Impermeability of the ascending limb of the Henle loop for water is traditionally regarded as essential for countercurrent multiplication in the kidney. Similar claims have been made about permeability properties of the collecting duct and some other epithelia. It is not clear, however, how a structure based on phospholipid bilayers can be water-impermeant if phospholipid bilayers themselves have measurable permeability. The presence of two membranes separated by the cytoplasm may only account for a several-fold reduction in permeability compared to a single bilayer. By analyzing published data, we conclude that these tubules do have a finite water permeability, especially the collecting duct. Although the results on isolated ascending limbs vary among authors, osmotic shock experiments clearly indicate that both the collecting duct and the ascending Henle loop are sufficiently water-permeable to observe volume regulation effects. We conclude that these epithelia by themselves do not display unusual resistance to water flow; it can be estimated that 20-50% of the fluid entering the tubules can be reabsorbed into a strongly hypertonic medulla. It is possible, however, that unstirred layers in the intact kidney may contribute to the apparent low permeability of the tubules.
The belief that the ascending loop of Henle and unstimulated collecting duct are impermeable to water is widely shared by physiologists; it is stated as a matter of course in most textbooks [1-3] and in scientific literature [4-6]. The biological significance of water-impermeant epithelia is that while ions are being removed from the filtrate by various transporters, water is unable to follow ions by osmosis, and the fluid becomes hypotonic. A similar process is responsible for the production of hypotonic saliva [7].
But even pure lipid membranes are permeable to water to some extent, with osmotic permeabilities Pf in the range of 10 – 160 µm/s [8, 9] (see [10] for the explanation of permeability); how can it be that some epithelia are not? This question has drawn interest from researchers, who pointed out the potential importance of membrane asymmetry, integral proteins, and mucins [11-13]. To that, we would like to add a few additional considerations.
1. Single membrane vs. epithelium.
When comparing the permeability of a phospholipid bilayer with that of a simple epithelium, we must
account for
the presence of two membranes in the latter (apical and basolateral) separated by cytoplasm. For
simplicity, we
can assume that both membranes are identical and separated by the distance h = 7 µm [14]; the
diffusion
coefficient of water Dw in the cytoplasm has been estimated at 400
µm2/s
[15-17]. For a single membrane, the steady state water flux is expressed as
$${{\Phi_m = P_f (C_0 - C_i)}}$$
In the case of a three-layer epithelium, the expression changes to
$${{\Phi_e = \frac{P_f D_w}{P_f h + 2 D_w}(C_o-C_i)}}$$
which can be derived by equating water fluxes through each of the three compartments or by using the rule
for
calculating the combined permeability of membranes in series [18]:
$${{\frac{1}{P_{\text{total}}}= \sum_i \frac{1}{P_i}}}$$
Therefore, the additional barriers present in the epithelium slow down water transport by the factor of
$${{ \frac{\Phi_m}{\Phi_e} = 2+ h \frac{P_f}{D_w} }}$$
For the values of Pf listed above, this ratio ranges from 2.2 to 4.8.
Although this may partly explain the difference between a single membrane and an epithelium (for example,
higher
values in vesicles isolated from the medullary thick ascending limb (MTAL) than in intact MTAL [18]), most
data
on kidney permeability have been obtained on isolated tubules.
2. What values of permeabilities would qualify a membrane as water-impermeant?
The rate of water permeation through a tube with length L and radius R and subjected to an
osmolarity gradient DP is
$${{ \Phi_{\text{out}} = P_f \cdot 2 \pi R L \cdot v_w \cdot \Delta \Pi }}$$
where vw is the molar volume of water equal to 18 cm3/mol. The typical value
of
Pf for the medullary collecting duct (MCD) in the absence of vasopressin is 20
µm/s
[18-20]; some authors have obtained slightly higher [21] or slightly lower [22, 23] values. The
permeability of
20 µm/s is regarded as low: “Pf greater than 0.01 cm/sec (at 25–37°C) is considered to
be high
and suggests the involvement of molecular water channels, whereas Pf less than 0.005
cm/sec is
consistent with water diffusion through the lipid portion of a membrane” [24]. However, assuming R
= 12
µm, L = 104 µm [25], and DP = 0.5 mol/L during diuresis [26], we find the rate of water
removal by a single collecting duct on the order of 6 nl/min, which is comparable to the estimated flow
rate
Fin = 14 nl/min [25]. In other words, almost half of the fluid entering the MCD and destined
for
elimination is expected to be reabsorbed, even in the “water-impermeable” state of the duct.
