Renal_transport (L4-L6) Flashcards
What is the equation of the Fick’s Law?
J = -D dc/dx
Φ = D/X (C2-C1) = molescm-2sec-1
c = concentration gradient
x = thickness
D = diffusion constant
Φ = flux = rate of solute movement per unit of cross sectional area
what is the partition coefficient?
Kmw = Cmembrane/Cwater
Higher (0.1) for less polar molecules
Lower (10^-7) for more polar partition
*partition coefficient is directly proportional to permeability (smaller particles, for same K, have higher permeability)
What actually/in practice determines simple diffusion through a membrane?
Φ = Dapparent /Xmembrane (C2 - C1)
D apparent = Dm*Kmw
Kmw = C membrane/Cwater
*The rate of simple diffusion depends on the ratio of solute centration in the membrane to that in the aqueous phase
Usually, Dm, Kmw and Xm are generally not known for biological membrane. How is simple diffusion calculated?
P = permeability coefficient empirically-determined
Φ = P (C2 - C1)
What determines the net diffusion flux of molecules?
Diffusion molecules see no force, but on average they tend to move to the region of lower concentration
*Not driven by an actual force towards lower concentration
They move due to Brownian motion (thermal agitation)
How is water permeability regulated in the kidney?
Water permeability can be regulated by Antidiuretic hormones (ADH) → allow water movement, but not solute movement in the corical collecting duct ~ 4-fold
What equation describes the membrane’s permeability to water?
*In the cortical collecting duct
*Driven by osmosis (following Na reabsorption)
J(H2O) = P ( π eff2 - π eff1)
P = permeability ~ 10^-4 or 10^-5 cm/sec
π eff = RTσC
R = gas constant
T = temperature (K)
σ = reflection coefficient (0-1, no units) → selectivity of the semi-permeable membrane (1 = actual osmotic pressure, 0 = 100% leaky, no membrane)
C = osmotic concentration (mOsm/l)
What is free diffusion of CO2 across the membrane important for?
(As a dissolved gas)
*Important for acid-base balance
Bicarbonate is reabsorbed by proximal tubule as CO2 dissolved gas (catalyzed by carbonic anhydrase, HCO3- could not diffuse freely)
Found in lumen and in blood as H+ + HCO3-, but diffuses as CO2
In plasma ~ 25 mmol HCO3-
Cells of the proximal tubules secrete H+ (Na+/H+ active transport) into lumen of tubule → combines with the bicarbonate that was filtered → CO2 + H2O (catalyzed by c.a.) → CO2 dissolved gas → diffuses → c.a. remakes H+ + HCO3- in the cells
*Reabsorption of HCO3- driven by excretion of protons
What is the value of D for small molecules in aqueous solution?
D = diffusion coefficient
D = 1x 10^-5 cm2/sec
In actual membrane, D is reduced by the ratio of solute cocentration in the membrane vs in aqueous phase (partition coefficient)
What happens is you expose cells to a solution of NH4+ (ammonium) (ex: ammonium chloride)?
- NH4+ will dissociate → NH3 + H+ (to an equilibrium)
- NH3 will diffuse freely through the cell membrane
- Inside the cell NH3 will assemble with H+ → NH4+ → increase the pH ~7.4
- Regulatory mechanisms will bring it back down a bit (~7.2)
When remove the NH4+ solution: - The NH3 in the cell diffuses back outside the cell to reassemble outside the cell to form NH4+ leaving the H+ ions inside the cell → big decrease in intracellular pH
What happens to the intracellular pH proximal tubule cells when we temporarily put a 5% CO2/ 25mmol HCO3- solution in the lumen
- HCO3 doesn’t diffuse, but CO2 will diffuse into the cells
- Will dissociate into H+ + HCO3- inside the cells bringing the pH down (< 7.0)
- Activate intracellular mechanisms to bring pH back up → secrete protons (Na+/H+ transporter)
- pH goes back up (a bit of overshoot)
When we remove the solution: - Intracellular CO2 diffuse back into the lumen to compensate for the loss of solution, but bicarbonate is left inside the cell → pH goes up (~7.6)
- Some mechanisms, kick in to bring it back down a bit
*Carbonic Anhydrase 4 in the lumen, C.A. 2 inside the cells of the proximal tubule
How do many drugs diffuse through the lipid bilayer?
Through non-ionic diffusion → as the undissociated acid form (HA)
How does clearance of neutral weak acids and neutral weak bases vary depending on pH?
