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
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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