The permeability of MTAL has also been a focus of much research. Rocha and Kokko [27] found its
permeability
close to zero; however, the standard error of their measurements was 4-6 µm/s. The results of Sasaki and
Imai
[28] were similar. The accuracy of those estimates may have been compromised by using fast perfusion rates
[29].
Other measurements have produced a wide range of values from 0.07 µm/s [30] to 5 µm/s [23] or between 6
µm/s and
23 µm/s (cited in [29]).
Using the conservative estimate Pf = 1 µm/s and the parameters R = 15 µm, L =
2∙104 µm, and DP = 1 mol/L, we find that the leak through the walls would amount to 2 nl/min,
or
about a quarter of the total flow of 8 nl/min [25]. Here, once again, epithelial impermeability does not
directly follow from the data.
3. Cell volume regulation
A separate body of work has focused on cell volume regulation - restoration of cell water content
following
either osmotic swelling (regulatory volume decrease, or RVD) or osmotic shrinkage (regulatory volume
increase,
RVI). The RVI and RVD are secondary responses to osmolarity changes caused by the activation of membrane
channels for ions or organic osmolytes [31]; but to initiate these responses, the membrane must be
permeable to
water in the first place.
The numerous reports of volume regulatory responses in the collecting duct [19, 32-35] and in the
ascending loop
[36 – 40] provide compelling evidence that these cells are sufficiently permeable to water, as are all
other
mammalian cells. A single publication claiming the lack of swelling of cheek epithelial cells in hypotonic
solutions [41] may have resulted from unnoticed rapid RVD that develops and subsides within a minute
(according
to our unpublished observations).
The presented brief review suggests that “water-impermeable” kidney epithelia do not possess watertight properties significantly beyond those expected from phospholipid bilayers. Their permeabilities are indeed two orders of magnitude less than the permeability of the thin descending loop [42, 43], which can be due to the lack of water channels, but are similar to those of many other cell types [8, 18, 44-48]. When comparing Pf values for epithelia and isolated lipid layers, the presence of an additional membrane and the cytoplasm should be taken into account; additionally, the effects of unstirred layers can be significant even in perfused tubule preparations [18, 43] and are expected to be particularly prominent in the interstitium of the kidney [49]. Indeed, a several-fold difference between in vivo and in vitro permeabilities of the salivary duct has been reported [50]. Conceivably, slow convection and dissipation of osmolality gradients can be a factor in water retention.
The main question, however, is whether the diluting function of the nephron and the effective diuresis require that MTAL and unstimulated MCD be strictly water-impermeable. It seems that the existing mathematical models assume zero permeabilities of both the thin and thick ascending loops [51-55], and the possibility of deviations from zero have not been considered. That would be an interesting question to investigate.
The authors are grateful to Alan Verkman (University of California San Francisco) for clarifying the concept of water permeability, to Eric Delpire (Vanderbilt University) for directing us to essential literature, and to Paul Fidel (Louisiana State University) for his advice on experiments with oral epithelia (which led us to undertake this investigation). The work was supported by the Kent State University Research Council.
The authors have no conflicts of interest.
| 1 | Hall JE (2016) Guyton and Hall textbook of medical physiology, 13th ed. Elsevier, Philadelphia.
|
| 2 | Widmaier EP, Raff H, Strang KT (2019) Vander's human physiology, 15th ed. McGraw Hill, New York.
|
| 3 | Silverthorn DU (2019) Human physiology: an integrated approach, 8th ed. Pearson, San Francisco.
|
| 4 | Staruschenko A (2012) Regulation of transport in the connecting tubule and cortical collecting
duct. Compr Physiol 2:1541.