Neutral weak acid: (A- + H+)
At low urine pH → association as HA → diffusion → low clearance/GFR ratio
At high urine pH (~7.6) → not much association with H+ → no-reabsorption → high clearance/GFR ratio (~2.0) and high conjugated base/acid ratio (A-/HA)
Ex: Salicylate
Neutral weak base: (B + H+)
At low urine pH → lots of H+ → association as BH+ → no reabsorption → high excretion → high C/GFR ratio
At high urine pH (~8) → not much H+ in filtrate → no association → reabsorption → C/GFR ~ 0
Ex: Quinine
What are the 3 characteristics of mediated transport?
Both carriers and channels exhibit these characteristics:
- Specificity → only 1 substance, or small group can bind to the transporter and permeate
- Saturation → transport rates reach some maximum (Vmax) when concentration of solute is elevated
- Competition → a second solute may also bind the transporter site although it isn’t necessarily transported
What is the difference between carriers and channels?
Channels ~ holes in the membranes, passive, but still specific
Carriers → involve a conformational change
*Both for large, hydrophilic, charged molecules
What is the importance/formula of the Michaelis-Menten equations?
*Concentration dependence of initial rate of transport
initial flux rate = Vmax/(1 + Km/[A])
Km = concentration of solute A at which the flux rate is 1/2-maximal
C = competitior for the transport site of A → reduces the flux rate for same [A]
What is the difference between Fick’s law diffusion (not mediated) and mediated transport curves ?
*Solute concentration (X-axis) vs Flux rate (Y-axis)
Fick’s law → linear increase, lower slope (lower flux rate than mediated at lower concentration)
Mediated transport → sharp increase at start → plateau when the transporter saturates
How is the turnover number/mechanisms of carriers different than of channels?
*Both are intrinsic membrane proteins
Carriers → lower turnover number (thousands/sec) because conformational change for every molecule that passes through the membrane
ex: Anion exchanger ~ 50,000/sec
Na-Glucose cotransporter ~ 5/sec
Channels → pores and gates
Conformational change in the protein opens the “gate” → allows many ions to pass through per second
Higher turnover number ~ 2-100 millions/sec
What is the structure of AQP1?
N-term-H1-H2-intramembrane loop (NPA)-H3-H4-H5-intramembrane loop (NPA)-H6-C-term
*Both N- and C-term are in the cytoplasm
2 subunits made of the 6 TM segments → 1,2,6 + 3,4,5 with the 2 loops in the middle forming the pore where H2O molecules can pass (NPANPA)
**Found as a tetramer → 1 subunit is glycosylated
Where are aquaporins? What are they inhibited by?
- Abundant in red cells and renal cortex
- Ancient protein family in bacteria and plants
- Initially called CHIP28 → Channel forming Integral membrane Protein of 28 kDa
- Increases water diffusional permeability by 8-fold
- AQP1 is present in apical and basolateral membrane of the proximal tubule
- Very specific to water → not permeable to urea, small ions, H+
- Inhhibited by HgCl2 (reacts at cysteine pore)
Where in the kidney is water permeability high?
- In S1, S2 and S3 of the proximal tubule
- In the Cortical collecting duct and Medullary collecting duct in presence of ADH
What are the 2 types of mediated transport?
Passive transport:
- Facilitated diffusion through carriers and channels
- Usually uncoupled flux
- Substrates move down their chemical (if uncharged) or net electrochemical gradient (charged)
Active transport:
- Net flux occurs against opposing gradient of electrochimcal potential (∆u → potential energy stored in the concentration and electrical gradients)
What is the electrochmical potential?
(equation)
μi = μ’ + RTlnCi + zFψi + RTlnfi
μi = partial molar free energy inside (would the same formula for outside) = chemical potential
μ’ = free energy in standard state
RTlnCi = chemical work
zFψi = electrical work (charged particles)
RTlnfi = work of interactions between solute molecules
ψ = electrical field (potential difference, if there is voltage)
R = gas constant
F = Faraday’s number
T = absolute temperature
What is the activity of a solute?
It combines the chemical work and interactions between solute particles → allows to replace Concentration by Activity
RTlnCi + RTlnfi = RTlnAi
*It is always lower than the solutes concentration, each ion has a different activity coefficient
K+ = 82 mM, Na+ = 78 mM (Na+ don’t interact as much)
pH measures the activity of H+, not its concentration
Activities can be determined using selective electrodes experimentally
What equation gives us ∆μ standard in volts?
*Divide ∆μ by F to have it in volts instead of joules
For monvalent cations:
∆μ/F = RT/zF*ln(Ai/Ao) + Vm
*divide by 1mol of charge
Vm = electrical component, rest = chemical component
If there is no net gradient, at equilibrium → ∆μ = 0 → Vm = -RT/zF*ln(Ai/Ao)
What are the 2 types of active transport?
