Renal Physiology and pH Flashcards
Give the equation for filtration rate:
Filtration rate = filtration pressure x SA x hydraulic permeability
How would you measure GFR? Why use inulin?
- Infuse inulin at a steady rate until [arterial] is constant
- GFR = rate of infusion/plasma concentration (effectively measures efficiency of filtration)
- Use inulin as: freely filtered, not synthesised or metabolised, non-toxic, does not alter renal function.
- Can also use creatine (released from muscles) using difference between blood and urine concentration.
What is clearance? How would you calculate it?
The volume of plasma that would have to be fully cleared of substance to give the excretory rate
- I.e. high clearance means removed quickly by kidneys
Equation: Clearance of X = ѵ (urine flow rate) x [X]u/[X]a in ml/min
How could you measure total bodily fluid volume?
Single Injection method:
- Injection into compartment and concentration measured at intervals
- Liner decay (on log graph) means concentration can be extrapolated back
- Using Evan’s blue dye
Constant perfusion method:
- Priming injection given then infused at a constant rate
- Concentration of marker = concentration in ECF when perfusion rate = excretion rate
Suggest markers used to measure bodily fluid volumes and why:
Requirements:
- Non-toxic
- Not metabolised or produced by body
- Doesn’t cross into other areas
- Easily measurable
- Distribute evenly
- Volume should be fixed
Examples: inulin, mannitol, thiosulphate, Na+ radioisotopes.
How can renal plasma flow rate be measured?
Using clearance: if completely cleared then clearance = renal flow rate
- Measure para-amino Hippurate (PAH) levels, since filtered and actively secreted
What is effective renal plasma flow?
Takes into account that PAH is filters in peritubular capillaries which get 90% of blood flow so RPF≈ERPF/0.9
Blood flow worked out using Fick’s principle (blood flow worked out using concentration in blood and urine of known marker concentration)
What evidence is there for isosmotic fluid reabsorption?
Simple micropuncture:
- Sampling early and late PCT fluid shows no osmotic change
- Measured using inulin since freely filtered so change to [inulin] proportional to change in volume of fluid
- [Inulin] decreases so volume decreases
Split oil drop experiments:
- Mineral oil injected into Bowman’s capsule to stop PCT flow
- Second injection of test fluid (e.g. isotonic NaCl) splitting oil drop
- With time, drops move closer together = shows decrease
- Test fluid resampled and still isotonic with plasma
What evidence is there for the medullary osmotic gradient?
- A kidney from a dehydrated (urine concentrating) animal is frozen and sectioned
- Solute concentrations estimated from melting points of different regions
- Suggests osmotic pressure constant in the cortex and rises from medullary boundary to inner medulla.
What evidence is there that cell volume changes allow ECF osmolality detection?
Experiments on magnocellular cells in hypothalamus:
- Cell can be placed in hypotonic solution but suction applied intracellularly to decrease pressure and cause cell shrinkage
- Stretch-inactivation of Na+ channels reduced so depolarisation stimulated.
What evidence is there for aldosterone being the major [K+] controller?
- Adrenalectomized dogs are infused with aldosterone at a constant rate
- Can regulate their Na+ concentration when [Na+] in food differed
- Cannot regulate [K+] when altered in food
What are the 4 general functions of the kidneys? (Broad catagories)
- Extracellular fluid regulation (pH, electrolyte balance and osmotic pressure)
- Long-term blood pressure regulation
- Excretion of metabolic waste
- Regulation of erythropoiesis
Describe how ultrafiltration occurs (including anatomy):
- Fluid under pressure is pushed through fenestrations in the capillary, basement membrane and then podocyte foot processes
- Filters sizes between 7,000-70,0000Da
- Filters charge: filters have -ve charge which repels protein (anionic at physiological pH) which retains cations in plasma.
What is a Donnan equilibrium?
- During ultrafiltration, cations are retained since proteins are repelled by -ve filtration barrier
- Retains cations at physiological pH
- Anions are too small to be directly affected by -ve filtration barrier
What is the Van’t Hoff law?
Colloid osmotic pressure is proportional to the concentration of particles.
- Proteins do not obey this law (particularly albumin)
What factors increase GFR? (Think Starling’s equation).
