6. Acid Secretion by the Kidney

HH++ SECRETION SECRETION AND ACID BASE AND ACID BASE

BALANCEBALANCEProf Harbindar Jeet SinghProf Harbindar Jeet Singh

Faculty of MedicineFaculty of MedicineUniversiti Teknologi MARAUniversiti Teknologi MARA

OBJECTIVES

1. Explain the importance of regulation of pH and the role of the kidneys and lungs

2. Explain the Henderson Hasselbach equation

3. Renal handling of H+

4. Explain acidosis and alkalosis.

Acid base balance

This in essence actually means regulating the H+ concentration.

An increase or decrease in the H+ concentration can affect normalcell function.

The normal H+ concentration in arterial plasma is 0.00004 mmol/L.

This is usually expressed as pH, which is a negative logarithm.

A H+ conc of 0.0004 mmol/L is equal to a pH of 7.4.

pH equivalent to – log10[H+]

A change in pH from 7.4 to 6.4 e.g. represents a 10-fold increase in [H+ ]

When pH is below 7.4 , acidosis is present, and when pH is above 7.4 alkalosis is present.

Consequences of disturbances in pH

Most biologically important molecules contain chemical groups that either donate or accept an H+. The have a net electrical charge.

A pH shift will affect their ability to donate or accept H+ and consequently their net electrical charge or valency.

This in turn will alter a molecule’s conformation and thus its biological activity.

Some pH sensitive molecules include,

a variety of enzymesreceptors and their ligandsion channelstransportersstructural proteins

e.g. the activity of the Na-K pump falls by about half when the pH shifts by 1 unit. the activity of phosphofructokinase falls by 90% when pH decreases by 0.1

Buffers to minimise change in pH

A buffer is a substance that has the ability to reversibly bind or release H+ in solution, thus keeping the pH relatively constant.

The general equation for a buffer system is

HA H+ + A-

According to the law of mass action, the product of the conc of products of the chemical reaction divided by the product of the conc of reactants at equilibrium is a constant (equilibrium constant, K)

[H+][A-] = K HA

[A-]Rearrange this formula, pH = pK + log

[HA]

This is the Henderson-Hasselbalch equation.pH = pK + log [HCO3

-] [PaCO2]

Buffers in blood

a) Plasma proteinsb) Haemoglobinc) Carbonic acid-bicarbonate system

Interstitial fluid

a) Carbonic acid-bicarbonate systemb) proteins

Intracellulara) Intracellular proteinsb) H2PO4

- H+ + HPO42-

The pH of the extracellular body fluids is controlled by three major systems

1. The chemical acid-base buffering by the body fluids that immediately

combine with acids or base to prevent excessive changes in pH.

2. The respiratory center, which regulates the removal of volatile CO2 as a gas in the expired air from the plasma and therefore also regulates bicarbonate (HCO3

-) from the body fluids via the pulmonary circulation (open system).

This response occurs in minutes.

3. The kidneys, which can excrete either acid or alkaline urine, thereby adjusting the pH of the blood (controls plasma HCO3

-).

This response takes place over hours or even days, but represents a more powerful regulatory system.

4. Phosphate buffering

While the phosphate buffering system is not important in buffering the extracellular fluid, it is very important in buffering intracellular pH and also in buffering renal tubular fluid.

The system is composed of H2PO4- and HPO4

-2

For example, addition of a strong acid like HCL leads to

HCl + Na2HPO4 - NaH2PO4 + NaCl

Replacing the strong acid with a weak acid minimizing the change in pH.

The pK of this buffer system is 6.8, so it operates near its maximum buffering power at normal pH.

Although the lungs excrete a large amount of CO2, a potential acid formed by metabolism, the kidneys are crucial for excreting non-volatile acids, such assulphuric acid, phosphoric acid, and various organic acids.

Metabolism also generates non-volatile base, like HCO3-, but subtracting

these from the acids still leaves a net endogenous H+ production of 40 mmole/day in a 70 kg man.

