Although the concentrating process in the
renal outer medulla is well understood, the concentrating mechanism in
the renal inner medulla remains an enigma. The purposes of this review
are fourfold. 1) We summarize a theoretical basis for
classifying all possible steady-state inner medullary countercurrent
concentrating mechanisms based on mass balance principles.
2) We review the major hypotheses that have been proposed to
explain the axial osmolality gradient in the interstitium of the renal
inner medulla. 3) We summarize and expand on the
Schmidt-Nielsen hypothesis that the contractions of the renal
pelvocalyceal wall may provide an important energy source for
concentration in the inner medulla. 4) We discuss the
special properties of hyaluronan, a glycosaminoglycan that is the chief
component of a gel-like renal inner medullary interstitial matrix,
which may allow it to function as a mechano-osmotic transducer,
converting energy from the contractions of the pelvic wall to an axial
osmolality gradient in the medulla. These considerations set the stage
for renewed experimental investigation of the urinary concentrating
process and a new generation of mathematical models of the renal
concentrating mechanism, which treat the inner medullary interstitium
as a viscoelastic system rather than a purely hydraulic system.
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INTRODUCTION |
IN STATES OF FLUID DEPRIVATION or
nonrenal water loss, the kidney can conserve water while maintaining
excretion of solutes. It does this by concentrating the solutes in the
urine to osmolalities that markedly exceed the osmolality of plasma. A
large number of studies, exemplified by the data shown in Fig.
1, have demonstrated that the urinary
concentrating process is associated with the generation of a
corticomedullary osmolality gradient in the medullary tissue, oriented
with the maximum osmolality in the deepest part of the inner medulla,
i.e., at the papillary tip. The classic micropuncture studies of
Gottschalk and Mylle (17) have established that the
medullary hypertonicity is due to solute accumulation in all structures
in the medulla, including loops of Henle, vasculature, and collecting
ducts. The high medullary interstitial osmolality provides a driving
force for osmotic water flow across the collecting ducts, which are
rendered permeable to water through the action of vasopressin
(33). The high water permeability allows osmotic equilibration of urine with the medullary interstitial fluid.
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Fig. 1.
Osmolality gradient in renal medullary tissue of
antidiuretic rabbit. Measurements were made by vapor pressure osmometry
of slices from different levels of rabbit kidney after quick freezing.
The figure is drawn from data from Ref. 32.
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In 1959, Kuhn and Ramel (43) proposed a model to explain
concentration of solutes in the renal medulla based on countercurrent amplification of a small osmotic difference between the ascending limb
and the descending limb of Henle's loop, resulting from active solute
transport out of the ascending limb. Their version of Hargitay and
Kuhn's (22) countercurrent multiplier hypothesis has
become generally accepted as the mode of solute accumulation in the
renal outer medulla and is now supported by extensive experimental
evidence (reviewed in Ref. 55). The key evidence was the
demonstration that the thick ascending limb of Henle's loop is capable
of a high rate of active NaCl transport out of the lumen, which results in luminal dilution owing to the low osmotic water permeability of this
segment (3, 67).
Thus renal physiologists have developed a good understanding of the
process that concentrates solutes in the renal outer medulla. The same
cannot be said for the renal inner medulla, however. The ascending
portion of Henle's loop (the thin ascending limb) has been shown to
have extremely limited, if any, capacity for active transport in the
inner medulla (25, 26, 53, 54, 59, 80). Therefore, in the
inner medulla there is no energy source for a classic Kuhn-Ramel
countercurrent multiplier, and other explanations must be sought for
the medullary osmolality gradient in the renal inner medulla.
The objectives of this paper are 1) to summarize a simple
theoretical scheme for classification of all possible steady-state countercurrent concentrating models in the inner medulla; 2)
to review proposed steady-state models for concentration of solutes in
the inner medullary interstitium; 3) to readdress the
Schmidt-Nielsen hypothesis that energy from smooth muscle contractions
of the pelvocalyceal wall is responsible for concentration of solutes in the inner medulla; and 4) to discuss the possible role of
inner medullary interstitial hyaluronan as a mechano-osmotic energy transducer converting mechanical energy of renal pelvic contractions to
axial osmolality gradients in the inner medulla.
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MASS BALANCE REQUIREMENTS FOR URINARY CONCENTRATION |
Knepper and Stephenson (37) and Knepper et al.
(34) developed a mathematical analysis of concentrating
processes in the renal inner medulla that allows classification of all
possible steady-state countercurrent concentrating models based on mass balance requirements. The full mathematical analysis will not be
repeated here but is summarized concisely in APPENDIX A. This analysis assumes that solutions exhibit ideal behavior and that
chemical reactions have negligible effects. Possible repercussions of
these assumptions will be considered in later sections. We discuss the
approach and the principles that derive from the analysis in the
following paragraphs.
Figure 2A is a diagram of a
unipapillate kidney typical of a rat, rabbit, or mouse. It illustrates
the relative positions of the three major regions of the kidney: the
cortex, the outer medulla, and the inner medulla. The deepest portion
of the inner medulla is a tapering structure, the papilla, whose tip is
the site of exit of urine formed by the kidney. After exiting the kidney at the papillary tip, this urine is carried downward to the
urinary bladder via the ureter. To analyze the processes responsible for generation of the osmotic gradient in the inner medulla, we apply a
"control volume" for mass balance (Fig. 2B), which
creates a boundary to allow us to account for flows into and out of a portion of the inner medulla. The lower end of this control volume is
defined to be at or just beyond the papillary tip. The upper end of the
control volume is arbitrary; it can be drawn at any level of the inner
medulla, for example, as defined by the solid line shown in Fig.