- Kf : increased SA for filtration due to relaxation of mesangial cells
- Pc : increased renal arteriole pressure and decreased afferent arteriole resistance (both increase flow rate)
What factors decrease GFR?
- πc : Increase in colloid osmotic pressure (e.g. starvation) or decreased renal plasma flow
- Pb : increased intratubular pressure (obstruction by kidney stone)
- Pc : increased efferent arteriole resistance (decreased renal blood flow outweighs increased pressure for filtration)
What autoregulation mechanisms does the kidney have to minimise the effect of blood pressure changes on GFR?
Myogenic mechanisms:
- Smooth muscle in arteriole contracts when stretched using non selective cation channels (Na+/Ca2+) causing contraction
Tubuloglomerular feedback:
- Increased GFR increases rate of Na+ and Cl- to macula densa
- Causes ATP release which constricts efferent arteriole
- Adenosine may also result in afferent and mesangial cell control
Renin-angiotensin system: controls ratio of afferent/efferent arteriole constriction.
Why is [Cl-] the controlling variable for GFR feedback to the macula densa?
- Though increased GFR will lead to more Na+ and Cl-, [Cl-] is controlling factor
- Since NKCC2 pumps always saturated with Na+ as high affinity but Cl- transporters not (lower affinity)
- Hence small changes in [Cl-] are more significant.
Which molecules are reabsorbed in the PCT?
PCT for CONSERVATION (reabsorption)
- Glucose
- Amino acids
- Protein
- Hydrogen carbonate
- Secretion of anions using low-specificity pumps (e.g. bile salts, oxalate, aspirin, PAH)
How is glucose reabsorbed in the PCT?
Na+ coupled secondary active transport (all filtered out unless hyperglycaemia occurs leading to glucosuria (=glucose in urine)):
- SGLT-2 transporters (1Na+/1 glucose)
- SGLT-1 transporters (1Na+/2 glucose)
- SGLT-2 density higher at start of PCT; SGLT-1 higher at end
- Na+/K+ pump created Na+ gradient; glucose moves down by facilitated diffusion
How are proteins (and amino acids) reabsorbed in the PCT?
Amino acid reabsorption:
- 5 different non-selective transporters depending on properties (acidic; basic; neutral; Imino acids; glycine)
- Creates competition for certain transporter types
Protein reabsorption:
- Partially degraded by peptidases on brush border then taken up by endocytosis
- Vesicles fuse with lysosomes then hydrolysed and ααs moved by f.diffusion
- Smaller peptides (e.g. ADH, angiotensin II) completely hydrolysed in tubule
How is HCO3- reabsorbed in the PCT?
- NHE3 transporters lower cell pH and allow carbonic acid formation in lumen
- Carbonic anhydrase aids CO2 and H2O formation
- CO2 absorbed and remade into HCO3-
- Secondary active transport out of cell (NBC1 (uses Na+ to symport which is rare) and HCO3-/Cl- antiport pump AE1)
What evidence is there for isosmotic fluid reabsorption in PCT being driven by Na+/K+ pump?
Treatment with 2,4-DNP:
- Leads to very reduced water reabsorption
- Since 2,4-DNP uncouples oxidation from ATP synthase so no active transport occurs.
Treatment with ouabain:
- Shows Na+ drives it since blocks Na+ transporter specifically.
How does the LoH uncouple water and NaCl reabsorption? Describe transport in each section of LoH:
Using counter current multiplier effect:
- Modest transverse gradient (set by transporter maxima) to be multiplied into steep longitudinal gradient.
- Thin ascending limb = passive NaCl reabsorption into interstitium
- Thick ascending limb = NaCl actively transported out and water follows via leaky tight gap junctions in descending limb
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Describe movement of substances in the collecting duct:
Cortical CD:
- Fluid entering is hypoosmotic to plasma so water moves out
- Extra NaCl absorbed driving more water out
- Extent of NaCl absorption changed by ADH.
Medullary CD:
- When ADH is high and CD water permeable, water drawn out causing concentrated urine
What are vasa recta and what is their function?
Long capillary tubes allowing solute and water diffusion without destroying medullary gradient.
- Counter current exchange diffusion with blood
- higher colloid osmotic pressure of blood and incomplete equilibration allows blood to carry away reabsorbed solute.
What is the effect of urea in the kidney?