There is a net acid uptake of 30 mmole/day from the gut due to a 10 mmole/dayof obligatory loss of bases in the stool.

On the average the body is faced with a total load of non-volatile acids of about70 mmole/day.

Or 1 mmole/kg body weight.

The kidneys handle this by excreting 70 mmole of H+ into the urine and simultaneously adding 70 mmole/day of new HCO3

- into the blood.

In the blood this HCO3- neutralises the 70 mmole of nonvolatile acid

Acid base balance for a 70 kg man on a typical western diet

The kidneys have two major roles in acid-base homeostasis

1. Reabsorption of bicarbonate filtered at the glomerulus(4000-4500 mmol/day)

2. Excretion of acid (by the A-type/α intercalated cells of the collecting duct), and ammonium NH4

+ (by the proximal tubule) to accomplish production of new HCO3

-

Both these functions rely on H+ secretion

Reabsorption of filtered bicarbonate

Under normal circumstance, the kidney reabsorbs all of the filtered bicarbonate.

The proximal tubule accounts for nearly/over 80% of the reabsorption.

Without this reabsorption, as happens in proximal renal tubular acidosis(Type 2), HCO3

- is lost in the urine, lowering plasma HCO3- and causing

metabolic acidosis.

Tubular reabsorption of bicarbonate

The proximal tubule reabsorbs 80 % of the filtered HCO-3

Absorption of filtered HCO-3 as follows

1. Apical secretion of H+ by either Na+/H+ exchanger (NHE3) and an H+-ATPase into the tubular lumen

2. Reaction of H+ with HCO-3 to form CO2 and H2O in the lumen

3. Diffusion of CO2 into the cell

4. Reaction of CO2 with H2O in the cell to produce H+ and HCO-

3

5. Basolateral transport of HCO-3 into the blood

Each of the reaction between CO2 + H2O H+ + HCO-3

or H+ + HCO-3 CO2 + H2O is catalysed by membrane

bound or cytosolic carbonic anhydrase

The source for the H+ is either from the dissociation of H2CO3 in the cell or the hydrolysis of H2O, the former being the more likely.

CO2 + H2O H+ + HCO3-

Na+

The apical membrane Na+/ H+ exchanger is NHE3

It is competitively inhibited by amiloride

Factors affecting proximal tubular bicarbonate reabsorption

i) Decrease in peritubular pH increases HCO3- reabsorption

ii) Potassium depletion increases HCO3- reabsorption

? Low K+ inducing intracellular acidosis causing increases in apical and basolateral transporters

iii) Increases in luminal HCO3- concentration, accompanied by

increased luminal pH, increases HCO3- reabsorption through

increase in the rate of H+ secretion leading to increase in cell pH and increased basolateral Na+/ HCO3

- co-transport

iv) Increased luminal flow increases HCO3- reabsorption

v) Volume expansion leads to a reduction in HCO3-

reabsorption

vi) Hormones

a) ET-1-Acidosis and decreases in intracellular pH increase ET-1 synthesis in the microvascular and proximal tubular cells.

By binding to the ETB receptor ET-1 in low concentration stimulate apical Na+/H+ exchange, as well basolateral Na+/ HCO3

-

b) Glucorticoids -Increase Na+/H+ exchange by possibly increasing NHE3 expression

c) Parathyroid Hormone - acutely decreases proximal tubular HCO3

- reabsorption via increases in cAMP

Chronically, PTH increases acid excretion but more in the loop of Henle and distal tubule

d) Angiotensin II – Increases HCO3- reabsorption

Ang II stimulates HCO3- reabsorption and increases basolateral

Na+/HCO3- transport

The mechanisms of these responses include decreased cAMP,activation of PKC, and activation of the tyrosine kinase pathways

The thick ascending limb of Henle (TAL) is also involved in the reabsorption of HCO3

-

HCO3- reabsorption is Na+ dependent and is mediated by

Na+/H+ exchanger, which is inhibited by amiloride

About 10-20% of the filtered load of HCO3- is reabsorbed

here

Bicarbonate reabsorption in the TAL

iv) Hypertonicity inhibits HCO3- reabsorption

iii) Ang II inhibits HCO3- reabsorption in the TAL

ii) Increases in dietary sodium increase TAL HCO3-

reabsorption

i) TAL HCO3- reabsorption increases as luminal HCO3

- conc increases

A number of factors affect HCO3- reabsorption in the TAL

STEP 1.

