Renal_L7- Flashcards
Where is the Na+/K+ ATPase?
It is ubiquitous → everywhere in the kidney
On the basolateral membrane → creates a gradient for Na entry in the cell → drives other cotransports
*Acts as the battery for Na transport systems
What is the concentration of Na in the ECF and in the ICF?
ECF → 130-140 mM (66% of Na in the body)
ICF → 10-25 mM (10%)
Rest in the bones
Where is the rate of transport/cm of tubule length the highest across all the kidney?
In the medullary thick ascending limb (MAL) → 2nd site of greatest reabsorption → recovery ~15% of filtered NaCl (8% remaining after it at the macula densa)
But the largest fraction of Na reabsorption occurs in the proximal tubule (> 60%) → but concentration remains almost constant bc water is also greatly rabsorbed at the same time
How does the potential of the lumen change along the proximal tubule length?
S1 → negative due to eletrogenic Na-Glucose cotransport
S2, S3 → positive Due to chlorides diffusion potential (but not in juxtamedullary nephrons, only in superficial nephrons (majority))
What is the concentration and quantity of Na urinary excretion/day?
- Urinary excretion of Na+ ~ 100 mmole/day
- 0.4% of filtered load remaining in the urine
Funfact:
V = 1500mL/day → Una = 67 mM
What is the backleak of Na+?
In proximal tubule:
It is the paracellular diffusion of Na+ through tight junctions from interstitial space → lumen
*Downhill because cellular Na+ transport build gradient in the interstitial space
Gradient ~ -3 mV → 0 mV (small but leaky membrane)
(vs -70 mV intracellularly)
What are the specific Na+ transporters present in the PROXIMAL TUBULE?
*In S1
Apical membrane:
- Organic solute cotransporters (both in, ex: glucose, AA)
- Na+/H+ exchangers (Na+ in/H+ out) → important because allow entry of Na to drive efflux of H+, when H+ gets in lumen → CO2 + H2O (because 25 mM bicarbonate in the lumen)
Basolateral membrane:
- Na+, CO3(2-), HCO3(-) cotransporter (all → out, Na transported by HCO3- gradient which favours efflux)
- 3Na+/Ca(2+) exchangers (Na in/Ca out)
*Carbonic Anydrase inside the cells take H2O + CO2 → H+ and HCO3- for the pumps/transporters
Paracellular diffusion of sodium chloride and water
In proximal tubule, small increase in Na only
What are the characteristics of NHE3?
Predominant in proximal tubule:
- 13 isoforms (originally 9)
- 12 TM alpha-helices
- In the renal brush border
- 1 Na+/ 1 H+ exchanged (2ndary active transport)
- Km for Na ~ 10 mM
- 2x H+ sites → 1 substrate binding site (for exchange) + 1 modifier sites (regulates activity, activates exchange when intracellular pH falls below 7.0)
- Electroneutral
*Inhibited by amiloride analogs → inhibits at high Na concentrations
C-term:
- Inhibited by PKA and PKC-mediated phosphorylation
- If cut, half reduced activity
What is the importance of the thin descending limb vs thin ascending limb in Na/Water reabsorption? (general)
*Not many mitochondria, no brush border, not much active reabsorption (low Na/K ATPase) in both
Thin Descending Limb:
Little active transport
- HIGH water permeability → fluid reabsorption (driven by high osmotic pressure in the insterstitium)
- NaCl and urea both diffuse into the lumen (epithelium tight junctions)
- Lots of aquaporins → important counter current multiplier
Thin Ascending Limb:
Little active reabsorption
- LOW water permeability but NaCl passively reabsorbed (leaks, but not H2O)
- Osmotic equilibration occurs by NaCl diffusion instead of water entry
What transport occurs in the THICK ascending limb of the kidney?
How is the lumen?
Also called the diluting segment → reabsorb salts, but IMpermeable to H2O → [NaCl] reduced to 30 mM by end
Apical membrane:
- NKCC2 → 1Na+/1K+/2Cl- (all → intracellular, gradient for Na and Cl import)
- K+ transports to recycle K+ to the lumen (if not, K+ runs out)
- Na/H exchange (because not all HCO3- was reabsorbed in the proximal tubule so can combine H+ to form CO2) → NHE3
Basolateral membrane:
- K+ channel
- Cl- channel
- Na/K ATPase (3out/2in)
- 3HCO3-/Cl- antiport (Cl-in, HCO3- out)
Lumenal TAL is +ive (because 2 Cl- in for every Na+, K+ is recycled) → drives passive reabsorption of divalent (espacially Mg2+) by leakage through tight junctions + transcellularly
Na+ paracellular diffusion
*Relatively low electrical resistance (2-fold higher than proximal tubule) → epithelia gets tighter, less leaks → less work to maintain gradients
What segment is the main site of energy input for the counter curent multiplier?
Thick ascending limb
*TAL has medullary and cortical segments
What is the NKCC2 sensitive to?
Found in Thick Ascending Limb
Sensitive to loop diuretics → furosemide, bumetanide
*Site of action of many loop diuretics → Cl- binding site on NKCC2 → prevents Na reabsorption → prevents H2O reabsorption
What are the characteristics/structure of Na+/K+ ATPase?
1 ATP → 3 Na out/2K+ in
- In the basolateral membrane
- Translocation = conformational change from E1 (cytoplasm) → E2 (facing interstitum)
- P-type ATPase → tranfer of the ATP phosphate on a binding site where it is phosphorylated
- a subunit → catalytic site (translocation of the ions) + contains ouabain binding site (inhibitor)
- beta subunit → essential for assembly and export of the pump from the ER to the plasma membrane (not involved in actual pump function)
- gamma subunit → FXYD subunit modulates pump function
Inhibited by:
- Ouabain
- cardiac glycosides digoxin (foxglove) → increase contractility of the heart by inhibiting Na import → more Ca import
What is the cycle of the Na/K ATPase pump?
