Building a Bioartificial Kidney: From Silicon Chips to .... Paul Brakeman.pdf · Paul Brakeman, MD, PhD ... USRDS Annual Report, 2009 Survival Probability ... Urea Urea Waste H 2O

Building a Bioartificial Kidney: From Silicon Chips to

Renal Clearance

Paul Brakeman, MD, PhDMedical Director, Pediatric Dialysis Unit

Assistant ProfessorDepartment of Pediatrics

UCSF

Currently There Are > 540,000 Patients with End Stage Renal Disease in the United States

USRDS Annual Report, 2009

Surv

ival

Pro

babi

lity

Months



Surv

ival

Pro

babi

lity

> 350,000 patients receive dialysis

Months



Surv

ival

Pro

babi

lity


> 70,000 patients waiting for a renal transplant

Months



Surv

ival

Pro

babi

lity


> 70,000 patients waiting for a renal transplant

Only 18,000 transplants per yearMonths

Strategies to Increase Numbers of Available Organs

• Publicity campaigns to increase organ donation• Extended criteria for identifying usable organs (Audard Trans Int 2007)

• Desensitized patients so they are more likely to be able to find a compatible kidney (Montgomery NEJM 2011)

Strategies to Increase Numbers of Available Organs

• Publicity campaigns to increase organ donation• Extended criteria for identifying usable organs (Audard Trans Int 2007)

• Desensitized patients so they are more likely to be able to find a compatible kidney (Montgomery NEJM 2011)

• Engineer replacement organs

Strategies for Engineering Replacement Organs

• Generate humanized animals and use them as a source for organs

• Grow an organ using stem cells• Use human kidney cells to reconstitute renal tissue

Strategies for Engineering Replacement Organs

• Generate humanized animals and use them as a source for organs

• Grow an organ using stem cells• Use human kidney cells to reconstitute renal tissue • Engineer a bioartificial organ using novel technology for hemofiltration and renal cells to provide cellular function

The Bioartificial Kidney Project


Frank Partridge 2009 Johnson Research


The Renal Filter Unit: the Nephron

Peritubular Capillary

ProximalTubule

Loop of Henle

DistalTubule

CollectingDuct

Glomerulus


ProximalTubule

Loop of Henle

DistalTubule

CollectingDuct

Glomerulus~500,000-1,000,000 per kidneyGenerate ~150L of filtrate per day



Loop of Henle

DistalTubule

CollectingDuct

GlomerulusProximal TubuleSelectively reabsorbs ~80% of most solutesReabsorbs ~80% of filtered water1,25-(OH)2-Vit D3 activation



ProximalTubule

Loop of Henle

Distal TubuleFine tunes solute excretionFurther water excretion

CollectingDuct

Glomerulus


H2O

Schematic of the Bioartificial Kidney

Blood

Filtrate

H2O Na+

Na+

Urea

Urea

Waste

H2O Na+ Urea

H2O Na+ Urea

H2O

Blood H2O Na+

Na+ Urea

H2O Na+

H2O Na+ Urea

H2O

Blood

Filtrate

H2O Na+

Na+

Urea

Urea

WasteHemofilter

H2O Na+ Urea

H2O Na+ Urea

H2O

Blood H2O Na+

Na+ Urea

H2O Na+

H2O Na+ Urea


H2O

Blood

Filtrate

H2O Na+

Na+

Urea

Urea

Waste

H2O Na+ Urea

H2O Na+ Urea

H2O

Blood H2O Na+

Na+ Urea

H2O Na+

H2O Na+ Urea

o Based on hemofiltration to eliminate need for dialysateo Implantable to allow for continuous blood cleansing o Incorporate of renal cells to reabsorb solutes and exclude toxinso Renal cells provide some metabolic and hormonal functions

Bioreactor - Human renal tubular cells


Innovations Required for Implantation of the RAD

Humes et al., Univ. of Michigan

Innovations Required for Implantation of the RAD

Humes et al., Univ. of Michigan

o Elimination of high pressure pumps

o Miniaturization of the hemofilter

o Better biocompatibility of the filter and blood path

o Intrinsic feedback to monitor patient‘s homeostasis

o Stable function for months to years

o Adequate reabsorptionof salt and water

• Targets– Hemofiltration‐

• 30 liters per day of filtrate produced

Design Targets for the Bioartificial Kidney


• 30 liters per day of filtrate produced – why?


Improvement in Survival with Intensive Daily Dialysis

Johansen K, Kidney Int, 2009

Conventional hemodialysis

Nocturnal hemodialysis


• 30 liters per day of filtrate produced – how?