Neutralization of the H+ load by extracellular HCO3-.

HCO3- + H+ = CO2 + H2O Equation -1

Consequently the bicarbonate levels decrease in proportion to the H+ load producing CO2 in the process, which is eliminated by the lungs.

The remaining H+ load is neutralised by the non-HCO3- buffers.

B- + H+ = BH Equation 2

Consequently these buffers too decrease.

A small fraction of H+ escapes (0.001%) buffering, and is responsible for the drop in blood pH.

After reabsorbing most of the filtered bicarbonate the kidney still has to deal with the 70 mmole of acid that has been produced.

The body’s response to the extra 70 mmole/day of acid challenge occurs in 3 major steps

STEP 2

The lungs excrete the CO2 formed in Equation 1

The body does not excrete BH formed in Equation 2 but rather converts it back to B-, where more bicarbonate is used.

BH + HCO3- = B- + CO2 + H2O

The lungs excrete the CO2.

STEP 3

The kidneys regenerate the HCO3- to replenish the used HCO3

-

Thus in a day approximately 70 mmole more HCO3- exits the kidneys

via the renal vein than that which entered the kidney via the renal artery.

How does the kidney generate HCO3-?

Ammonium secretion and bicarbonate synthesis

Although all nephron segments are capable of producing NH4+,

the proximal tubule is quantitatively the most important.

It is a segment where there is adaptation to acidosis, in terms of NH4

+ production and the presence of the key ammoniagenesis enzymes

NH4+ synthesis results predominantly from the metabolism of

glutamine

During this process the kidneys not only produce NH3/NH4+ but

also for each NH3 /NH4- produced the kidney returns one HCO3

-

to the blood.

The kidneys generate bicarbonate through ammonium synthesis

Major pathway for ammoniagenesis in the proximal tubule

GA - Glutaminase-1GDH - Glutamate dehydrogenaseOAA - OxaloacetateTCA - Tricarboxylic acid cyclePEPCK – phosphoenolpyruvate carboxykinaseKG - -ketoglutarate

Acid excretion

In addition to the regeneration of bicarbonate by the kidney, the kidney also secretes H+.

Secretion of H+ occurs in the proximal tubule, the distal tubule and collecting duct.

The secreted H+ can combine with filtered HCO3-, filtered phosphate

to form the titratable acid, and combine to NH3- to form NH4

-

H+ moves across the apical membrane from tubule to lumen by three mechanisms:

i) Na-H Exchanger (NHE)ii) Electrogenic H+ pumpiii) H-K exchange pump

Na-H exchanger (NHE)

It is responsible for the largest fraction of net H+ secretion in particularNHE3.

It is present in the proximal tubule, TAL and DCT.

The steep lumen to cell Na gradient drives this exchange process, butSecretion ultimately depends upon the basolateral Na-K pump.

Electrogenic H+ pump

This is mainly located in the intercalated cells (α cells) of the CCT, cells of the IMCD, OMCD. They are however also found in the apical membrane of the proximal tubular cells and TALH.

H-K exchange pump

This is present in the CCT and OMCD

If we were to add 70 mmole of nonvolatile acid to 1.5 litres of urine, the urinepH will be down to 1.3.

The lowest urine pH possible is 4.4. How does the kidney manage this?

It solves this by binding the H+ to buffers in the urine.

Examples of these buffers include phosphate, creatinine, urate and NH3/NH4+

Through adaptive increases in the synthesis of NH3 and excretion of NH4+ the

kidneys can respond to the body’s need to excrete increased loads of H+.