https://doi.org/10.1002/j.2040-4603.2012.tb00434.x |
| 5 | Palm F, Carlsson PO (2005) Thick ascending tubular cells in the loop of Henle: regulation of
electrolyte homeostasis. Int J Biochem Cell Biol 37:1554-1559.
https://doi.org/10.1016/j.biocel.2005.02.007 |
| 6 | Randall Thomas S (2009) Kidney modeling and systems physiology. WIREs Syst Biol Med 1:172-190.
https://doi.org/10.1002/wsbm.14 |
| 7 | Catalán MA, Nakamoto T, Melvin JE (2009) The salivary gland fluid secretion mechanism. J Med
Investig 56(Suppl):192-196.
https://doi.org/10.2152/jmi.56.192 |
| 8 | Fettiplace R, Haydon DA (1980) Water permeability of lipid membranes. Physiol Rev 60:510-550.
https://doi.org/10.1152/physrev.1980.60.2.510 |
| 9 | Mathai JC, Tristram-Nagle S, Nagle JF, Zeidel ML (2008) Structural determinants of water
permeability through the lipid membrane. J Gen Physiol 131:69-76.
https://doi.org/10.1085/jgp.200709848 |
| 10 | Manning GS, Kay AR (2023) The physical basis of osmosis. J Gen Physiol 155:e202313332.
https://doi.org/10.1085/jgp.202313332 |
| 11 | Zeidel M (1996) Low permeabilities of apical membranes of barrier epithelia: what makes watertight
membranes watertight? Am J Physiol Renal Physiol 271:F243-F245.
https://doi.org/10.1152/ajprenal.1996.271.2.F243 |
| 12 | Verkman AS (1989) Mechanisms and regulation of water permeability in renal epithelia. Am J Physiol
Cell Physiol 257:C837-C850.
https://doi.org/10.1152/ajpcell.1989.257.5.C837 |
| 13 | Hu P, Meyers S, Liang FX, Deng FM, Kachar B, Zeidel M, Sun TT (2002) Role of membrane proteins in
barrier function: uroplakin deficit compromises the urothelial permeability barrier. Am J Physiol
283:F1200-1207.
https://doi.org/10.1152/ajprenal.00043.2002 |
| 14 | Kumaran GK, Hanukoglu I (2020) Identification and classification of epithelial cells in nephron
segments by actin cytoskeleton patterns. FEBS J 287:1176-1194.
https://doi.org/10.1111/febs.15088 |
| 15 | Tanner JE (1983) Intracellular diffusion of water. Arch Biochem Biophys 224:416-428.
https://doi.org/10.1016/0003-9861(83)90228-X |
| 16 | Rorschach HE, Lin C, Hazlewood CF (1991) Diffusion of water in biological tissues. Scanning
Microsc 1991:1.
|
| 17 | Mastro AM, Keith AD (1984) Diffusion in the aqueous compartment. J Cell Biol 99:180s-187s.
https://doi.org/10.1083/jcb.99.1.180s |
| 18 | Rivers R, Blanchard A, Eladari D, Leviel F, Paillard M, Podevin RA, Zeidel ML (1998) Water and
solute permeabilities of medullary thick ascending limb apical and basolateral membranes. Am J
Physiol Renal Physiol 274:F453-F462.
https://doi.org/10.1152/ajprenal.1998.274.3.F453 |
| 19 | Strange K, Spring KR (1987) Cell membrane water permeability of rabbit cortical collecting duct. J
Membr Biol 96:27-43.
https://doi.org/10.1007/BF01869332 |
| 20 | Hebert SC, Schafer JA, Andreoli TE (1981) The effects of antidiuretic hormone (ADH) on solute and
water transport in the mammalian nephron. J Membr Biol 58:1-9.
https://doi.org/10.1007/BF01871030 |
| 21 | Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA (1995) Vasopressin increases
water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water
channels to plasma membrane. Proc Natl Acad Sci USA 92:1013-1017.
https://doi.org/10.1073/pnas.92.4.1013 |
| 22 | Schafer JA, Andreoli TE (1972) Cellular constraints to diffusion: The effect of antidiuretic
hormone on water flows in isolated mammalian collecting tubules. J Clin Invest 51:1264-1278.