- Nephron mostly poorly permeable to urea (concentration in tubular fluid rises)
- ADH increases urea permeability of inner medullary collecting duct (IMCD) allowing passive diffusion into interstitial fluid.
- Urea recycling between LoH and IMCD allows high [urea] to build up, increasing osmotic pressure
- Can contribute 50% of osmotic pressure in concentrating kidney
How does a change in blood pressure result in renin release?
Afferent arteriole acts as internal baroreceptor:
- Fall in pressure leads to renin release
Macula densa senses change in flow rate:
- Decrease in flow rate stimulates renin release (as suggests upstream constriction to maintain filtration pressure given reduced volume)
Why is constriction of the afferent arteriole not sufficient to increase filtration rate?
- Increases filtration fraction
- However, also increases pressure in peritubular capillaries which reduces reabsorption
- Also increases back leakage at Bowman’s capsule due to raised interstitial pressure
How can sympathetic nerves increase water retention?
Release of hormones:
- Renin (and then angiotensin II and aldosterone)
Constriction of renal arteries:
- makes efferent resistance > afferent resistance
- Increases filtration fraction
- Increased reabsorption back into blood in peritubular capillaries (increased COP)
Directly stimulates Na+ reabsorption in PCT by adding NHE3 transporters.
How is renin release stimulated?
When BP is low (retention mode)
- Stimulation by sympathetic nerves through NA secretion
- On β1 adrenoreceptors (Gs) on specialised smooth muscle cells
[Often in conjunction with direct stimulation of PCT for Na+ reabsorption via α1-adrenoreceptors (Gq)
to add NHE3 transporters]
How does renin result in angiotensin II formation?
- Renin secreted by smooth muscle cells in afferent arteriole
- Renin catalyses the production of angiotensin I from angiotensinogen
- Angiotensin I to II catalysed by ACE in lungs
Detail the effects of angiotensin II:
- Na+ absorption in PCT (binds AT1 receptor to increase NHE transporter density)
- Stimulates aldosterone synthesis and secretion
- Stimulates hypovolaemic thirst
- Stimulates sodium appetite
- Vasoconstriction of efferent arteriole
What are the effects of vasoconstricting the efferent renal arteriole?
- Reduced RBF since resistance increased overall
- Therefore a fall in pressure in peritubular capillaries and vasa recta (promotes Na+ reabsorption)
- Slight increase in filtration fraction but GFR reduced due to lower blood flow
- Stabilisation of GFR (important in severe hypovolaemia)
Detail the effects of aldosterone:
Acts on CD by stimulating new protein synthesis in principle cells:
- Increased transepithelial potential
- Promote Na+ reabsorption through ENaC channels and in long term Na+/K+ATPase
- Promote H+ and K+ secretion (through small conductance SK channels
How is aldosterone secretion stimulated?
Angiotensin II:
- Binds to AT2 receptors in distal nephron stimulating aldosterone synthesis
Increased [K+]:
- Secreted by zona glomerulosa of adrenal gland
What is ANP? Where is it produced?
Atrial natriuretic peptide:
- Produced by atrial myocytes when stretch increases
- Suggests high BP
Detail the effects of ANP:
Acts to reduce BP by reducing ECFV:
- Inhibition of Na+ reabsorption in collecting duct (increases cGMP which phosphorylates ENaC channels reducing their activity)
- Inhibition of Na+ reabsorption in PCT: cells locally secrete dopamine which inhibits reabsorption
Inhibition of renin - Dilation of glomerular mesangial cells: increased SA for filtration
- Vasodilation of afferent and efferent arterioles (efferent more) raising relative glomerular hydrostatic pressure
- Inhibition of ADH secretion in supra optic nucleus (SON)
What is Addison’s disease?
Adrenal insufficiency:
- Aldosterone deficiency causing reduced ECF volume and low BP
- Can be improved by intaking large amount fo salt.
How is Ca2+ reabsorbed in the PCT and LoH?
Fixed reabsorption in PCT
- Paracellularly due to +ve transepithelial potential; increases solvent drag
Transcellular absorption:
- Entry into cells through TRPV5/6 and moved through by calbindin-D
- Exits through Ca2+/ATpase and NCX (Na+/Ca2+) transporter.
