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1

What are the components of the loop of henle? What percentage of the filtered water and NaCl and reabsorbed there? What causes changes in plasma osmolality? How does the loop of Henle handle this? Why is hypoosmolality dangerous?

The loop of Henle consists of the thin descending limb, thin
ascending limb, and the thick ascending limb. The thick limb
is further subdivided into medullary (mTAL) and cortical
segments (cTAL). In the interest of clarity, this chapter will
assume that the ascending limb is physiologically homogenous
(hereafter referred to as the mTAL). The loop reabsorbs
25% of the filtered NaCl but only 15% of the filtered water.
The dissociation of NaCl and water reabsorption underlies the
excretion of urine with an osmolality that differs from plasma.
Figure 7.1 illustrates the importance of urine dilution and concentration
in the regulation of plasma osmolality. Ingestion
of a water load will transiently reduce the plasma osmolality
(referred to as hypoosmolality). The kidney preserves water
balance by producing dilute urine. The generation of dilute
urine is a consequence of active NaCl reabsorption in the
mTAL, as discussed below. In contrast, water loss (eg, sweating,
respiration, stool losses) increases the plasma osmolality
(hyperosmolality). Importantly, the kidney defends against an
increase in osmolality by reducing urinary water loss (i.e., urinary
concentration).


44 C l i n i c a l Impl i c at i o n
Alterations in plasma osmolality can be life threatening.
For example, plasma hypoosmolality promotes movement
of water from the extracellular compartment to the
intracellular compartment resulting in cell swelling.
Although many cells can tolerate acute increases in cell
volume, this is not the case with brain cells. Since the
cranium is a rigid compartment, a relatively small increase
in brain water (5%-10%) engenders a large increase in
intracranial pressure. Elevated intracranial pressure can
precipitate a variety of neurological disturbances including,
lethargy, disorientation, nausea, seizures, and coma. A
10%-20% increase in brain volume can result in brain
herniation and death

2

What allows the loop of Henle to change the osmololality of urine? Explain how it works. What modulates the concentration of urine?

Production of urine with an osmolality differing from plasma
derives from the unique structural and functional properties
of the descending and ascending limbs of the loop of Henle,
respectively (Fig. 7.2). The descending limb is highly permeable
to water, but impermeable to NaCl. The ascending limb
actively reabsorbs NaCl, via the Na-K-2Cl cotransporter
(NKCC2, in Fig. 7.3), but is impermeable to water. The excretion
of dilute or concentrated urine ultimately depends on the
circulating concentration of antidiuretic hormone (ADH),
which, when increased, significantly increases water permeability
of the collecting duct (described below).

3

Describe ion transport on the mTAL. What are the channels involved, what might prevent them from working correctly, and what are the results of that? How does the mTAL contribute to dilution of urine? How does it contribute to acid base balance? What effect does calcium have and how?

The epithelial cells lining the mTAL are endowed with unique
transport proteins and ion channels (Fig. 7.3). Sodium entry
(energized by the basolateral Na/K-ATPase) occurs via the
NKCC2, an electroneutral, and furosemide-sensitive cotransporter,
located on the apical membrane. Loop diuretics (furosemide, bumetanide, torsemide) inhibit NKCC2 by
binding to the chloride site and inducing a conformational
change in the protein. NKCC2 is a member of the solute
carrier family of proteins, specifically SLC12. Sodium entry
requires the simultaneous operation of several transport proteins
in addition to NKCC2, including
• The renal outer medullar potassium channel (ROMK).
• Kidney specific chloride channel-B (CLC-KB).
Potassium ions are recycled back into the tubular lumen
via ROMK. Recycling of potassium plays a pivotal role in
the function of NKCC2. Since the potassium concentration
of the tubular fluid is low (~4 mEq/L), continuous recycling
is essential to prevent depleting luminal potassium to critically
low levels resulting in NKCC2 pump failure. Loss of
function mutations in ROMK are associated with salt-wasting,
polyuria, volume contraction, and low blood pressure.
Potassium recycling also creates a positive luminal potential
(nearly +10 mV relative to the basolateral membrane),
which, in turn, promotes paracellular reabsorption of various
cations, including magnesium, calcium, sodium, and potassium.
These ions undoubtedly pass through specific tight junction ion channels (ie, paracellin-1/claudin-16). Intracellular
chloride exits the cell via CLC-KB, which consists of
two subunits. Since the mTAL epithelial cells do not express
aquaporins, they are impermeable to water. Thus, urinary
dilution occurs in this segment (the mTAL is referred to as
the diluting segment of the nephron). The mTAL also contributes
to acid-base homeostasis since ammonium can substitute
for potassium on the NKCC2 (see Chap. 13 ). It has
long been known that an increase in serum calcium inhibits
NKCC2 function and engenders a diuresis. Recent studies
suggest that the calcium sensing receptor (CaSR), a member
of the class C superfamily of G-protein coupled receptors, is
expressed on the basolateral membrane of the mTAL epithelial
cells.