Improved Membrane Technology is Critical for Miniaturizing the Bioartificial Kidney

Standard dialysis membrane Silicon nanopore membrane

William Fissell and Shuvo Roy, J Memb Sci, 2010

• Very consistent pore sizes create highly selective pores and allow for exclusion of antibodies and medium to large size proteins for better biocompatibility

• High hydraulic permeability• Smooth surface allows for coating

the membranes to allow for low bioreactivity

Silicon nanopore membrane after 48 hours of in vitroexposure to whole blood

Fissell, J Memb Sci, 2010


• Very consistent pore sizes create highly selective pores and allow for exclusion of antibodies and medium to large size proteins for better biocompatibility

• High hydraulic permeability• Smooth surface allows for coating

the membranes to allow for low bioreactivity

Silicon nanopore membrane after 48 hours of in vitroexposure to whole blood

Fissell, J Memb Sci, 2010


3‐Dimensional CAD Rendering of the Bioartificial Kidney

Hemofilterblood in blood out

ultrafiltrate

Membrane chips bondedback-to-back

}

ultrafiltrateblood

membranesilicon substratebonding layer

Bioreactor Current hydraulic

permeability of the silicon membrane is 10 l/cm2/min/PSI.

At this porosity the hemofilter requires 380 cm2 of filter area to generate 30L per day of filtrate – About the size of two decks of cards

Ex‐vivo Hemofilter Design Testing

Computer Assisted Design

ManufacturingPrototype

TestingPrototype


• 30 liters per day of filtrate produced• Biocompatibility of the filter and blood path

– Bioreactor‐• Barrier function to prevent reabsorption of toxins• 25 liters of water reabsorption• 3500 mM of sodium reabsorption• Metabolic function


Properties of an Ideal Renal Cell for the Bioreactor

o Barrier to reabsorption of toxinso Adequate reabsorption of sodium, potassium,

phosphorus and watero Provides metabolic function including 1,25 OH

Vitamin D productiono Stable for 3-4 monthso Non-immunogenic

Analysis of Cells in a LaminarFlow Bioreactor

Standard Tissue Culture Tissue Culture Under Flow Conditions

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

OK LLC-PK1

CreatinineRetention

Urea Retention

Barrier Function of Commercially Available Proximal Tubule Cell Lines

n = 6 n = 6 n = 6 n = 6

Water Transport of Proximal Tubule Cell Lines

Tran

sport R

ate L

/cm

2 /day

Ferrell et al. 2011, submitted

0

10

20

30

40

50

60

OK LLC-PK1 HCTC

n = 4 n = 4

Immortalized Human Proximal Tubule Cells ‐ RPTEC/TERT1 Cells

Primary human proximal tubule cells were transfected with human telomerase and clones were selected and characterized

Green = Acetylated Tubulin Green = -catenin

Wieser, Grillari, AJPR, 2008

Barrier Function of RPTEC/TERT1 Cells

n = 6 n = 6 n = 6 n = 697.0%

97.5%

98.0%

98.5%

99.0%

One Month Two Months Three Months

Perc

ent r

etai

ned

crea

tinin

e

Salt Reabsorption of RPTEC/TERT1 CellsTran

sport R

ate

mEq

/cm

2 /day

n = 6 n = 6

20181614121086420

Salt Reabsorption of RPTEC/TERT1 Cells

n = 6

25 kD

20 kD

-tubulin

ZO-1

10uM

Water Transport of Other Immortalized Human Renal Proximal Tubule Cells

n = 4 n = 4

Sanechika, NDT, 2011

Water Transport of Other Immortalized Human Renal Proximal Tubule Cells

n = 4 n = 4

** Actual reabsorption 17 uL/cm2/day

Sanechika, NDT, 2011

Salt and Water Transport in Renal Proximal Tubule Cells

Bioartificial Kidney Project

Silicon Nanofabrication Cell Engineering

UCSF Bioartificial Kidney Team

• UCSF Team memberso Brakeman Lab

o Chao‐Zong Leeo Natalie Spivako Daniel Kaplan

o Shuvo Roy Labo Rishi Kanto Alex Hellero Peter Solero Torin Yeager

o Mark Wilson Labo Steven Hettso Loi Doo Mathem Saeedo Jeremy Durack

Bioartificial Kidney

• Non‐UCSF Team memberso William Fissell Lab (Vanderbilt School of Medicine)

o Nicholas Ferrell, PhDo David Humes, MD (University of Michigan)

Project Team

Bioartificial Kidney Project

Silicon Nanofabrication Cell Engineering

Documents

Building a Bioartificial Kidney: From Silicon Chips to .... Paul Brakeman.pdf · Paul Brakeman, MD, PhD ... USRDS Annual Report, 2009 Survival Probability ... Urea Urea Waste H 2O