The contribution of creatinine and urate is small.

Similarly, the amount of phosphate filtered is limited or relatively fixed, and only afraction of the secreted H+ can be buffered by the phosphate.

Therefore the more important urine buffer is NH3

Problem

Acid base balance for a 70 kg man on a typical western diet

Arterial Plasma

Cause of the pH Pco2 [HCO3-] Type of disturbance (mm Hg) (mM/L) disturbance

Prolonged 7.55 44 37 Met. Alkalosisvomiting

Ingestion of 7.18 28* 10 Met. AcidosisNH4Cl

Hysterical 7.57 24 21 Resp. Alkalosishyperventilation

Emphysema 7.33 68 34 Resp. Acidosis

Normal values: pH = 7.37-7.42; [HCO3-] = 23 – 25 mM/L; Pco2 = 37-43 mm Hg

The major pH disturbances are (i) respiratory acidosis and alkalosis, and (ii) metabolic acidosis and alkalosis.

* Kussmaul respirations

Renal response to acid base disturbances

Respiratory acidosis

Where the primary disturbance is an increase in arterial Pco2, the compensatory response is

- increased NH4- secretion

- increased production of new HCO3-

- increased secretion of H+

Respiratory acidosis stimulates H+ secretion in two ways

- elevated Pco2 directly stimulates H+ secretion- upregulation of acid-base transporters

in chronic acidosis

Respiratory alkalosis

The opposite occurs in respiratory alkalosis. There is

- decreased H+ secretion- decreased NH3/NH4

- secretion.

Metabolic acidosis

The first response to metabolic acidosis is increased alveolar ventilation.

The kidneys also participate in the compensatory mechanism, providedthe acidosis is not due to renal failure or renal tubular acidosis, etc.

In acute - increased proximal H+ secretion- enhanced HCO3

- fluxes- reduced HCO3

- backleak

In chronic metabolic acidosis - similar to changes for respiratory acidosis

Metabolic alkalosis

The response in acute

- decreased HCO3- reabsorption

- decreased HCO3- generation

- decreased H+ secretion- increased paracellular HCO3

- backleak intotubular lumen.

In more severe or chronic metabolic alkalosis, there may also beHCO3

- secretion.

Metabolic alkalosis over a period of days shifts the intercalated-cellpopulation from α to β cells.

The β-intercalated cells will secrete HCO3- into the lumen

Anion gap (AG)

Plasma like all other body fluids is neutral; total anions match total cations.

The major plasma cation is Na+ and the major cations are Cl- and HCO3-.

AG is calculated from the following formula:

Anion gap = [Na+] - [Cl-] - [HCO3-]

Normal range is 8 to 12 mmol/l.

AG represents the concentration of all the unmeasured anions in the plasma. The negatively charged proteins account for about 10% of plasma anions and make up the majority of the unmeasured anion represented by the anion gap under normal circumstances. The acid anions (e.g. lactate, acetoacetate, sulphate) produced during a metabolic acidosis are not measured as part of the usual laboratory biochemical profile. The H+ produced reacts with bicarbonate anions (buffering) and the CO2 produced is excreted via the lungs (respiratory compensation). The net effect is a decrease in the concentration of measured anions (i.e. HCO3) and an increase in the concentration of unmeasured anions (the acid anions) so the anion gap increases.

Major Clinical Uses of the Anion Gap 1. To signal the presence of a metabolic acidosis and confirm other findings

2. Help differentiate between causes of a metabolic acidosis: high anion gap versus normal anion gap metabolic acidosis. In an inorganic metabolic

acidosis (e.g. due HCl infusion), the infused Cl- replaces HCO3- and the anion

gap remains normal. In an organic acidosis, the lost bicarbonate is replaced by the acid anion which is not normally measured. This means that the AG is increased.

3. To assist in assessing the biochemical severity of the acidosis and follow the response to treatment

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