4

What are the symptoms of Bartter Sydrome? What are the etiologies?

In 1962, Bartter described a syndrome characterized
by salt-wasting, hypokalemia, hypomagnesemia and
metabolic alkalosis. The clinical manifestations of the
syndrome were analogous to those described in patients
abusing diuretics, suggesting impaired function of NKCC 2. The molecular basis of the Bartter syndrome has recently
been characterized and includes inactivating mutations
of NKCC 2 (Bartter type 1), ROMK (Bartter type II), and CLC -
KB (Bartter type III and IV). Activating mutations of CaSR
produce Bartter type V. Since each of these mutations
produces a similar phenotype, it is likely that impaired
function of NKCC 2 is ultimately responsible (see Fig. 7.3).

5

How great is the range of urine osmolality? How does osmolality change in the different parts of the kidney? What is the tonicity of the interstitium like? How is this established? What is the final factor in determining the osmolality of urine? Explain?

Urine osmolality in humans can vary from as low as 50 mOsm/kg
H2O, to as high as 1200 mOsm/kg H2O (approximately fourfold
higher than plasma osmolality (~290 mOsm/kg H2O).
These adaptations occur rapidly. For example, after an overnight
fast the urine osmolality may exceed 1000 mOsm/kg
H2O. In contrast, within hours of consuming water, the urine
osmolality may fall to as low as 50 mOsm/kg H2O. In mammalian
kidneys, the osmolality progressively increases from
the cortex to the papillary tip (see Fig. 7.5). The corticomedullary
gradient is less during diuresis compared to antidiuresis
(also see Fig. 7.7)

The generation of a hypertonic medullary interstitium
requires active transport of NaCl (without water) in the mTAL,
which creates a transverse concentration gradient between the
lumen and surrounding interstitium at each level (known as the
single effect, Fig. 7.4). The hairpin configuration of the loop
of Henle augments the transverse gradient (see countercurrent
multiplication, later in this chapter). In general, the longer
loop nephrons have the greatest capacity to concentrate the
urine. The final urine osmolality is ultimately determined by
the water permeability of the collecting duct, which, in turn, is
regulated by the circulating level of vasopressin (also known
as antidiuretic hormone). In the absence of ADH, the urine is
excreted osmotically unchanged (dilute). In the presence of
ADH, water moves down its concentration gradient into the
surrounding interstitium, producing concentrated urine.

6

Explain the single effect.

Figure 7.4 depicts the steps involved in generating dilute urine
in tandem with a concentrated interstitium and descending
limb. Although fluid is flowing continuously through the loop
(up and down arrows), the illustrations in Figs. 7.4 and 7.5
employ stop flow images (horizontal dashed line) to simplify
visualizing the process. At time zero the fluid in the entire loop
(as well as the surrounding interstitium) is osmotically equivalent
to plasma. In step 1, a transverse concentration gradient
is generated by extrusion of NaCl by the NKCC2 into the
surrounding interstitium. The transverse gradient established
is approximately 200 mOsm/kg H2O in humans. Because the
mTAL is impermeable to water the tubular fluid concentration
falls relative to plasma. In step 2, the water in the thin
descending limb equilibrates with the hypertonic surrounding
interstitium. Notably, the expression of aquaporin (specifically,
AQP-1) is abundant in the descending limb, but absent
in the ascending limb. Dilution of the interstitium (because
of passive water movement from the descending limb) is prevented
by ongoing active NaCl transport. The net effect of
these steps is the generation of dilute urine in the ascending
limb, while concentrating the surrounding interstitium and
descending limb (relative to plasma!).

7

Explain countercurrent flow.