2B or by the horizontal dashed line just below it. The
analysis that we present applies to all such control volumes. Figure
2C shows the same control volume identifying all of the relevant flows into and out of it. Entering flows include those in the
descending vasa recta, the descending limb of Henle's loop, and the
collecting ducts. Exiting flows include those in the ascending vasa
recta, the ascending limbs of Henle's loop, and the final urinary flow
exiting the papillary tip. At steady state, the flow of water, NaCl,
and urea into the control volume must exactly equal the flows out of
the control volume.
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Fig. 2.
Structural basis of steady-state mass balance analysis of
renal inner medulla of a unipapillate kidney. A:
diagrammatic representation of kidney structure. See text for
description. B: cutaway view showing a "control volume"
for analysis of mass balance defined by the heavy solid rectangle. A
series of such control volumes can be defined by moving the upper
border of the control volume upward or downward. An alternative choice
for the upper border is illustrated by the horizontal dashed line. For
steady-state operation of the inner medulla, mass balance must be
maintained for all such control volumes. C: enumeration of
the individual flows into and out of an inner medullary control volume
represented by the rectangle. Each flow stream is the aggregate of
flows in all individual structures of a given type. For example, if the
upper border of the control volume is assumed to be at the inner-outer
medullary junction, the descending limb stream is the aggregate of
11,000 individual descending limbs (35). Asc., ascending;
Desc., descending.
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Figure 3 is a detailed view of the
control volume for a portion of the inner medulla defining the
terminology used. Here, the subscripts have been modified to indicate
explicitly the structure being considered [e.g., descending vasa recta
(DV); ascending vasa recta (AV); descending limb (DL); ascending limb
of Henle's loop (AL); collecting duct (CD); final urine (U),
peritubular interstitium (P)]. Total solute concentrations are
represented by Cj, where the subscript
j designates the structure. Volume flow rates are
represented by Qj. The products Cj · Qj
represent the total solute flow rates into and out of the control
volume. The total solute concentrations in the interstitium are given
by the Cj terms. Using this terminology, the
mass-blance equation (APPENDIX A, Eq. A7) can be
written in terms of the individual structures involved. This equation
can be arranged so that the left-hand side expresses the interstitial
total solute gradient from a given point (point x) along the
inner medulla to the papillary tip L as follows
![<AR><R><C>C<SUB>P</SUB>(<IT>L</IT>) − C<SUB>P</SUB>(<IT>x</IT>) = </C></R><R><C> </C></R><R><C> </C></R><R><C> </C></R><R><C> </C></R></AR><AR><R><C>[Q<SUB>DV</SUB>(<IT>x</IT>)/Q<SUB>u</SUB>] [C<SUB>DV</SUB>(<IT>x</IT>) − C<SUB>P</SUB>(<IT>x</IT>)]</C></R><R><C>+ [Q<SUB>AV</SUB>(<IT>x</IT>)/Q<SUB>u</SUB>] [C<SUB>AV</SUB>(<IT>x</IT>) − C<SUB>P</SUB>(<IT>x</IT>)]</C></R><R><C>+ [Q<SUB>DL</SUB>(<IT>x</IT>)/Q<SUB>u</SUB>] [C<SUB>DL</SUB>(<IT>x</IT>) − C<SUB>P</SUB>(<IT>x</IT>)]</C></R><R><C>+ [Q<SUB>AL</SUB>(<IT>x</IT>)/Q<SUB>u</SUB>] [C<SUB>AL</SUB>(<IT>x</IT>) − C<SUB>P</SUB>(<IT>x</IT>)]</C></R><R><C>+ [Q<SUB>CD</SUB>(<IT>x</IT>)/Q<SUB>u</SUB>] [C<SUB>CD</SUB>(<IT>x</IT>) − C<SUB>P</SUB>(<IT>x</IT>)]</C></R></AR>](/content/vol284/issue3/fulltext/F433/img001.gif)
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(1)
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To have a positive osmotic gradient between point x and
the papillary tip [CP(L) 
CP(x) > 0], at least one of the terms on
the right-hand side must be positive. Each of these terms consists of a
normalized flow multiplied by a total solute concentration difference
("osmolality difference") across a given structure. Such a
transverse osmolality difference has been referred to in the
physiological literature as a "single effect," a literal
translation of the German term "Einzeleffekt" (22,
43). The normalized flow is the absolute flow divided by the
final urinary flow. The flow represents an aggregate of all flows for a
given structure; e.g., QAL(x) is the sum of
flows in all individual ascending limbs at level x. Each
total solute concentration difference in the equation is a potential
single effect, which could account for the positive gradient in the
medulla.
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Fig. 3.
Control volume for analysis of mass balance requirement
for steady-state countercurrent multiplier mechanisms in the renal
medulla. See text for definiton of terms.
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The direction of the concentration difference (single effect) that is
necessary for medullary interstitial concentration is dependent on the
direction of flow in the structure. The flows oriented in the direction
of the papillary tip (DV, DL, and CD) are positive and therefore
require a positive value of
Ci(x) CP(x). The flows oriented away from the
papillary tip (AV and AL) are negative and therefore require a negative
value of Ci(x) CP(x) to obtain a positive axial gradient. For
the ascending limb, the requirement for a single effect is
[CAL(x) CP(x)] < 0, as is the case in the outer medulla due to active NaCl transport out of the thick ascending limb. Table 1
summarizes all possible single effects that could account for a
positive axial interstitial gradient in the inner medulla for
steady-state operation. Potential concentrating models can therefore be
analyzed on the basis of their ability to generate one or more of the
required single effects indicated in Table 1. In summary, steady-state
concentrating models must dilute the ascending limb of Henle or the
ascending vasa recta relative to the surrounding interstitium or,
alternatively, must concentrate the descending limb of Henle, the
descending vasa recta, or the collecting duct relative to the
surrounding interstitium. In the next section, we discuss some of the
models that have been proposed in the context of the mass balance
requirements summarized in Table 1.