Primary active transport:
- Occurs by pumps (move solutes energetically uphill) → take their energy directly from a chemical reaction, usually ATP hydrolysis
ex: Na+/K+ ATPase (ubiquitous), H+ ATPase (proximal tubue, apical membrane, distal nephron), H+/K+ ATPase (distal nephron, H+ out, K+ in)
Secondary active transport:
- Occurs by transporters that couple solute movements
- Energy from the downhill flux of one molecules/ion drives the uphill transport of another
Ex: Na+-Glucose cotransporter , Na+/H+ exchanger, etc.
How does glucose reabsorption occur?
*Na+/Glucose cotransporter → Na+ entry from lumen to the inside of the cell is favoured (chemically and electrically)
Mostly in S1 of the proximal tubule
Lumen → Na+/G cotransporter (at apical membrane) → cell → diffusion (basolateral membrane) → peritubular capillaries
APICAL membrane
SGLT1 → S3 → 2 Na : 1 Gc, D-glucose and D-galactose, in kidney and intestine, very high affinity
SGLT2 → S1, S2 → 1 Na : 1Gc, only D-glucose (nothing else), Km ~ 15mM for Na and 6mM for glucose, only in kidneys, less affinity (lumenal concentration ~1/100 of that in the cell)
BASOLATERAL membrane
Facilitated diffusion by Glut1/Glut2 carrier
How can we study glucose transport across renal tubule membrane?
- Take parts of the brush border (microvilli) that pinch off to form vesicles
*vesicles have the transporters on their membranes - Put these vesicles in a solution of low/high Na + marker for glucose
In low/no Na concentrations → low increase in intravesicular glucose to equilibrium (glucose slowly leaks into the vesicles and equilibrates)
In High Na → peak in the glucose uptake at first, than back to equilibrium
Which drugs are responsible for inhibiting Glucose reabsorption ?
Phlorizin competitively binds SGLT at the apical membrane
Phloritin blocks the Glut2 passive carrier
What would be the effect of a mutation in Glut2?
Would cause Glucose build up inside the cells → would change the electrochemical gradient → could inhibit entry of glucose
What is glucose galactose malabsorption disease caused by?
Caused by a mutation in SGLT1:
D → N in the 1st TM segment
Little effect on renal glucose reabsorption, mostly effect on intestine
What is the effect of a defective SGLT2?
*In S1, S2
Relatively benign → reduced plasma glucose threshold (Tm) → just eat more sugar as more is excreted
How are different amino acids reabsorbed?
Plasma has ~2.5 mM [L-Amino Acids] → 50g/day
Almost all reabsorbed in similar way as glucose (Na-dependent)
*Also at proximal tubule
- Neutral AA → Same as glucose, Na cotransport
- Acidic AA (anions) → Coupled to Na+ and H+ (keep electrical gradient)
- Basic (cationic) + cystine → By exchanger process for intracellular neutral AA (independent of Na+)
- Glycine, proline, hydroxyproline → Reabsorbed with H+
- Beta-alanine → cotransport with H+ in the early S1 and with Na+ in late S3 of the proximal tubule
What are the consequences of a defect in neutral amino acid reabsorption?
- Hartnup disease
- Skin rash
- Cerebellar ataxia
What are the consequences of a defect in cystine reabsorption?
*Cystine = 2 cysteines + disulfide bridge between them
- Not soluble → precipitation → Kidney stones
- Low grade chronic pain
- Tiredness
- Depression
- Unquenchable thirst
- Irritability
- Mood swings
How does reabsorption of carboxylic acids occur?
(anions)
Apical membrane: (co-transport, with Na entering)
- Monovalent (ex: lactate) → 2 Na+ : 1 anion (electrogenic)
- Di- trivalent (a-ketoglutarate2-, citrate3- → kreb cycle intermediates): 3Na+ : 1 anion
Basolateral membrane:
- H+ cotransport
- Organic anion exchange
*Lactic acid produced by exercise, epilepsy, hypothermia
Ketoacids produced by fasting, diabetes mellitus
What is the purpose of formate (HCOO-) reabsorption?
Present in low concentrations in the plasma and ultrafiltrate (~0.3 mM) → enters the cells with H+ on a carrier
- HCOO-/H+ cotransport (both in)
- NHE3: Na+/H+ cotransporter (Na+ in, H+ out)
- CFEX: HCOO-/Cl- cotransport (HCOO- out, Cl- in)
Allow small amount of formate to maintain NaCl reabsorption at high rates
How does urate transport in the kidney occurs?
