Renal_L7-

Question 1

Where is the Na+/K+ ATPase?

Answer 1

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

Question 2

What is the concentration of Na in the ECF and in the ICF?

Answer 2

ECF → 130-140 mM (66% of Na in the body)

ICF → 10-25 mM (10%)

Rest in the bones

Question 3

Where is the rate of transport/cm of tubule length the highest across all the kidney?

Answer 3

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

Question 4

How does the potential of the lumen change along the proximal tubule length?

Answer 4

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))

Question 5

What is the concentration and quantity of Na urinary excretion/day?

Answer 5

• Urinary excretion of Na+ ~ 100 mmole/day
• 0.4% of filtered load remaining in the urine

Funfact:
V = 1500mL/day → Una = 67 mM

Question 6

What is the backleak of Na+?

Answer 6

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)

Question 7

What are the specific Na+ transporters present in the PROXIMAL TUBULE?

Answer 7

*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

Question 8

What are the characteristics of NHE3?

Answer 8

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

Question 9

What is the importance of the thin descending limb vs thin ascending limb in Na/Water reabsorption? (general)

Answer 9

*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

Question 10

What transport occurs in the THICK ascending limb of the kidney?
How is the lumen?

Answer 10

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

Question 11

What segment is the main site of energy input for the counter curent multiplier?

Answer 11

Thick ascending limb
*TAL has medullary and cortical segments

Question 12

What is the NKCC2 sensitive to?

Answer 12

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

Question 13

What are the characteristics/structure of Na+/K+ ATPase?

Answer 13

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

Question 14

What is the cycle of the Na/K ATPase pump?

Answer 14

1. E1-ATP → Cytoplasmic Na+ binds the pump (3 sites)
2. E1-P → Na+ binding stimulates phosphorylation by ATP → P is tranfered from ATP to phosphate site on the pump
3. E2-P → Phosphorylation causes protein to change conformation expelling Na+ to the outside of the cell
4. E2-P → Extracellular K+ binds to the protein (2 sites) → triggers release of the phosphate group
5. E1 → Loss of the phosphate → retores protein original conformational (to facing the cytoplasm)
6. E1 → K+ released in cytoplasm, Na+ sites are receptive again
   Cycle repeats

Question 15

What is the importance of the distal convoluted tubules (after TAL) in the kidneys?

Answer 15

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

Question 16

What is the difference between the transepithelial potential in the Thick Ascending Limb and in the Distal Convoluted Tubule?

Answer 16

As we move along TAL → transepithelial potential becomes more positive
VS
As we move along the DCT → transepithelial potential becomes increasingly more negative

Question 17

What are the characteristics of NCC?

Answer 17

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

Question 18

What cells are present in the cortical collecting duct?

Answer 18

1. 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
2. Intercalated cells → acid/base regulation

The 2 types of cells are not coupled to each other by gap junctions

*Vasopressing srimulates water permeability

Question 19

How does the luminal voltage of the cortical collecting duct vary?

Answer 19

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

Question 20

What sodium channel is responsible for Na reabsorption in the Proximal tubule? ThinD/AL? ThickAL? DCT? In the CCD?
*On the apical membrane only

Answer 20

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

Question 21

What is the Koefoed-Johnson and Ussing model?

Answer 21

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+

Question 22

What is the importance/mechanism of the patch clamp technique?

Answer 22

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

Question 23

What are the different patch clamp configuration that can be used?

Answer 23

Depends on the purpose of the experiment

1. Cell attached → record single channel currents in intact cell
2. Whole cell → A pulse of suction → break the patch while leaving rest of the cell intact → record whole cell current (pipette continuous with cytoplasm)
3. 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
4. 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

Question 24

What are the characteristics of the ENaC channel?
(Epithelial Na Channel)

Answer 24

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

Question 25

What is Liddle disease?

Answer 25

It is caused by mutations in beta and gamma subunits of the ENaC channel
Rare type of hypertension cause by Na hyperabsorption

Associated with hypokalemia and low plasma aldosterone levels

Question 26

Why is amiloride considered to be a potassium-sparing diuretics?

Answer 26

Amiloride has positively charged groups on it → binds and blocks flow of Na ions through ENaC
When Na is not reabsorbed → hyperpolarization of the membrane → holds K+ inthe cells/ reduced efflux of K+ (no potaassium wastes

Question 27

How does Na reabsorption occur in the Medullary collecting duct?

Answer 27

• similar channel (not the same), but with higher conductance
• less Na+ selective
• regulated by G proteins
• Inhibited by atrial natriuretic peptide through its 2nd messenger cGMP → 2 separate actions of cGMP:
  1. direct blockage of the channel
  2. Activation of a cGMP-dependent protein kinase → deactivates a G protein that normally stimulates the cation channel

Question 28

What are the characteristics of the outer and inner stripes of the Medullary Collecting Duct?