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Table 1.
Classification of steady-state inner medullary concentrating models
based on structure responsible for concentrating "single
effect"
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PROPOSED STEADY-STATE MODELS |
Single Effect in the Thin Ascending Limb of Henle
As noted in Table 1, a positive axial osmolality gradient in the
inner medulla could be generated as a result of any process that
dilutes the lumen of the thin ascending limb relative to the
interstitium. The possibility that the thin ascending limb functions
like the thick ascending limb to dilute its lumen relative to the
interstitium by active NaCl transport has been ruled out, as discussed
above. It has been proposed that urea may be actively reabsorbed from
the thin ascending limb (45), although this hypothesis
lacks experimental verification.
A model by which the luminal fluid in the thin ascending limb could be
diluted by purely passive means has been proposed by Kokko and Rector
(39) and by Stephenson (77) (Fig.
4). This model assumes that the luminal
fluid at the bend of the loop contains NaCl as the predominant solute
and that the inner medullary interstitium contains a fluid in which
urea is the predominant solute. It has been noted that the permeability
to NaCl is higher than the permeability to urea in the thin ascending
limb (25). These permeability characteristics predict that
NaCl would escape the lumen more rapidly than urea would enter,
resulting in passive dilution, a prediction that was bourne out by
perfused tubule experiments in vitro (25). Although this
model appeared promising at first, thorough quantitative analysis did
not support an important contribution of this process to the generation
of an inner medullary osmolality gradient (5, 6, 46, 81, 83,
92). Furthermore, a physiological analysis on the basis of the
known permeability properties, medullary solute concentrations, and
flow rates in medullary structures has led to the conclusion that the
Kokko-Rector-Stephenson passive model could account for only a modest
axial osmolality gradient in the inner medulla (55). One
important discrepancy between the experimental data and the
requirements of the Kokko-Rector-Stephenson model is the urea
permeability of the ascending thin limb epithelium. Measurements in
isolated perfused rodent thin ascending limbs yielded extremely high
values, in the range 38-170 × 10
5 cm/s
(7, 24), which is too high to permit sustained net luminal
dilution along the length of the thin ascending limb. In addition,
measurements of the urea permeability of the descending limb epithelium
in the inner medulla demonstrated values too high to prevent
substantial urea entry into the descending limb (46). It
is beyond the scope of this article to analyze the full evidence in
detail; see Masilamani et al. (55) for a thorough analysis of the feasibility of the Kokko-Rector-Stephenson model.
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Fig. 4.
Proposed function of the thin ascending limb of Henle in
the Kokko-Rector-Stephenson passive model for concentration of the
inner medullary interstitium. The entering fluid and peritubular fluid
are assumed to have the same osmolality (osm) but to have equal and
opposite transepithelial gradients for urea and NaCl. If the thin
ascending limb has the special properties indicated in the text (high
NaCl permeability, low urea permeability, low water permeability),
rapid NaCl efflux would occur without a balancing entry of urea, thus
lowering luminal osmolality below that of the peritubular fluid.
Measurements of urea permeability of the thin ascending limb have
yielded relatively high values, seemingly ruling out the hypothesis
(see text).
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Although the existing literature raises considerable doubt about the
view that the single effect for inner medullary concentration resides
in the thin ascending limb, recent evidence from studies of gene
knockouts of the ClC-K1 chloride channel in mice emphasizes that the
thin ascending limb is nonetheless important in the inner medullary
concentrating mechanism (1, 56). ClC-K1 is expressed exclusively in the thin ascending limb, where it is responsible for
extraordinarily high chloride permeabilities in that segment (85,
86). The knockout mice exhibited a severe concentrating defect
and a failure to substantially concentrate the inner medullary interstitium in association with a low chloride permeability in its
thin ascending limbs. The basis of the defect can be understood from
Eq. A1 in APPENDIX A. As can be
appreciated, if the transepithelial osmolality gradient across the thin
ascending limb were oriented lumen > interstitium, this would
provide a negative term in the equation calculating the axial
osmolality gradient (compare with single-effect condition, Table 1).
This would create essentially a "negative single effect" as fluid
flowed upward from the highly concentrated papillary tip to the
less-concentrated outer medulla. Thus failure of osmotic equilibration
across the thin ascending limb would decrease the axial gradient that
could be generated in the inner medulla by any mechanism. Therefore, high permeabilities to NaCl and urea in the thin ascending limb are
extremely important as a means of preventing dissipation of the inner
medullary solutes by the upward flow in thin ascending limbs as
illustrated by the ClC-K1 knockout studies.