https://doi.org/10.1172/JCI106921 |
| 23 | Morgan T, Berliner RW (1968) Permeability of the loop of Henle, vasa recta, and collecting duct to
water, urea, and sodium. Am J Physiol (Legacy Content) 215:108-115.
https://doi.org/10.1152/ajplegacy.1968.215.1.108 |
| 24 | Verkman AS (2000) Water permeability measurement in living cells and complex tissues. J Membr Biol
173:73-87.
https://doi.org/10.1007/s002320001009 |
| 25 | Gilmer GG, Deshpande VG, Chou CL, Knepper M (2018) Flow resistance along the rat renal tubule. Am
J Physiol Renal Physiol 315:F1398-F1405.
https://doi.org/10.1152/ajprenal.00219.2018 |
| 26 | Sadowski J, Dobrowolski L (2003) The renal medullary interstitium: focus on osmotic hypertonicity.
Clin Exp Pharmacol Physiol 30:119-126.
https://doi.org/10.1046/j.1440-1681.2003.03810.x |
| 27 | Rocha AS, Kokko JP (1973) Sodium chloride and water transport in the medullary thick ascending
limb of Henle. Evidence for active chloride transport. J Clin Invest 52:612-623.
https://doi.org/10.1172/JCI107223 |
| 28 | Sasaki S, Imai M (1980) Effects of vasopressin on water and NaCl transport across the in vitro
perfused medullary thick ascending limb of Henle's loop of mouse, rat, and rabbit kidneys. Pflügers
Arc 383:215-21.
https://doi.org/10.1007/BF00587521 |
| 29 | Burg MB (1982) Thick ascending limb of Henle's loop. Kidney Int 22:454-464.
https://doi.org/10.1038/ki.1982.198 |
| 30 | Burg MB, Green NO (1973) Function of the thick ascending limb of Henle's loop. Am J Physiol
(Legacy Content) 224:659-668.
https://doi.org/10.1152/ajplegacy.1973.224.3.659 |
| 31 | Lang F (2007) Mechanisms and significance of cell volume regulation. J Am Coll Nutr 26:613S-623S.
https://doi.org/10.1080/07315724.2007.10719667 |
| 32 | Fu WJ, Kuwahara M, Gragoe Jr EJ, Marumo F (1993) Mechanisms of regulatory volume increase in
collecting duct cells. Jpn J Physiol 43:745-757.
https://doi.org/10.2170/jjphysiol.43.745 |
| 33 | Sun A, Hebert SC (1989) Rapid hypertonic cell volume regulation in the perfused inner medullary
collecting duct. Kidney Int 36:831-842.
https://doi.org/10.1038/ki.1989.269 |
| 34 | Ford P, Rivarola V, Chara O, Blot‐Chabaud M, Cluzeaud F, Farman N, Parisi M, Capurro C (2005)
Volume regulation in cortical collecting duct cells: role of AQP2. Biol Cell 97:687-697.
https://doi.org/10.1042/BC20040116 |
| 35 | Solenov EI (2008) Cell volume and sodium content in rat kidney collecting duct principal cells
during hypotonic shock. J Biophys 2008:420963.
https://doi.org/10.1155/2008/420963 |
| 36 | Roger F, Martin PY, Rousselot M, Favre H, Féraille E (1999) Cell shrinkage triggers the activation
of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending
limb of the Henle's loop: requirement of p38 kinase for the regulatory volume increase response. J
Biol Chem 274:34103-34110.
https://doi.org/10.1074/jbc.274.48.34103 |
| 37 | Onuchic LF, Arenstein IR, Lopes AG (1992) Cell volume regulation in rat thin ascending limb of
Henle's loop. Am J Physiol Renal Physiol 263:F353-F362.
https://doi.org/10.1152/ajprenal.1992.263.3.F353 |
| 38 | Montrose-Rafizadeh CH, Guggino WB (1991) Role of intracellular calcium in volume regulation by
rabbit medullary thick ascending limb cells. Am J Physiol Renal Physiol 260:F402-F409.
https://doi.org/10.1152/ajprenal.1991.260.3.F402 |
| 39 | Grunewald RW, Fahr M, Fiedler GM, Jehle PM, Müller GA (2001) Volume regulation of thick ascending
limb of Henle cells: significance of organic osmolytes. Exp Nephrol 9:81-89.
