Mechanisms of intervention to reduce proteinuria

&Biomarkers: beyond proteinuria

Jeffrey Kopp, MDKidney 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 stabilizationRansom KI 2005

- Anti-apoptoticWada JASN 2005

- Transport from ERFuji KI 2006

Retinoids-reverse FPE

- nephrin, podocinVaughan 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 2008Rask- 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

Macro Micro

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

0 40 s 14 min

Gene therapy reduces tubulointerstitial injury in rat overload proteinuria model

IB

Takase, KI 2005Shimizu, 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, IB) 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

TreatmentClinical end

point

B

B SI

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

CKD and biomarkers

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 20070

250

500

750

1000

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)

Cystatin and kidney function

1998 1999 2000 2001 2002 2003 2004 2005 2006 20070

50

100

150

200

250

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 2007Song NDT 2006

ACEI + ARB

Urinary exosomes

Derived from podocytes, RTEC, and lower tract cells

Sample various cellular compartments, including nucleus

Zhou KI in press

6.1 8.4 10.9 15.0 g/day proteinuria

Female

Normal FSGS

WT-1

2.2 2.8 4.0 6.4 8.4 20 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