSI

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

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

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

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

Two biomarkers are better than one Hewitt, JASN 2004

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)

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

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

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

Lupus nephritis: urinary cytokines Li Autoimmunity Rev 2006

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

Urinary exosomes  Derived from podocytes, RTEC, and lower tract cells  Sample various cellular compartments, including nucleus Zhou KI in press g/day proteinuria Female Normal FSGS WT g/day proteinuria Male Normal FSGS WT-1

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 CKD progression Glomerular microalbuminuria diabetic vs metabolic Glomerular macroalbuminuria Tubular microalbuminuria Glomerular hypertension Endothelial injury Podocyte injury GBM abnormalities Proximal tubule dysfunction Tubular macroproteinnuria Strength of association