https://doi.org/10.1159/000052598 |
| 40 | Hebert SC, Sun A (1988) Hypotonic cell volume regulation in mouse medullary thick ascending limb:
effects of ADH. Am J Physiol Renal Physiol 255:F962-F969.
https://doi.org/10.1152/ajprenal.1988.255.5.F962 |
| 41 | Lee EJ, Patten GS, Burnard SL, McMurchie EJ (1994) Osmotic and other properties of isolated human
cheek epithelial cells. Am J Physiol Cell Physiol 267:C75-C83.
https://doi.org/10.1152/ajpcell.1994.267.1.C75 |
| 42 | Chou CL, Knepper MA (1992) In vitro perfusion of chinchilla thin limb segments: segmentation and
osmotic water permeability. Am J Physiol Renal Physiol 263:F417-F426.
https://doi.org/10.1152/ajprenal.1992.263.3.F417 |
| 43 | Berry CA (1983) Water permeability and pathways in the proximal tubule. Am J Physiol Renal Physiol
245:F279-F294.
https://doi.org/10.1152/ajprenal.1983.245.3.F279 |
| 44 | Capurro C, Escobar E, Ibarra C, Porta M, Parisi M (1989) Water permeability in different
epithelial barriers. Biol Cell 66:145-148.
https://doi.org/10.1111/j.1768-322X.1989.tb00827.x |
| 45 | Garrick RA, Polefka TG, Cua WO, Chinard FP (1986) Water permeability of alveolar macrophages. Am J
Physiol Cell Physiol 251:C524-C528.
https://doi.org/10.1152/ajpcell.1986.251.4.C524 |
| 46 | Garrick RA, Ryan US, Chinard FP (1988) Water permeability of isolated endothelial cells at
different temperatures. Am J Physiol Cell Physiol 255:C311-C314.
https://doi.org/10.1152/ajpcell.1988.255.3.C311 |
| 47 | Folkesson HG, Matthay MA, Frigeri A, Verkman AS (1996) Transepithelial water permeability in
microperfused distal airways. Evidence for channel-mediated water transport. J Clin Invest
97:664-671.
https://doi.org/10.1172/JCI118463 |
| 48 | Squier CA, Cox P, Wertz PW (1991) Lipid content and water permeability of skin and oral mucosa. J
Invest Dermatol. 96:123-126.
https://doi.org/10.1111/1523-1747.ep12515931 |
| 49 | Whitledge V (1998) Influence of unstirred layers on membrane transport in the mammalian kidney.
State University of New York at Stony Brook.
|
| 50 | Field MJ, Young JA (1973) Kinetics of Na transport in the rat submaxillary main duct perfused in
vitro. Pflügers Arch 345:207-220.
https://doi.org/10.1007/BF00586335 |
| 51 | Layton HE, Knepper MA, Chou CL (1996) Permeability criteria for effective function of passive
countercurrent multiplier. Am J Physiol Renal Physiol270:F9-F20.
https://doi.org/10.1152/ajprenal.1996.270.1.F9 |
| 52 | Layton HE (1986) A mathematical model of the urine concentrating mechanism (kidney,
counter-current system, integral equations); (Doctoral dissertation, Duke University).
|
| 53 | Jen JF, Stephenson JL (1994) Externally driven countercurrent multiplication in a mathematical
model of the urinary concentrating mechanism of the renal inner medulla. Bull Math Biol 56:491-514.
https://doi.org/10.1007/BF02460468 |
| 54 | Wexler AS, Kalaba RE, Marsh DJ (1987) Passive, one-dimensional countercurrent models do not
simulate hypertonic urine formation. Am J Physiol Renal Physiol 253:F1020-F1030.
https://doi.org/10.1152/ajprenal.1987.253.5.F1020 |
| 55 | Nawata CM, Pannabecker TL (2018) Mammalian urine concentration: a review of renal medullary
architecture and membrane transporters. J Comp Physiol B 188:899-918.
https://doi.org/10.1007/s00360-018-1164-3 |