Single Effect in the Descending Limb of Henle
As noted in Table 1, a positive axial osmolality gradient in the
inner medulla could be generated as a result of any process that
increases the osmolality of the luminal fluid in the thin descending
limb relative to the interstitium. If active transport of solute in
this segment were to be implicated in generation of a single effect,
the direction of transport would have to be in the secretory direction,
i.e., into the lumen. Kriz and colleagues (41, 42) have
proposed such a solute secretory mechanism as part of a "cascade
model" of inner medullary concentration, but there is thus far no
evidence to support the presence of active solute transport into the
thin descending limb. Bonventre and Lechene (2) have also
presented a similar concept. They suggested that the tubule fluid of
the long descending limbs in the outer medulla may be hypertonic to the
interstitium of the upper part of the inner medulla because of
selective interaction with the interstitial subregion that surrounds
the thick ascending limbs in the outer medulla. Mathematical modeling
studies by Lory (48) have predicted that even with a
substantial rate of solute transport into the descending limb, other
concomitant processes would decrease the axial osmolality gradient in
the renal medulla. Apparently, the extremely high water permeability of
the descending limb would make it impossible for the thin limb to
sustain the required transepithelial osmolality difference. The high
water permeability of this segment owes to the abundant expression of
the aquaporin-1 water channel in the plasma membranes of the thin
descending limb cells (8, 50).
Given the high water permeability of the thin descending limb, it has
also been proposed that a single effect (luminal osmolality > interstitial osmolality) could be generated as a result of unequal osmotic reflection coefficients for urea and NaCl (34).
The urea concentration in the interstitium is higher than that in the
descending limb lumen, whereas the NaCl concentration in the lumen is
higher than that in the interstitium (15, 29, 52). Under
these circumstances, the urea gradient would tend to drive water out of
the lumen and the NaCl gradient would tend to drive water inward. If
the reflection coefficient for NaCl were lower than that for urea,
osmotic equilibration would occur with a higher NaCl gradient than the
opposing urea gradient, resulting in an osmolality that is higher in
the lumen than the interstitium. That is, a single effect would be
generated (Table 1). This hypothesis has not been experimentally tested
for thin descending limbs from the inner medulla. However, the
hypothesis seems somewhat questionable based on characterization of the
aquaporin-1 water channel, the main pathway for water movement across
the thin descending limb. Fundamentally, a reflection coefficient of
<1 for a given solute requires that the water pathway across the
barrier membranes be permeable to that solute (30).
Measurements of solute permeability of aquaporin-1 heterologously
expressed in Xenopus laevis oocytes or reconstituted into
artificial lipid vesicles indicate that the urea and NaCl
permeabilities are extremely low (64, 95). However,
passive cation fluxes associated with aquaporin-1 have been reported in
X. laevis oocyte expression studies, and these fluxes have
been noted to be increased by cAMP treatment (94). Nevertheless, the chloride permeability remained very low, seemingly ruling out substantial net penetration of NaCl through aquaporin-1. Because the reservations to this model are based on theoretical considerations only, direct measurements in isolated perfused inner
medullary descending limbs will be necessary to rule out the hypothesis
with certainty.
Single Effect in the Collecting Duct
As summarized in Table 1, a single effect accounting for an axial
osmolality gradient in the inner medulla could theoretically be
generated in the collecting duct if the luminal osmolality were
maintained greater than that of the surrounding interstitium throughout
most of the inner medulla. Wexler et al. (93) have proposed one means by which this could happen. Specifically, an extremely hyperosmotic fluid may be generated in the outer medullary collecting duct and delivered to the inner medullary collecting duct
(IMCD). Based on the prior studies of Lemley and Kriz
(47), it has been concluded that the outer medullary
collecting ducts are segregated with the thick ascending limbs in the
outer medulla. Theoretically, the rapid active NaCl transport from the
thick ascending limbs would concentrate the interstitium adjacent to the collecting ducts to a level that greatly exceeds the average osmolality of the outer medullary tissue. This would raise the osmolality to a high level in the collecting duct lumen, and this highly concentrated fluid would enter the IMCDs, providing a single effect (luminal osmolality > interstitial osmolality). A
mathematical model devised by Wexler and colleagues (93)
showed that such a process could result in concentration of the inner
medullary interstitium, but this model required a special condition:
the osmotic water permeability of the initial portion of the IMCD was
required to be very low to prevent the high luminal osmolality from
being dissipated by the water secretion that would otherwise occur.
Experimental studies by Han and colleagues (21) did not confirm that key assumption and instead found a high osmotic water permeability in the initial IMCD, apparently ruling out the model (21). Subsequent papers by Wang and colleagues (87,
88) suggested that the requirement for a low water permeability
in the initial IMCD could be relaxed somewhat if a high rate of rapid active NaCl transport occurs out of the initial IMCD. However, based on
further analysis using a detailed three-dimensional model of the
medullary concentrating process, Thomas and Wexler (83) concluded that
if realistic values of urea permeability in the inner medullary
descending limbs and water permeability in the upper inner medullary
section of the collecting ducts are taken into account, even a model
including the three-dimensional vascular bundle structures fails to
explain the experimentally observed inner medullary osmolality gradient.
A different model for generation of a single effect (luminal
osmolality > interstitial osmolality) in the IMCD, based on a difference in reflection coefficients for urea and NaCl, has been proposed several times (2, 5, 6, 20, 27, 65, 68). The
principle is similar to that proposed for generation of a single effect
in the thin descending limb as discussed above, except that the
directions of the transepithelial urea and NaCl gradients in the IMCD
are opposite those seen in the descending limb. Therefore, the proposed
model depends on the assumption that in the IMCD, the reflection
coefficient for urea is much lower than that for NaCl. Indeed, early
measurements of reflection coefficients in the IMCD seemed consistent
with this assumption [see Morgan and Berliner (58) and
Imai et al. (27), for example]. However,
subsequently the transport proteins responsible for water transport
[aquaporin-2 (14)] and urea transport [UT-A1
(74)] across the apical plasma membrane of the IMCD have
been identified by molecular cloning, providing direct evidence for
independent, highly selective transport pathways for water and urea.