Figure 7.5 depicts the effect of countercurrent flow on the
transverse gradient discussed above. In essence, countercurrent
flow multiplies the single effect as follows. A transverse
gradient of 200 mOsm/kg H2O is established in step 1. In step
2, a hyperosmotic column of fluid from the descending limb
moves into the ascending limb. In addition, isoosmotic fluid
from the proximal tubule enters the descending limb. In step 3,
the transverse osmotic gradient of 200 mOsm has been reestablished
by active NaCl transport in the mTAL and passive water
movement from the descending limb. Notably, the fluid exiting
the ascending limb is more dilute after step 3 (compared to
step 1), in concert with a higher osmolality in the surrounding
interstitium and descending limb. Steps 4-5 represent another iteration of the process. The maximum osmolality at the papillary
tip is dependent on the length of the descending limb and
the size of the transverse gradient (which is approximately 200
mOsm at each level of the loop). For example, chinchillas have
very long loops of Henle and can produce a urinary osmolality
that exceeds 8000 mOsm/kg H2O.

8

Generally, explain what determines the final urine osmolality.

The urine exiting the ascending limb is dilute with respect to
plasma (~100 mOsm/Kg H2O). The early distal convoluted
tubule may further dilute the urine to as low as 50 mOsm/kg
H2O (via active NaCl transport). However, the final urine
osmolality is dependent on the water permeability of the collecting
duct. In the absence of ADH the water permeability of
the entire collecting duct is very low. Under these conditions
the urine will be excreted osmotically unchanged (dilute).
This is the appropriate response to a water load. Elevations in
circulating ADH produce a marked rise in the water permeability
of the collecting duct. Water will equilibrate with the
surrounding hyperosmotic interstitium resulting in excretion
of concentrated urine. ADH secretion is sensitive to changes in
plasma osmolality, which trigger “osmoreceptors” that reside
in the hypothalamus (see Chap. 9). An increase in osmolality
activates these osmoreceptors and elicits an increase in plasma
ADH, whereas a decrease in plasma osmolality suppresses
ADH release.

9

Explain the molecular (cellular) mechanism of ADH. Explain the pathophysiology of diabetes insipidus.

The cellular mechanism by which ADH increases the water
permeability of the collecting duct is depicted in Fig. 7.6.
Circulating ADH binds to a G-protein linked receptor (vasopressin-
2 receptor or V2R) on the basolateral membrane of
the principal cell. Other vasopressin receptor isoforms include
V1a and V1b/V3. These receptors are primarily expressed in
vascular smooth muscle, whereas, V2R, is only expressed in
kidney and inner ear. Activation of V2R stimulates adenylyl
cyclase yielding intracellular cyclic AMP. Cyclic AMP
induces a cascade of protein phosphorylation reactions, which
culminate in the recruitment of water channels (aquaporin-2,
AQP2) into the apical membrane. The specific proteins
involved in this shuttling mechanism remain incompletely
understood, although, SNAP/SNARE proteins are almost certainly
involved. When ADH levels are low, AQP2 is recycled
back into the cell via an endocytic mechanism.

Diabetes insipidus is a clinical syndrome characterized by
failure to maximally concentrate the urine. This syndrome is
associated with a defect in vasopressin release or synthesis,
dysregulation of AQP2 shuttling (the drug Lithium is a well
known offender), and loss of function mutations in AQP2 or
V2R. V2R mutations are predominately X-linked, while the
AQP2 mutations are typically autosomal recessive.

10

Explain how urea affects the concentration of urea.

Urea is a small molecule that is generated in the liver during
protein catabolism. It has long been known that nitrogenous waste is primarily excreted as urea. In addition, several studies
have shown that protein deprivation (which reduces urea
generation) is associated with impaired urinary concentration.
These early observations permitted expression cloning of the
kidney urea transporter in the early 1990s. Two urea transporter
genes have now been cloned:
• Urea transporter-A (UT-A)
• Urea transporter-B (UT-B)
Multiple isoforms of the each gene have also been characterized,
although, the functional significance of the isoforms
remains incompletely understood. UT-A1 is expressed in the
inner medullary collecting duct and UT-A2 is confined to the
descending limb of Henle. UT-A3 is primarily expressed on
the basolateral membrane of the inner medullary collecting
duct cells. ADH markedly increases the urea permeability of the inner medullary collecting duct by inducing recruitment of
UT-A1 to the apical membrane (analogous to the AQP shuttling
mechanism). This leads to the accumulation of urea in
the inner medulla because of passive diffusion of urea. Passive
urea diffusion occurs during antidiuresis because of the unique
permeability characteristics of the collecting duct (Fig. 7.7). In
the cortical and outer medullary collecting duct, water is reabsorbed
under the influence of ADH; however, urea permeability
is low. Under these conditions, the urea concentration of tubular
fluid entering the inner medullary collecting duct is high. This
favors passive diffusion into the surrounding inner medulla.
Urea accumulation in the inner medullary interstitium increases
interstitial osmolality. Loss of function mutations affecting
UT-A1, decrease the urea concentration in the inner medulla
and produce an osmotic diuresis (urinary excretion of urea obligates
water loss) and decrease maximal urine concentration.