Urate = end product of the purine metabolism → 90% reabsorbed (10% excreted)
Reabsorption → S1, S3 by apical URAT1 (R for reabsorption, Urate/anion exchanger) + simple passive diffusion (paracellularly)
Secretion (not always) in S2 by the basolateral exchanger OAT1 and OAT3 (organic anion transporter) and apical channel UAT1 (passive)
What is the effect of a mutation in URAT1 vs UAT?
URAT1 → apical reabsorption of urate
URAT1 mutation → hypouricemia (deficiency of urate in the body)
UAT → apical secretion of urate
UAT mutation → gout (excess urate in plasma, low solubility of urate at low pH of low T˚ → crystallization in the extremities)
Tm for uric acid is increased (net reabsorption) → hyperuricemia
How does transport of PAH in the kidney occurs?
*PAH not normally produced by the body
Secreted by the proximal and late tubule (S2, S3)
Clearance of PAH = Renal Plasma Flow → flow-limited
Prototype for physiological ORGANIC ANIONS: hippurate, cAMP, bile salts, antibiotics, drugs
Basolateral membrane: (uphill step, saturable carrier-mediated process)
3 Na+: CD2- (both interstitial → intracellular) → DC2-:PAH- (PAH into the cell, antiport)
Apical membrane:
Anion/PAH- antiport (PAH- intracellular → tubule lumen)
*DC2- = dicarboxylic acid
How does organic cations transport occur?
Organic cations are secreted in S3 segment
Basolateral membrane → facilitated diffusion through OCT1
Apical membrane efflux → H+/organic cation exchange (H+ in, O.C. out)
Why is active secretion of organic anions under their acid form necessary?
Because only 20% is filtrated and some bind to albumin and are not filtered
How does transport of urea occur in the kidneys?
Urea is filtered freely and then partially reabsorbed → urea clearance < GFR
Urea reabsorption in the S3 of the proximal tubule and medullary collecting duct (increased indirectly by ADH) → simple passive diffusion (after H2O reabsorption)
*permeability of tubular wall for urea < than for water → more H2O leaves than urea → more concentrated → greater concentration gradient favouring reabsorption
More excretion of H2O → more excretion of urea
What is the physiological range for urea concentration in the kidneys?
3 - 9 mM
Urea in plasma ~ Blood Urea Nitrogen (BUN)
H2N-C(=O)-NH2
What does the efficiency at which urea is cleared depend on?
Depends on the urine flow rate (because its passively reabsorbed)
- low flow rates ~ 20% of filtered load excreted (more reabsorbed)
- high flow rates ~ 70% of filtered urea excreted (less reabsorbed)
*In volume depletion → fall in urine flow rate → less excretion of urea → fall in GFR
What is the significance of the plasma urea:creatinine concentration ratio?
- Creatinine is filtered but not reabsorbed → clearance ~ GFR → independent on urine flow rate
- Urea filtered, then reabsorbed following H2O reabsorption (passively) → dependent on urine flow rate
Urea:creatinine ratio → Provides a useful indicator of effective blood volume and renal perfusion
Elevation of BUM/creatinine ration is a useful clinical sign of reduced effective blood volume
*in volume depletion, clearance of urea is more impaired than clearance of creatinine
What is creatinine? And its importance in the kidney?
Creatinine = product of muscle metabolism → filtrered, but not reabsorbed
Clearance of creatinine ~ GFR, independent urine flow rate
Plasma creatinine ~ 0.5 - 1.3 mg/10mL → rises if fall in GFR
How does reabsorption of small proteins and oligopeptides occur?
Glomerular barrier restricts proteins > 10 kDa → some albumins, immunoglobulins, small proteins (hormones) are filtered and must be reabsorbed
Reabsorption in the proximal collecting tubule:
1. Small peptides are borken → AA (enzymes at the brush border) → apical Na+/AA symport
2. Short peptides (2-4AA) → by oligopeptide cotransporter as symport with H+
3. Receptors on apical membrane bind full proteins non-specifically → endocytosis → endosomes → fused to lysosomes → AA → facilitated diffusion on the basolateral side (saturable, non-selective)
What are the names f the receptors on the apical membrane responsible for reabsorption by endocytosis of small proteins in the PCT?
Megalin and cubilin = receptors → saturable, non-selective endocytosis
What is the structure/mechanism of action of megalin and cubilin?
Endocytic receptors on proximal tubule apical membrane
Megalin → TM domain + cytosolic C-term + 4x complement-type repeat with spacer region (containing YWTD) + EGF-type repeats between the complement-type repeats
Cubulin → anchor protein with NH2 + EGF-type repeats + CUB domains
Both receptors work as a complex, stick out of the brush border and move along the microvilli → pit → pinches off → endosome → recycled to the brush border by Dense apical tubules
