Theme C lecture 5

Question 1

What are the methods used to examine kidney development?

Answer 1

organ culture.

Genetically modified animals (KO or Knock in mice)

Gene microarray or RNA sequencing (compare gene expression at different levels of development)

Molecular regulation of metanephric development

Question 2

What are the methods by which organ cultures can be used to examine kidney development?

Answer 2

Whole metanephroi from rats and mice are grown in vitro from E13.5/11.5 respectively.

Addition of growth factors to media to examine effect of kidney differentiation.

Glomeruli can be visualised using immunohistochemistry (WT-1 which stains podocytes or PNA)

Ureteric tree can be visualised by staining with calbindin-28 or cytokeratin

Question 3

What staining can be used to visualise ureteric branching?

Answer 3

calbindin-28 or cytokeratin

Question 4

What are BMPs?

Answer 4

BMPs are important regulators of kidney development and function.

Question 5

What does dexamethosone (DEX) do to kidney growth?

Answer 5

It inhibits branching morphogenesis (growth)

Question 6

What is dexamethosone?

Answer 6

A type of glucocorticoid

Question 7

What happens to renal development in the presence of a glucocorticoid vehicle or corticosterone?

Answer 7

vehicle and corticosterone can inhibit renal branching and development.

Question 8

What do glucocorticoids do during development of the kidneys in general?

Answer 8

Many factors are regulated during kidney development by glucocorticoids.

Question 9

What are the pros and cons of using genetically modified animals for experiment?

Answer 9

Pros:
Large sample size can be used.

Knock out/knock in allow identification of the role of a gene in vivo.

Many technologies available

Cons:
Difficult to eliminate effect of other genes that are upregulated or downregulated as a result.

Question 10

What are some specific gene families and systems that are important in kidney development?

Answer 10

TGF-B

GDNF (glial cell line-derived neurotrophic factor)

BMP

WnT

Renin-Angiotensin system

Question 11

Where is it expressed? What is the function of GDNF?

Answer 11

Expressed in intermediate mesoderm (then wolfiann duct) and acts on metanpehric mesenchyme to signal invasion of ureteric bud into metanephric mesenchyme.

Question 12

What receptors does GDNF act on? Where are these receptors located?

Answer 12

c-RET tyrosine kinase receptor and the GPI-linked co-receptor (GFRalpha)

In the wolfiann duct

Question 13

What happens when GDNF is knocked out?

Answer 13

When GDNF is completely knocked out there is no production of kidneys.

When only one allele is knocked out there is 30% reduction in nephrons

Question 14

What do external factors do to branching conducted by GDNF?

Answer 14

They inhibit GDNF and inhibit branching.

Question 15

Name some external factors that act on the ureteric branching conducted by GNDF?

Answer 15

Robo2

Slit2

BMP-4

Question 16

What do internal factors do to branching conducted by GDNF?

Answer 16

It stimulates branching

Question 17

What are BMPs?

Answer 17

BMPs are members of the transforming growth factor B (TGF-B) family.

They are secreted proteins.

BMP-4 is important for growth of UB and branching morphogenesis (negative regulation)

Question 18

Which receptors do BMPs act on?

Answer 18

BMPs act on activin like kinase (ALK) cell surface receptors.

Question 19

What is the “big picture” behind GDNF expression and reciprocal action from the metanephric mesenchyme?

Answer 19

GDNF is being expressed within mesenchyme acting on cRET within ureteric tip which is met by reciprocal action from utreteric tip.

GDNF signalling from metanephric mesenchyme as well as gremlin regulating its expression.

Wherever you have GDNF signalling you have a branch point forming.

Question 20

Where are Wnt11 and Wnt4 expressed?

Answer 20

Wnt11 is expressed at the tip of the ureteric bud. Wnt4 is expressed in the pretubular agreggate. These hormones interact

Question 21

What happens when you knock out Wnt genes?

Answer 21

reduction in the kidney’s size.

Question 22

What happens when TGF-b2 is knocked out?

Answer 22

Dilate renal pelvis and hypoplastic kidneys.

Question 23

What happens when Wnt11 is knocked out?

Answer 23

There is a 30% nephron deficit.

Question 24

What is the result of Agtr1 receptor being knocked out?

Answer 24

vascular thickening and an abnormal medulla

Question 25

What is the result of Agtr2 receptor being knocked out?

Answer 25

CAKUT

Question 26

What is the normal function of Agtr receptors?

Answer 26

regulates renal development in utero and causes additional consequences in renal function during adult life when knocked out.

Question 27

What happens when Odd-1 is knocked out?

Answer 27

Renal agenesis due to lack of metanephric mesenchyme formation.

Question 28

What happens when you knock out Slit2?

Answer 28

Multiple collecting ducts form

Question 29

What are some perturbations that cause changes to renal RAS expression?

Answer 29

low protein in diet

nutrient restriction

maternal hypoxia

uteroplacental insufficiency

corticosterone.

Question 30

How does a decrease in nephron number result in a higher blood pressure?

Answer 30

glomeruli regulate systemic homeostasis (BP) decrease in nephron number increases blood pressure

Question 31

How may the kidneys be involved in programming?

Answer 31

Nephron number

Regulation of fluid/Na homeostasis

Impaired regulation of RAS

Question 32

How does regulation of fluid/Na homeostasis change in people with fetal programming to have higher blood pressures?

Answer 32

Shift occurs in renal-pressure natriuresis to higher blood pressures

Question 33

How does impaired regulation of RAS affect fetal programming?

Answer 33

programmed increase in renin activity can have hypertensive effects on multiple organs

Question 34

How does a low nephron number cause further decrease in nephron number throughout life? (Brenner hypothesis)

Answer 34

if you’re born with a low nephron number there is a lower surface area of filtration in the kidneys which decreases filtered load and so there is more sodium and fluid being retained and ECF volume is larger and a higher arterial pressure followed by increased glomerular capillary pressure.

Increased glomerular pressure results in increased nephron GFR and so glomerulus hypertrophies until a stage where it dies and this further decreases the number of nephrons.

Question 35

What is the effect that dexamethosone had on sheep?

Answer 35

dexamethosone caused decrease in nephron number at birth and a further increase in blood pressure with aging

Question 36

Do all individuals with a low nephron number develop hypertension?

Answer 36

No, Not all individuals with low nephron number develop hypertension.

Question 37

What are the changes observed in renal development in response to glucocorticoid treatment?

Answer 37

Glucocorticoids dysregulated lots of factors affecting renal morphogenesis. This inludes a reduction in nephron number. This effect is often seen in males but not females. Males develop albuminuria whereas females do not. More hyperactive RAS system in males with glucocorticoid receptors.

Question 38

What do glucocorticoids do to the body?

Answer 38

glucocorticoids cause altered adrenal development, altered cardiac function

Question 39

What do sex specific adaptations tell us about fetal programming for cardiovascular disease?

Answer 39

Sex specific adaptations to maternal insult may help understand mechanisms leading to disease. These sex specific outcomes are likely due to problems with the placenta.

Question 40

What do studies suggest about the systemic nature of CV disease?

Answer 40

problems with cardiovascular system often occur to the system as a whole rather than individual organs and should be observed as a whole.