11

Explain urea recycling.

Two urea recycling pathways contribute to the high urea
concentration in the inner medulla (see Fig. 7.7). The majority
of urea recycling occurs from inner medullary collecting
duct (UT-A1/UT-A3) to the thin descending limbs (UT-A2).
UT-B (which is identical to the Kidd blood group antigen)
is expressed mainly in the descending vasa recta endothelial
cells. Urea diffuses into the interstitium from the descending
vasa recta, ensuring a high concentration of urea in the inner
medulla. Interestingly, humans that do not express the Kidd
antigen have impaired maximal urinary concentration (usually
less that 800 mOsm/kg H2O).

12

Explain countercurrent exchange.

The descending and ascending vasa recta deliver blood to the
inner medulla. These capillaries are arranged in a countercurrent
configuration that envelops the loop of Henle. These
vessels are highly permeable to solute and water. Passive diffusion
of solute from the surrounding hypertonic interstitium
to capillary, and water from the capillary to the surrounding
interstitium occurs in the descending limb of the vasa recta.
This results in osmotic equilibration within the capillary
(Fig. 7.8). If hypertonic blood were to exit the kidney at the
papillary tip, substantial solute would be removed and the
effects of countercurrent multiplication would be dissipated.
However, the countercurrent configuration of the vasa recta permits solutes and water to re-equilibrate as the capillary
ascends the surrounding interstitium (see Fig. 7.8). The
exchange of water and solute between the vasa recta and surrounding
interstitium is known as countercurrent exchange.

13

Explain how solute and water is reabsorbed in the loop of Henle and why its important.

Approximately 25% of the filtered load of sodium (1500
mEq/d) and 15% of the filtered load of water (27 L) are reabsorbed
in the loop of Henle. The vasa recta transports solute
and water back to the systemic circulation. Since the equilibration
of solute and water in the descending and ascending
vasa recta proceeds at a slightly different rate the osmolality
of fluid in the ascending vasa recta (as it exits the medulla) is
higher than the fluid that enters via the descending vasa recta.
Moreover, the bulk flow rate of fluid exiting the ascending
limb is greater than that in the descending limb. These disparities
in osmolality and flow rate account for the ongoing
removal of the solute and water by the loop of Henle. Steadystate
removal of solute and water from the loop is important to
prevent uncontrolled swelling of the medulla.

14

Why is oxygen tension reduced in the medulla? What are the clinical consequences?

In all mammalian species (including humans), renal medullary
oxygen tension is remarkably low. For example, the partial
pressure of oxygen in the renal medulla averages only 10-20
mm Hg as compared to 50-60 in the renal cortex. The mechanism
responsible for renal medullary hypoxia include
• High oxygen consumption because of the robust energy
requirements of the mTAL.
• Countercurrent exchange reduces oxygen tension by
promoting oxygen diffusion from the descending vasa
recta into the ascending vasa recta.
• Relatively meager blood supply to the renal medulla.
The blood flow in the medulla averages 1.9 cc/min/g
relative to cortex, which is 4.2 cc/min/g.

The renal medulla is uniquely susceptible to ischemic injury
(also known as acute kidney injury). This type of injury
is characterized by necrosis and apoptosis of the mTAL
epithelium. Interestingly, inhibitors of transport in the thick
ascending limb, such as loop diuretics, increase medullary
partial pressure of oxygen from approximately 20 to
>40 mm Hg, and have proven useful in animal models of
ischemic acute kidney injury. Unfortunately, human clinical
trials have not been able to confirm these promising
animal studies.