Theme C lecture 5 Flashcards
What are the methods used to examine kidney development?
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
What are the methods by which organ cultures can be used to examine kidney development?
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
What staining can be used to visualise ureteric branching?
calbindin-28 or cytokeratin
What are BMPs?
BMPs are important regulators of kidney development and function.
What does dexamethosone (DEX) do to kidney growth?
It inhibits branching morphogenesis (growth)
What is dexamethosone?
A type of glucocorticoid
What happens to renal development in the presence of a glucocorticoid vehicle or corticosterone?
vehicle and corticosterone can inhibit renal branching and development.
What do glucocorticoids do during development of the kidneys in general?
Many factors are regulated during kidney development by glucocorticoids.
What are the pros and cons of using genetically modified animals for experiment?
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.
What are some specific gene families and systems that are important in kidney development?
TGF-B
GDNF (glial cell line-derived neurotrophic factor)
BMP
WnT
Renin-Angiotensin system
Where is it expressed? What is the function of GDNF?
Expressed in intermediate mesoderm (then wolfiann duct) and acts on metanpehric mesenchyme to signal invasion of ureteric bud into metanephric mesenchyme.
What receptors does GDNF act on? Where are these receptors located?
c-RET tyrosine kinase receptor and the GPI-linked co-receptor (GFRalpha)
In the wolfiann duct
What happens when GDNF is knocked out?
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
What do external factors do to branching conducted by GDNF?
They inhibit GDNF and inhibit branching.
Name some external factors that act on the ureteric branching conducted by GNDF?
Robo2
Slit2
BMP-4
What do internal factors do to branching conducted by GDNF?
It stimulates branching
What are BMPs?
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)
Which receptors do BMPs act on?
BMPs act on activin like kinase (ALK) cell surface receptors.
What is the “big picture” behind GDNF expression and reciprocal action from the metanephric mesenchyme?
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.
Where are Wnt11 and Wnt4 expressed?
Wnt11 is expressed at the tip of the ureteric bud. Wnt4 is expressed in the pretubular agreggate. These hormones interact
What happens when you knock out Wnt genes?
reduction in the kidney’s size.
What happens when TGF-b2 is knocked out?
Dilate renal pelvis and hypoplastic kidneys.
What happens when Wnt11 is knocked out?
There is a 30% nephron deficit.
What is the result of Agtr1 receptor being knocked out?
vascular thickening and an abnormal medulla
What is the result of Agtr2 receptor being knocked out?
CAKUT
What is the normal function of Agtr receptors?
regulates renal development in utero and causes additional consequences in renal function during adult life when knocked out.
What happens when Odd-1 is knocked out?
Renal agenesis due to lack of metanephric mesenchyme formation.
What happens when you knock out Slit2?
Multiple collecting ducts form
What are some perturbations that cause changes to renal RAS expression?
low protein in diet
nutrient restriction
maternal hypoxia
uteroplacental insufficiency
corticosterone.
How does a decrease in nephron number result in a higher blood pressure?
glomeruli regulate systemic homeostasis (BP) decrease in nephron number increases blood pressure
How may the kidneys be involved in programming?
Nephron number
Regulation of fluid/Na homeostasis
Impaired regulation of RAS
How does regulation of fluid/Na homeostasis change in people with fetal programming to have higher blood pressures?
Shift occurs in renal-pressure natriuresis to higher blood pressures
How does impaired regulation of RAS affect fetal programming?
programmed increase in renin activity can have hypertensive effects on multiple organs
How does a low nephron number cause further decrease in nephron number throughout life? (Brenner hypothesis)
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.
What is the effect that dexamethosone had on sheep?
dexamethosone caused decrease in nephron number at birth and a further increase in blood pressure with aging
Do all individuals with a low nephron number develop hypertension?
No, Not all individuals with low nephron number develop hypertension.
What are the changes observed in renal development in response to glucocorticoid treatment?
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.
What do glucocorticoids do to the body?
glucocorticoids cause altered adrenal development, altered cardiac function
What do sex specific adaptations tell us about fetal programming for cardiovascular disease?
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.
What do studies suggest about the systemic nature of CV disease?
problems with cardiovascular system often occur to the system as a whole rather than individual organs and should be observed as a whole.