These results provided no evidence for a shared pathway for water and urea transport as required for a true reflection coefficient of <1
(30). Indeed, careful measurements in isolated perfused
tubules demonstrated that the reflection coefficient for urea is
virtually 1 and that the apparent low value of the reflection
coefficient was due to rapid dissipation of imposed urea gradients by
facilitated urea transport (9, 36). A mathematical
analysis of transport of solutes and water across the IMCD indicates
that the presence of unstirred layers (chiefly in the cytoplasm) can
contribute to the presence of an apparent reflection coefficient for
urea of <1 without having nonunity reflection coefficients for
transport across the plasma membranes (19).
In general, models that depend on a single effect in the IMCD are at a
theoretical disadvantage relative to models that depend on a single
effect in the loop of Henle because of the low aggregate tubule fluid
flow rate in collecting ducts relative to the loop of Henle. As can be
seen in Eq. A1 in APPENDIX A, the degree of
countercurrent multiplication is directly proportional to both the
normalized tubule fluid flow rate and the magnitude of the single effect.
Proposed Single-Effect Mechanisms in the Vasa Recta
The ascending vasa recta are lined by fenestrated endothelial
cells (57), which presumably permit free exchange of
solutes and water between the interstitium and the ascending vas rectum lumen. Consequently, the composition of the interstitial fluid has
generally been assumed to be similar to that of the blood plasma in the
ascending vas rectum. In contrast, the endothelium of the descending
vasa recta is continuous, and transendothelial gradients are a
possibility. Thus it is conceivable that the inner medullary
concentrating process could be driven by generation of a single effect
in the descending vasa recta, i.e., a process that maintains the
osmolality of the lumen greater than that of surrounding interstitium
(Table 1). The water permeability of the thin descending limbs is very
high due to the expression of very high levels of the water channel
aquaporin-1 in the plasma membranes of the endothelial cells
(61). Therefore, steady-state models that depend on active
or passive solute transport are unlikely to generate sustained
osmolality gradients, as discussed above with regard to the descending
limb of Henle's loop. Nonetheless, it is conceivable that a process
based on a difference in reflection coefficients for NaCl and urea
could contribute to the concentration of solutes in the inner medullary
interstitium by increasing the luminal osmolality above that of the
surrounding interstitium. The same reservations can be made about this
model in the descending vasa recta as were made for the descending limb
of Henle (see above).
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NONCONVENTIONAL MODELS |
Conventional models of the medullary concentrating process have
assumed steady-state conditions, ideal solutions, and negligible solute
production by chemical reactions (see APPENDIX A). However,
the appropriateness of these assumptions has been questioned. In the
remainder of this review, we examine potential concentrating models
based on reconsideration of these assumptions.
Possible Role of Solute Generation Via Chemical Reactions
In 1994, Jen and Stephenson (28) provided theoretical
justification for the view that generation of some "external
osmolyte" in the inner medulla could provide the driving force for
the inner medullary concentrating process. In their formulation, an
unspecified solute is assumed to be added de novo and continuously to
the inner medullary interstitium. A subsequent mathematical modeling study by Thomas and Wexler (83) using a complex
three-dimensional model of the renal medulla confirmed that addition of
such a solute to the inner medullary interstitium could potentially
explain the axial concentration gradient in the inner medulla by
driving water efflux from the thin descending limb. This would
concentrate NaCl in the descending limb, setting up a favorable
gradient for NaCl efflux from the ascending limb and dilution of the
ascending limb lumen relative to the interstitium. In effect, this
external solute substitutes for urea in the Kokko-Rector-Stephenson
passive model.
What could be the identity of this "external solute"? The model
requires a chemical reaction that generates more osmotically active
particles than it consumes. Thomas (82) has proposed that
the external solute is lactate, which is generated by anaerobic glycolysis (the predominant means of ATP generation in the inner medulla) in the proportion of 2 lactate ions/glucose molecule consumed:
The feasibility of the proposed model depends on the fate of the
H+ ions that are generated. If the H+ ions
titrate HCO
, they will remove two osmotically active
particles (HCO ions), resulting in a net
disappearance of osmotically active particles
Because CO2 readily permeates lipid bilayers, it is
unlikely to be osmotically effective. Alternatively, if the
H+ ions titrate buffers other than HCO, e.g., NH3, phosphate, and proteins with relatively neutral
isoelectric points, a net generation of osmotically active particles
can be expected.
In proposing that lactate generation provides a driving force for the
inner medullary concentrating process, Thomas (82) raises
critical questions about lactate transport in the inner medulla. For
the model to explain the axial osmolality gradient in the inner
medulla, the lactate generated would need to be transported out of the
epithelial and endothelial cells in a polarized fashion, so as to
generate osmotic differences across individual renal tubule segments
(Table 1). Thomas proposes that lactate should be preferentially
transported across the basolateral plasma membranes of all cell types.
Possible Role of Solution Nonideality in the Renal
Medulla
Most models of the urinary concentrating mechanism have assumed
that the tubule fluid and interstitial fluids in the renal medulla
behave as ideal solutions. However, as pointed out by Wang et al.
(89), renal medullary fluids may deviate substantially from ideality under antidiuretic conditions. Their preliminary calculations using a complex multinephron model of the renal medulla indicate that compared with ideal solution models, decreased activity coefficients for urea tend to increase predicted urinary osmolalities, while decreased activity coefficients for NaCl tend to decrease predicted urinary osmolalities. Such nonideal effects probably should
be taken into consideration in any model of the urinary concentrating process.