- E1-ATP → Cytoplasmic Na+ binds the pump (3 sites)
- E1-P → Na+ binding stimulates phosphorylation by ATP → P is tranfered from ATP to phosphate site on the pump
- E2-P → Phosphorylation causes protein to change conformation expelling Na+ to the outside of the cell
- E2-P → Extracellular K+ binds to the protein (2 sites) → triggers release of the phosphate group
- E1 → Loss of the phosphate → retores protein original conformational (to facing the cytoplasm)
- E1 → K+ released in cytoplasm, Na+ sites are receptive again
Cycle repeats
What is the importance of the distal convoluted tubules (after TAL) in the kidneys?
Contains multiple cell types → mixture of cell types, gradually changes as we move along the DCT → smooth transition from distal tubule → collecting duct
- Transepithelial potential becomes more negative
- IMpermeable to H2O
- Apical membrane → NCC = Na+/Cl- cotransport (both in)
- Basolateral membrane → Na/K ATPase
- ROMK channels for K+ secretion
What is the difference between the transepithelial potential in the Thick Ascending Limb and in the Distal Convoluted Tubule?
As we move along TAL → transepithelial potential becomes more positive
VS
As we move along the DCT → transepithelial potential becomes increasingly more negative
What are the characteristics of NCC?
Na/Cl cotransporter in the Distal Convoluted Tubule
- Electroneutral → 1:1
- 12 TM segments
- MW ~ 112 kDa
- Sensitive to Thiazide diuretics (TSC)
- NOT sensitive to loop diuretics (furosemide and bumetanide)
- Gene sequence 47% identical as NKCC
What cells are present in the cortical collecting duct?
- Pincipal cells:
- Na+ (and K+) transport by Na/K-ATPase on basolateral membrane
- On apical membrane ENaC for Na reabsorption
- If lumen is negative, K+ goes out by channels
- Sensitive to potassium-sparing diuretic amiloride - Intercalated cells → acid/base regulation
The 2 types of cells are not coupled to each other by gap junctions
*Vasopressing srimulates water permeability
How does the luminal voltage of the cortical collecting duct vary?
Voltage may be positive in acidosis (due to H+ secretion), may be negative (due to Na+ reabsorption)
Under physiological conditions, it tends to be negative
What sodium channel is responsible for Na reabsorption in the Proximal tubule? ThinD/AL? ThickAL? DCT? In the CCD?
*On the apical membrane only
Proximal tubule → organic solute cotransporter + Na+/H+ exchangers (NHE3)
Thin descending and ascending limbs → passive diffusion reabsorption
TAL → NKCC2 (Na+/K+/2Cl-) + Na+/H+ exchanger
DCT → NCC (Na+/Cl-)
CCD → ENaC
MCD → Non-selective Na channel, not ENaC
What is the Koefoed-Johnson and Ussing model?
It is a model which dictates Na+ reabsorption in late distal tubule and CCD → model for Na absorption by frog skin and other tight epithelia
*Apical and basolateral membranes are independent electrically
Apical
When Na concentration of outer solution was increased by 10-fold, transepithelial potential (Vt - Eo - Ein) varied by -58 mV as predicted if the apical membrane was selective for Na+ (outside of skin became 58mV more negative with respect to the inside) → follows the Nernst equation
K+ variations don’t change Vt
Basolateral
By contrast, varying Na concentration of inner solution had no effect, but deacreasing the inner solution K+ concentration by 10-fold → -58mV for transepithelial potential *Basolateral membrane having predominant K+ conductance
Conclusion → Na going from lumen to interstitium by diffusion + Na/K ATPase is done in exchange for K+
What is the importance/mechanism of the patch clamp technique?
Patch clamp technique involved pressing a fine tipped, glass micropipette containing solution against the membrane of a cell → high resistance seal when suction applied → electrically isolates a small patch of membrane
Patch clamping → direct information about the conductance of a single cell + properties of individual channels
Investigator has complete control over membrane potential and composition of solutions of both sides
What are the different patch clamp configuration that can be used?
Depends on the purpose of the experiment
- Cell attached → record single channel currents in intact cell
- Whole cell → A pulse of suction → break the patch while leaving rest of the cell intact → record whole cell current (pipette continuous with cytoplasm)
- Outside-out patch → from the whole cell configuration, retract the pipette to detach the patch of membrane from the cell (will partly reform in outside-out configuration) → control on extracellular medium
- Inside-out patch → start from cell attached and pull the pipette out (pipette only attached to a patche of membrane, not the whole cell anymore) → can have control on inner medium
What are the characteristics of the ENaC channel?
(Epithelial Na Channel)
Found on the Apical membrane of the principal cells of the CCD
- Highly selective for Na+ (over K+), also to H+ and Li+ → H+ concentration is very low and Li is not a physiological ion
- Low single channel conductance ~ pS → not strongly voltage gated, but open probability slightly increased by hyperpolarization
- Km for Na ~ 5 mM
- Slow channel gating kinetics (opposit of neurons)
- Blocked by potassium-sparing amiloride (given when give Li drugs)
- Heterotrimer → 3 homologous subunits (a, b, y → 1:1:1) → each have 2 TM segments, MW < 75 kDa each, 630-650 AA