How to make a kidney

How to make a kidney Winyardmiles begins A journey ofPaul a thousand with a single step Nephro-Urology Unit UCL Institute of Child Health Chinese phil...
Author: Alexander Casey
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How to make a kidney Winyardmiles begins A journey ofPaul a thousand with a single step Nephro-Urology Unit UCL Institute of Child Health Chinese philosopher Laozi (604bc - 531bc) Tao Te Ching, chapter 100.

Why is kidney disease / development important? • 47,000 patients with chronic kidney disease in the UK • In the UK, £3billion of the whole NHS budget is used to treat patients with kidney disease; $1 trillion for ESRD in USA over next decade • 800+ children have end-stage renal disease, half of which are caused by congenital malformations • Support rather than cure, eventually progressing to dialysis or transplantation • Urgent need to discover new treatments

Kidney development

Human kidney development 5

6

mb 8

9

com lm

u u

cm

u lm

S

u

g

Dysplasia 5 weeks 6 8 9

Key events in nephrogenesis • Outgrowth of the ureteric bud • Contacts between the bud and mesenchyme, which cause the bud to branch and mesenchyme to condense • Mesenchyme-to-epithelial conversion • Differentiation of segment specific cells • Elongation and growth

The first step – ureteric bud ougtrowth No budding

Normal

Mutations in: GDNF, RET, GFRa Gremlin Pax2, EYA1 Six 1 Hox11 paralogues

Extra buds

Mutations in: Spry2, BMP4 Robo2, Slit2 Foxc1/c2 Adapted from Harshman & Brophy, 2012

Double knock out: 2 wrongs do make a right!

Potential causes of kidney malformations 1. Gene dysregulation » Mutations » Epigenetics

2. Obstruction of urinary flow 3. Teratogens

Model of dysplasia

Genetic factors Impaired External factors: urinary flow, teratogens, obstruction maternal diet

Epithelium

X

Abnormal branching / primitive ducts

PAX2

Cysts

Aberrant collecting system

Mesenchyme

Primitive nephrons

TGFβ1 Increased stroma

Formation of Failed nephron metaplastic cartilage differentiation

Nephron deficit

PAX2 Linked to proliferation and survival during development of many organ systems Gene dosage variation causes abnormal kidneys null: complete absence reduced: hypoplastic kidneys overexpression: cystic kidneys

PAX2 mutations: human renal-coloboma syndrome

PAX2 expression u

co

co cy v

Developing kidney

Dysplastic kidney

Abnormal smooth muscle

TGFb1 Expressed in murine nephrogenesis Affects epithelial cells in culture • reduces proliferation • promotes mesenchyme

Upregulated by urinary tract obstruction • (mature human kidneys) • animal models of dysplasia

TGFb1 expression

Developing kidney

Dysplastic kidney

Experimental model Background:

metanephros arises at 30 days ovine gestation is 145 days

Complete unilateral fetal ureteric obstruction at 90 days, when a few layers of glomeruli have formed. Kidneys examined 10 days later Our previous studies demonstrated: severe disruption of nephrogenesis dysregulation of proliferation and apoptosis increased expression of PAX2, a-SMA

Increased smooth muscle Sha m

Obst

m

u

g

p

m

g

Sham

Obst

g

v g

v

g g

Obstruction disrupts nephrogenesis

PAX2 TGFb1 Deregulated cell survival Aberrant differentiation Normal

‘Dysplastic’

PAX2 and TGFb1 in dysplasia Initiating factor (obstruction etc)

Epithelial PAX2 upregulation

TGFb1 upregulation

Epithelial growth

Epithelial to mesenchymal transformation

Epithelial cyst formation

"Loss" of potential renal epithelia

Aim The ultimate aim is to generate new nephrons or increase repair/regeneration capacity of existing structures

Objectives • Investigate human kidney cells • Get them to differentiate • Generate neo-kidneys

Mesenchymal cells isolated from normal embryonic kidneys (and postnatal dysplastic)

Romio L et al., J Am Soc Nephrol. 2003;14(3):680-9.

Price KL et al., Physiol Genomics. 2007 28(2):193-202

Cell lines • Human Developmental Biology Resource • Four normal human embryonic metanephroi N1 – 70d gestation N2 – 73d gestation N3 – 75d gestation N4 – 84d gestation • Two postnatal dysplastic kidneys D1 & D2

All have a mesenchymal appearance N1 N2 N3 N4 D1 D2

Mesenchymal to epithelial conversion in rat metanephros is induced by LIF Barasch, Yang, ……Aranoff, Oliver. Cell. 1999, 99:377-86.

TGFb2, LIF and FGF2 cooperate to induce nephrogenesis Plisov, Yoshino, ……Rubin, Perantoni. Development. 2001, 128:1045-57.

LIF system in human kidneys A

ub

ub

B

cm

v s LIF

LIFR

C

D

gp130

negative

Changes:but a load balls Changes not of epithelialisation A

B

C

D

LIF treatment – increased organisation? A

B

0 hr cont

0 hr LIF

C

D

72 hr cont

72 hr LIF

Effect of lithium on genes associated with mesenchyme-to-epithelial differentiation E-cadherin

*

Aminopeptidase A

*

Wnt4

*

Wnt9b

* 0.0

0.5

1.0

1.5

2.0

2.5

3.0

Gene expression (fold-change compared to control) * indicates p