Non-Steady-State, Periodic Models
The potential role of periodic contractions of the renal pelvic
wall as a source of energy for the concentrating process in the inner
medulla has been emphasized by Schmidt-Nielsen (70). The
renal inner medulla is surrounded by the renal pelvocalyceal wall (Fig.
2A), a structure comprised chiefly of two thick smooth muscle layers (75). The pelvocalyceal wall undergoes
intermittent contractions, which have been seen to compress the renal
medullary parenchyma (71) (Fig.
5). These compressions, occurring at a frequency of 15-40/min in rodents, have been seen to alter flow rates in tubule and vascular structures of the inner medulla (66, 73). The frequency of the contractions is regulated via both sympathetic and parasympathetic inputs (12, 40). Thus
pelvocalyceal wall contractions impart a periodic character to the
function of the inner medulla that has been largely ignored in formal
mathematical modeling studies of the urinary concentrating process. As
pointed out by Schmidt-Nielsen (70), the pelvic wall
contractions could provide an energy input to the concentrating process
itself. In support of this view is the longstanding observation that
disruption of the continuity of the pelvocalyceal wall markedly reduces
the tonicity of the inner medullary tissue and urine (10, 16, 60). Two studies have directly addressed this possibility with differing conclusions. Oliver et al. (60) tested the
effects of paralyzing the upper portion of the ureter (which surrounds the papillary tip) in young rats and found no impairment of
concentrating ability. In contrast, Schmidt-Nielsen et al.
(72) found in hamsters that paralysis of the pelvic wall
(or mechanical damage to the pelvic wall) significantly decreased
concentrating ability. In the remainder of this article, we present a
discussion of ways that periodic contractions of the renal pelvic wall
could concentrate the inner medulla, including consideration of the
role of hyaluronan in the interstitial matrix as a molecular
mechano-osmotic transducer for the concentrating process.
Before a consideration of specific models, it is important to note that
the formulation which defines possible single-effect mechanisms for the
inner medulla given in APPENDIX A applies only to the
steady state. Further work will be needed to extend the analysis to
periodic and other non-steady-state conditions. Nevertheless, it is
reasonable to assume that in the periodic case, single-effect
conditions similar to those presented in Table 1 will apply. In
particular, we assume that positive axial osmolality gradients will be
generated when the contents of the descending limbs (or descending vasa
recta) are concentrated relative to the surrounding interstitium or
when the contents of the ascending limbs (or ascending vasa recta) are
diluted relative to the surrounding interstitium.
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CONCENTRATING MODEL DRIVEN BY RENAL PELVIC WALL CONTRACTIONS:
HYALURONAN AS A MECHANICO-OSMOTIC TRANSDUCER |
Schmidt-Nielsen (70) was the first to emphasize the
remarkable spongelike properties of the renal interstitial hyaluronan matrix and the potential role of hyaluronan in concentration of the
medullary interstitium. In this section, we describe a concentrating model based on the view that the inner medullary interstitium consists
of a semisolid, viscoelastic hyaluronan gel rather than being a freely
flowing aqueous compartment. As outlined in detail below, it is
proposed that the hyaluronan matrix can store the mechanical energy
from the pelvic contractions by direct mechanical compression without
the need to generate high hydrostatic pressures and can utilize this
energy to lower interstitial pressure after completion of each
contraction of the pelvic wall to drive water efflux from the
descending limb of Henle. The latter process would increase the luminal
osmolality to above that of the interstitium, thereby generating a
single effect for concentration of urine. Before laying out this model,
we summarize the necessary background regarding the biochemistry of
hyaluronan and its physicochemical properties.
Hyaluronan (hyaluronic acid) is a member of a family of biomolecules
called glycosaminoglycans (GAGs), which are all unbranched polysaccharide chains composed of repeating disaccharide units. Aside
from hyaluronan, other mammalian GAGs include chondroitin sulfates,
dermatan sulfate, keratan sulfate, heparan sulfate, and heparin.
Hyaluronan differs from the other GAGs in that it is not generally
covalently linked to proteins to form proteoglycans and is not sulfated
(23). Furthermore, in contrast to the other GAGs that are
synthesized in the Golgi apparatus, hyaluronan is produced at the
plasma membrane by an integral membrane protein, hyaluronan synthase
(HAS) (84, 90). Three mammalian HAS genes have been
identified, namely, HAS1, HAS2, and
HAS3. All three produce hyaluronan on the cytoplasmic side
of the plasma membrane and transport it across the plasma membrane to
the extracellular fluid. Thus hyaluronan secretion does not directly
involve vesicular trafficking, in contrast to most other types of
secreted biomolecules. Because of the importance of GAGs in the
structure of connective tissues, such as cartilage, bone, synovial
fluid, intervertebral disks, tendon, skin, and cornea, the
physicochemical properties of these substances have been thoroughly
characterized (11).
Several studies have demonstrated that hyaluronan is highly abundant in
the interstitium of the renal inner medulla in contrast to the low
amounts seen in other regions of the kidney (4, 13, 18).
Figure 6 illustrates the high level of
hyaluronan accumulation in the rat inner medulla as revealed by Alcian
blue staining. Other GAGs are present in the inner medulla in much lower amounts. The hyaluronan in the inner medulla is believed to be
produced by a specialized interstitial cell (the so-called type 1 interstitial cell) that forms characteristic "bridges" between the
thin limbs of Henle and vasa recta (62).
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Fig. 6.