Answer 28

Outer stripe:
- Often has lumen-positive electrical potential due to H+ secretion (especially in acidosis)

Inner Stripe:
- No intercalated cells → no H+ secretion
- Na reabsorption → lumen negative potential
- Na reabsorption is inhibited by cGMP → allows correction of hypertension (more loss of salt)

Question 29

How does NaCl balance out when ingesting higher quantities of salt?

Answer 29

When the intake increases step-wise → positive-balance → gradual increase in Na excretion, then plateau when equilibrium
When intake decreases step-wise → negative-balance → gradual decrease in Na excretion to reach plateau

Question 30

What is the split droplet technique?

Answer 30

ANSWER AFTER VIDEO

Question 31

What is the Curran model of water transport?

Answer 31

Set up → 3 compartements side by side with 2 membrane to separate them, middle compartment has grater osmolality
Possible to produce a net volume flow where the rate of flow depends on:
- asymmetrical solute permeabilities
- hydraulic conductivities
of the 2 membrane

In reality:
Energy of H2O reabsorption driven from ATP hydrolysis by Na/K ATPase → Na transport
Membrane water permeability depends on aquaporins

Question 32

What happens to Na excretion when aldosterone is administered ? (not naturally produced)

Answer 32

Aldosterone → Stimulates Na reabsorption

Mineralocorticoid escape:
Aldosterone administration → step decrease in Na excretion (bc stimulate CCD to reabsorb Na) → increase in hydrostatic pressure → mineralocorticoid escape → increase excretion to come back to equilibrium even if aldosterone (to not have hypertension)

2 main mechanisms of mineralocorticoid escape:
1. Increase in extracellular fluid volume (ex: tumors in adrenal glands) → build up in hydrostatic pressure around proximal tubule → inhibit reabsorption of salt in PT
So even if GFR increases as a result of increase in extracellular fluid, reabsorption decreases, increased backflux of fluid crom interstitium to lumen
Causes more Na in the proximal tubule → goes downstream → overwhelm CCD to reabsorb Na → excrete Na

2. In the atria when get distension → release of atrial natriuretic peptide → increase GFR + inhibit reabsorption of salt in MCD (non-selective Na channels)

End of aldosterone administration → step increase in aldosterone excretion → gradual decrease to match intake

Question 33

In a 2 compartement model with an H2O permeable membrane between both, what is an efficient way to increase volume on only 1 side?

Answer 33

1. Add isotonic saline (same osmolarity) in the target compartement
2. Add NaCl in the target compartement → some water from the other compartement flows to the target compartement to equilibrate osmolarities

Inefficient way:
Adding just water in the target compartement → water will go on both sides to have same osmolarity

Question 34

How are NKCC and NCC trafficking and activation regulated?

Answer 34

Regulated by protein kinases in the WNK-SPAK pathway

WNK1 (with no lysine kinase type 1) and WNK3 stimulate salt reabsorption by phosphorylating SPAK (another kinase) → phosphorylates and activates NKCC and NCC

Low intracellular Cl- → WNK4 phosphorylates SPAK → positive regulator of NCC
High Cl- inhibits WNK4 → less phosphorylation of SPAK → less Na reabsorption
*Used so that more Na is reabsorbed later → help drive K+ secretion

Question 35

What is the effect of mutations in the WNKs (gain of function)

Answer 35

WNK = With no lysine kinase

Mutations lead to excessive salt absorption and familial hyperkalemic hypertension

Question 36

How does WNK4 helps correct hyperkalemia?

Answer 36

Hyperkalemia depolarizes the membrane → High intracellular Cl- → inhibits WNK4 → reduces Na+ absorption and increases Na delivery to CCD → electrogenic ENaC promotes K+ secretion → corrects hyperkalemia

Question 37

What determines Na excretion?

Answer 37

Na+ balance → Circulating volume

Experiment to prove it:
If someone sits in a water pit → increased pressure around vital organs → increase in Na excretion to bring the perceived volume down

Question 38

What is NHE1?

Answer 38

Is is a houskeeping exchanger that helps maintain intracellular pH.
- Present in most cells
- Expressed on the basolateral membrane of renal tubule cells

*vs NHE3 on the apical membrane

Question 39

Where is the largest portion of HCO3- reabsorbed?
When reaching the collecting duct, what is the % of filtered HCO3- left in the lumen?

Answer 39

Most reabsorption in the proximal tubule and loop of Henle

At the collecting duct 95% of filtered HCO3- has been reabsorbed

Question 40

What is the difference between basolateral reabsorption of HCO3- in the proximal tubule and in the TAL?

Answer 40

PT → Na+: CO3 (2-), HCO3- symport

TAL → Cl-: 3x HCO3- antiport

Question 41

When ∆u/F = 0 (no difference in electrochemical potential), what does Vm =?

Answer 41

Vm = - (RT/zF)*ln(Ao/Ai)

*∆u = RTln (Ao/Ai) + F(ψo - ψi)

Question 42

What is ∆u of the Na/K ATPase?

Answer 42

∆u = 3∆u Na + 2∆u K

Question 43

Which segment of the kidney is considered to be the diluting segment?

Answer 43

TAL (fluid coming to the DCT is always hyposomotic)