Fixed reabsorption in LoH: paracellular (in ascending limb) and transcellular
Describe the different properties of NCX and Ca2+/ATPase:
NCX is low affinity, high transport capacity
Ca2+/ATPase is high affinity, low capacity
How can PTH increase Ca2+ in the DCT and CD?
- Variable reabsorption in CD and DCT: increases Ca2+ reabsorption by phosphorylation of NCX transporters
- Increases 1,25-Dihydroxycholecalciferol (1,25-DHCC) which increases:
- TRPV5 and 6 expression
- Calbindin-D
- NCX and type II Na+/Pi channel densities.
- No paracellular transport as potential is wrong direction
How is phosphate reabsorbed in the PCT?
Taken into cells by:
- Type IIa (3Na+/HPO42- transporter)
- Type IIc (2Na+/HPO42- transporter)
- Type III 2Na+/H2PO4- transporter.
(Na+ gained from Na+/K+ transporter)
Exits cells through:
- Anion/HPO42-
- Anion/H2PO4- transporters (A- commonly oxalate)
How does PTH decrease phosphate reabsorption?
PTH can bind on both cell surfaces (luminal = Gq receptor and interstitial = Gs receptor) causing decreased type IIa and c receptor density:
- Phosphorylation of scaffold protein NHERF-1 causes its dissociation from channel
- Allows channel to be endocytosed
- Effect on type IIa is faster than for type IIc
Name some factors which can affect the ECF [K+]:
- Hormones (insulin, aldosterone, adrenaline)
- Acid-base balance (acidosis increases [K+] as H+ brought into cells and K+ pushed out)
- Plasma osmolality (cell shrinkage causes K+ excretion)
- Cell lysis (due to damage)
- Exercise (K+ leaves muscles during contraction - effect opposed by adrenaline)
How is K+ reabsorbed in the PCT and LoH?
PCT:
- Paracellular diffusion through leaky tight gap junctions
LoH thick ascending limb:
- Transcellular by secondary active transport
- Entry through NKCC2; exit via Cl- symporters
- Paracellular diffusion ascending limb due to transepithelial potential
How can the DCT/CD show such a wide range of K+ transport?
Can show either secretion or reabsorption! Depends on:
- Na+/K+ ATPase density and activity
- The electrochemical gradient for K+ loss
- Permeability for Na+ and K+ of the luminal membrane (SK channels; Na+/K+ pump)
- Reabsorption of K+ by type A intercalated cells (to maintain H+ homeostasis)
What hormonal and physical controls affect K+ homeostasis?
- High [K+] causes aldosterone synthesis and release
- Changes flow rate: increased flow rate increases secretion of K+
- ADH stimulates K+ secretion (though slows flow rate; increases luminal K+ permeability)
What is the Henderson-Hasselbalch equation? What is normal physiological pH?
Normal physiological pH: blood = 7.4; CSF = 7.3 within very small range.
Henderson-Hasselbalch: relates concentrations of conjugate pairs and pH:
pH = pKa + log10([base]/[acid])
How can ingestion affect pH?
Excess non-volatile fatty acids:
- Sulphuric acid from methionine and cysteine
- H2PO4- from phospholipids and phosphorylated proteins
What is the isohydric principle and how can it be applied to the HCO3-/H2CO3/CO2 system?
How is pH calculated from this buffer system?
Isohydric principle: if multiple buffers interacting; control of one component allows control of the whole system:
pH = composite pKa + log10([HCO3-]/[H2CO3]) = 6.1 + log10([HCO3-]/0.03 x pCO2)
Effectively: 6.1 + log10(kidneys/lungs)
Describe how [pCO2] is measured and resulting physiological buffering:
Measured by central chemoreceptors in IVth ventricle
- CO2 diffuses across BBB
- Lower pH increases AP frequency
- Stimulates increased breathing rate
Physiological buffering;
- CO2 formed is excreted from lungs
- HCO3- regulated by liver and kidneys
- Tissue buffering: hydroxyapatite in bone accepts protons in acute acidosis
How is [HCO3-] regulated by kidneys and liver?
Kidneys:
- New HCO3- produced from glutamine (produces 2 moles of HCO3- and NH4+)
- Excess is eliminated and excreted in urine through urea formation
Liver:
- Forms glutamine for breakdown in kidneys by combining NH4+ with α-ketoglutarate
- Low pH promotes more glutamine conversion
What is the urea cycle in the liver?