Alcian blue staining of normal rat kidney revealing distribution of
hyaluronan in inner medulla. Bar = 2 mm.
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Figure 6 illustrates that the hyaluronan-laden inner medulla is
contained within the renal pelvic wall with its thick smooth muscle
lamina. The compression of hyaluronan in the medullary interstitium by
the peristaltic contractions of the pelvic wall can hypothetically
serve to generate a single effect for inner medullary concentration in
two ways: 1) by lowering the osmolality of the surrounding
interstitial fluid; and 2) by storing mechanical energy,
which when released can create forces that drive water absorption from
the descending limb of Henle. We consider these two mechanisms in turn.
Hyaluronan Contraction May Lower Local Osmolality
Hyaluronan is a large, unbranched polysaccharide molecule composed
of repeating glucuronic acid/N-acetylglucosamine
disaccharide subunits (Fig.
7A).1
It is a polyanion, owing to the carboxylate groups of the glucuronic acid subunits. It is a huge molecule, typically with a molecular mass
in the range 1,000-10,000 kDa. It is strongly hydrophilic and
adopts highly expanded, stiffened random-coil conformations that occupy
a huge volume relative to their mass. In solutions of physiological
ionic strengths, the domains of individual molecules begin to overlap
at low concentrations (<5 mg/ml). The hydrodynamic domains are readily
compressed when concentrated under a mechanical load and expand when
the compressive force is removed. Thus the inner medullary interstitium
can be visualized as being composed of a compressible, viscoelastic
hyaluronan matrix. The extended state of hyaluronan owes partly to
repulsive electrostatic forces exerted by neighboring COO
groups, which maximize the distance between neighboring negative charges (Fig. 7B), and partly by the constraints of the
glycosidic bonds that prefer somewhat extended conformations. This
creates a swelling pressure (turgor) that allows the hyaluronan matrix to generate an elastic-like force (resiliance) that resists
compression. When HA is compressed, as may occur in a meniscus in the
knee joint under load-bearing conditions, the repulsive force of
neighboring COO groups is overcome in part by
immobilization (or "condensation") of cations (chiefly
Na+), forming a localized crystalloid structure (Fig.
7C). Thus compression of a hyaluronan gel results in a
lowering of the local Na+ ion activity in the gel. In
aqueous solutions in Donnan equilibrium with the gel, one can predict a
decrease in the NaCl concentration secondarily to the
compression-induced reduction in Na+ activity within the
gel. Thus the free fluid that can be expressed from such a gel would
have a lower total solute concentration than that of the gel as a
whole. Such an effect could be important in the renal inner medulla
when the force of pelvocalyceal contractions compresses the inner
medullary interstitial matrix. The slightly hypotonic fluid expressed
from the interstitial matrix would tend to escape the inner medulla via
the ascending vasa recta, which is the only structure that remains open
during the compressive phase of the contraction cycle
(49). Thus an ascending stream (the ascending vasa recta)
would have a lower total solute concentration than the interstitium as
a whole and therefore this would create a single effect for medullary
concentration (Table 1).
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Fig. 7.
Structure of hyaluronan. A: disaccharide
subunit. B: extended polyanion. Hyaluronan is a linear
polymer of the disaccharide shown in A, with a molecular
mass in the range 1,000-10,000 kDa. Polymer tends to remain in the
extended state because of repulsion of negative charges of carboxylate
groups. C: when the hyaluronan polyanion is compressed, free
cations are sequestered.
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Conversion of Mechanical Energy to Chemical Potential Energy Via
the Viscoelastic Properties of Hyaluronan
During the relaxation phase of the pelvocalyceal
contraction-relaxation cycle, two additional processes may contribute
to urinary concentration through creation of a single effect (luminal osmolality > interstitial osmolality) across the thin descending limb epithelium. Both processes result from relaxation of the compressed hyaluronan matrix: 1) water would be absorbed
from the descending limb as a result of a decrease in the hydrostatic pressure in the medullary interstitium; and 2) water would
be absorbed from the descending limb as a result of elastic forces exerted directly by the expanding medullary interstitial matrix. The
relevant forces have been described by Maroudas (51) in the analysis of water transport from articular cartilage, a
glycosaminoglycan-filled tissue similar in properties to the
hyaluronan-filled inner medullary interstitium. Fundamentally, the flux
of water into and out of cartilage (or the inner medullary
interstitium) may be viewed as being driven by three forces,
expressed as pressure differences: 
Posmotic,
Phydrostatic, and Pelastic. In
the context of forces determining water transport between the thin
descending limb and the hyaluronan matrix of the inner medulla,
Posmotic represents the osmolality difference between
the lumen and the interstitial matrix, Phydrostatic
represents the hydrostatic pressure difference between the lumen and
the interstitial matrix, and Pelastic represents the
force exerted due to the elastic deformation of the interstitial matrix
(given here as an equivalent pressure difference). According to this
formulation, during the relaxation phase after passage of the pelvic
peristaltic wave, elastic forces from expansion of the compressed
hyaluronan would increase water transport in two ways: 1)
Pelastic could directly draw water out of the descending limb (and other water-permeable structures); and 2) the
tendency to interstitial expansion due to the relaxation of the
compressed hyaluronan may lower the interstitial pressure below ambient
pressure levels to produce a hydrostatic pressure difference
Phydrostatic, increasing water withdrawal from the
descending limb and other water-permeable structures. The tendency of
the pressure drop in the interstitium to cause cavitation would be
countered by the gel structure. In the inner medulla, the flow of water
driven by the sum of elastic and hydrostatic pressure forces
(Pelastic + Phydrostatic ) would
concentrate the lumen of the descending limb relative to the
interstitium. As water flows out of the descending limb of Henle, the
limiting condition of no water flow is approached where
Posmotic = Pelastic + Phydrostatic. Here, Posmotic
represents a limiting single-effect value.