- Catabolism of primary carboxyl and amino acid groups produces NH4+ and HCO3-.
- NH4+ is toxic and is turned into glutamine by combination with α-ketoglutarate
- Glutamine converted to urea in kidneys:
2(HCO3- + NH4+) = CO2 + H2O + urea
- CO2 produced con be blown off by lungs, shifting equilibrium away from HCO3-.
How do plasma proteins and the phosphate system help to regulate pH?
Plasma proteins:
- Can reversibly bind CO2 to amine groups
- E.g. in Hb CO2 binds to imidazole groups on histidine amino residues of α/β chains
Phosphate system:
- Shifts equilibrium between H2PO4-/HPO42-
Together contribute approx. half of buffering capability
How are the products of glutamine degradation transported in the PCT?
NH4+ transport: Into lumen
- Closely resembles K+ in size and charge hence uses same transporters
- Replaces H+ in NHE3 transporter into lumen
- Diffusion through leaky gap junctions
HCO3- transport: into interstitium:
- Reabsorbed through conversion to CO2 and H2O
Why is the pH of tubular fluid limited?
Transepithelial potential:
- Acidity of PCT fluid limited as NH4+ (alkaline) can diffuse through gap junctions
How are the products of glutamine degradation transported in the LoH?
- HCO3- reabsorbed slowly (no CA) in thick ascending limb
- NH4+ reabsorbed by replacing K+ in NKCC2 and K+/Cl- transporters
- Transepithelial potential pushes out NH4+
- NH3 diffuses across cells (extra proton excreted
How are the products of glutamine degradation transported in the DCT/CD?
- Remaining HCO3- is reabsorbed by type A intercalated cells (see above sections)
- Ammonium trapping: NH4+ converted to NH3 then trapped as NH4+ once in lumen
- NH3 diffuses into lumen passively and is transported out by rhesus glycoprotein transporters (Rhbg and Rhcg from interstitium then only Rhcg to lumen)
- Conversion provides H+ ions for HCO3- cycle
- Leads to low pH of urine
How is H+ secretion stimulated? (During acidosis)
Increased HCO3- filtered load in PCT by increased H+ secretion from type A cells
Decreased plasma pH (and decreased HCO3-):
- Increases gradient for HCO3- efflux via NBC1 and Cl-/HCO3- exchanger
- Lowers pH inside cells which increases secretion
Increased pCO2
- Enters cells and lowers pH – causes H+ removal via NHE3
- K+/H+ ATPase insertion in type A cells.
Increased K+ reabsorption [DCT/CD]:
- K+/H+ ATPase insertion [CD] in type A cells.
Hypovolaemia
- Angiotensin II (Na+/H+ transporter stimulated)
How does HCO3- secretion occur during alkalosis?
- Type B intercalated cells (opposite to type A) secrete HCO3-
How might chronic respiratory alkalosis occur?
- High altitude results in decreased pO2
- Compensated for by hyperventilation
- Decreases pCO2
- Kidneys/liver compensate by increasing HCO3- secretion
- Not enough HCO3- left in blood to push equilibrium towards CO2 production
- Therefore pH of blood still high.
How does diabetic ketoacidosis occur?
- H+ released from prolonged incomplete oxidation of fatty acids
- Buffered by HCO3-
- More glutamine broken down into NH4+ and HCO3- to compensate
- More ammonium produced and therefore must be excreted
- If excess NH4+ cannot be excreted (limit) then acidosis results
What effect does vomiting have on plasma pH?
- Excreted gastric acid not united with HCO3- in duodenum
- Causes metabolic alkalosis
- Ventilation rate slowed (compensated metabolic alkalosis)
How might failure of K+ homeostasis cause alkalosis?
Hypokalaemia results in alkalosis
- K+/H+ are exchanged to maintain electrical neutrality in cells
- Less K+ induces more H+ in cells which reduces pH
- Increases H+ secretion and HCO3- reabsorption
What does a nomogram show?
Shows arterial blood pH against blood plasma pCO2 (or [HCO3-]).
Visualises where areas of alkalosis/acidosis are and whether they are primary pulmonary or metabolic.