It is possible that the sum Pelastic + Phydrostatic may be much larger than 1 atm, although
measurements of this force are not presently available. It is important
to reemphasize that this represents a very low pressure in the
interstitium rather than a very high pressure in the renal tubule
relative to ambient pressures. The fall in hydrostatic pressure in the
interstitial matrix would be expected to be bounded, if one assumes
that "absolute negative" hydrostatic pressures are an
impossibility, so that Phydrostatic would not exceed 1 atm. Nevertheless, negative absolute pressures have been reported, for
example, in the xylem of trees as a result of transpiration
(70). These pressures are believed to furnish the driving
force for the flow of water upward from the roots to the tree tops of
large deciduous trees, overcoming the weight of a 200-ft column of
water. Xylem pressures of 5 to 6 atm relative to ambient pressure
have been reported (63). For values of
Pelastic + Phydrostatic ranging from
1 to 6 atm, the value of the single-effect osmolality difference
across the thin descending limb would range from 40 to 240 mosmol/kgH2O [Cosm = (Pelastic + Phydrostatic)/
· RT].
As described in APPENDIX B, this would give a urinary osmolality in the range from 1,416 to 4,000 mosmol/kgH2O,
spanning the value of maximal urinary osmolality measured in normal
rats (2,900 mosmol/kgH2O) (31).
In summary, the chief new concept presented in this review is that the
inner medullary interstitium might best be modeled as a viscoelastic
system with stress-strain properties, rather than a purely hydraulic
system. This necessitates consideration of force terms other than
hydrostatic pressure and osmotic pressure. The main additional force
term that we add to the analysis is elastic force. During inner
medullary compression resulting from the contraction of the pelvic
wall, the compression of the hyaluronan matrix stores some of the
mechanical energy generated from the smooth muscle contraction. This
compression would not require an increase in hydrostatic pressure but
would simply require a direct mechanical compression of the hyaluronan
matrix as one would compress a steel spring. After passage of a
peristaltic wave, the compressed hyaluronan will tend to spring back
from its compressed state, exerting an elastic force and lowering
interstitial pressure, thereby driving water from the descending limb
and other water permeable structures. The water efflux would
concentrate solutes in the tubule lumina. This would complete an energy
conversion starting with ATP hydrolysis in smooth muscle cells of the
pelvic wall, leading to compression of the hyaluronan in the medullary interstitium and then to an increase in electrochemical potential due
to concentration of solutes in the tubule lumina. We have analyzed this
process here only with regard to mass balance requirements. Clearly,
further theoretical and experimental analysis is required to evaluate
the feasibility of these proposed energy transfers purely on the basis
of energy balance, as done previously for steady-state systems
(78, 79, 91). The single effect generated from this
process could add to single effects from other processes, e.g., lactate
generation in the renal medulla, to concentrate the urine.
An important question that must be addressed experimentally is whether
the rate of energy generation by ATP hydrolysis and contraction of the
pelvic wall is sufficient to account for the energy input needed to
concentrate the collecting duct urine as it flows along the inner
medullary axis. An additional question concerns the stress-stain
properties of the renal papilla and whether the modulus of elasticity
is sufficient to mediate the proposed mechanical energy transduction.
Finally, an important experimental question is the degree to which the
basement membrane of the thin limbs of Henle can withstand hypothetical
transepithelial pressure differences of 1 atm or more without
undergoing permanent deformation. Perhaps the small radius of these
tubules plays an important role in limiting the wall tension needed to
counter such pressure forces according to the Law of Laplace (P = T/r).
| |
CONCLUSION |
The identification of the process responsible for concentration of
solutes in the interstitium of the inner medulla has been elusive. The
lack of definition of these mechanisms undoubtedly owes to the
technical difficulty of studying processes in the intact renal medulla
without disrupting these processes. In recent years, interest in
investigation of this problem has flagged as renal physiologists have
turned their attention to individual genes and proteins, focusing on
the molecular aspects of transport regulation and especially on
processes that are amenable to study in cell culture. Nevertheless, the
purely integrative question of how the inner medullary interstitium is
concentrated remains as important as ever. This review has been
presented with the idea of stimulating further work on the problem. By
proposing specific hypotheses involving specific genes and gene
products, e.g., the hyalurononan synthase (HAS) genes and hyaluronan,
we hope to stimulate investigators to reexamine this problem with the
tools of 21st-century physiology.
For example, it may be possible to use transgenic and gene knockout
technology to address critical elements of the model such as
1) targeted/conditional knockouts of the HAS2
gene in the renal inner medulla; 2) targeted deletion of
interstitial cells of the renal inner medulla, which produce the
interstitial HA; and 3) targeted deletion of contractile
proteins of the pelvic wall.
The flows are assumed to be positive if oriented toward
the renal papillary tip and negative if oriented toward the cortex (see
Fig. 2 for definition of structures and control volumes).
These calculations ignore single effects that might be generated in the
descending vasa recta and collecting ducts by the same mechanism but
also ignores the fact that dissipative terms in text Eq. 1
would tend to reduce the gradient generated.
The authors gratefully acknowledge the career and contributions of
Bodil Schmidt-Nielsen; many of the concepts presented in this review
originated in her work.
TOP
ABSTRACT
INTRODUCTION
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