The loop of Henle, a turning-point in the history of kidney physiology

François Morel

Professeur honoraire au Collège de France

Correspondence and offprint requests to: 126 Rue Houdan, F-92330 Sceaux, France.

The purpose of this article is to analyse the historical reasons why nearly a hundred years elapsed between the discovery of the loop of Henle in the last century and our understanding of its physiological function.

In the middle of the nineteenth century, at a time when anatomical microscopy was rapidly developing in Germany—leading Rudolf Virchov to propose the cellular theory of tissue organisation in 1858—Jacob Henle (1809–1885) was investigating the histological anatomy of renal tissue; from 1862 onwards, he described in a series of publications the presence of tubular loops running perpendicular to the kidney surface, and penetrating at a variable depth in the medulla. The descending portion of these loops had a small outer diameter (thin limb) as compared to that of the ascending portion located in the outer medulla (thick limb). Figure 1Go reproduces illustrations published by Henle in 1866 [1]: note the remarkable accuracy of those hand-made drawings. They show a short portion of thin limb (Figure 1AGo), of thick limb (Figure 1BGo), and a thin-to-thick limb transition (C). Of course, Henle could not assign any function to these structures, at a time when even the basic principles of urine formation were still a matter of controversy between those supporting the `Filtration Theory' (so-called mechanists) and those supporting the `Secretion Theory' (so-called vitalists).



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Fig. 1. Tubular loops of the rabbit kidney, as described by Henle [1]. The figure on the right shows hairpin thin limbs penetrating at a variable depth in the papilla. (A) A piece of thin limb, (B) a piece of thick limb and (C) a thin-to-thick limb transition.

 
In 1859, Claude Bernard (1813–1878) [2] gave his personal thoughts about this controversy:
`We have not to pay attention to those theoretical views which consider urine formation as a vital process: they do not offer any hope for critical analysis; but we shall examine the relevance of mechanical theories of circulation; they are probably erroneous theories, since they neglect one aspect of the question, but they are clearly formulated and easy to understand'.

As stressed later by Homer Smith (1895–1962) [3; p. XVIII]

`During the next 50 years, the respective nature of glomerular and tubular activity was debated back and forth, with no conclusive evidence to settle the argument'.

From 1916 onwards, the English pharmacologist Arthur Cushny (1866–1926) contributed a great deal to appease this wrangle by proposing a unified theory which he called `The modern theory' [4]. According to his view, urine is formed by simple ultrafiltration in glomeruli, and its composition is then modified by selective reabsoption processes as it flows along the tubular portion. As stated by Homer Smith [3; p. XXI]:

`In principle, the theory as a whole was very attractive to most investigators because it treated the kidney as an organ of fixed and predictible function, like muscle and nerve'.

It should be mentioned at this point that in the meantime (1909) Klaus Peter [5] had reported that the loops of Henle are present in the kidney of mammals only, and that they are proportionally more developed in species living in a dry habitat and producing a concentrated urine.

During the two decades after the introduction by Cushny of the `filtration-reabsorption' theory, renal physiology progressed quite rapidly as a consequence of the fact that two kinds of new experimental approaches adapted to the analysis of kidney functions were successively made available.

Up until the end of the 1940s, the systematic use of these two complementary methods allowed the collection of a large number of experimental data which were summarized and discussed by Homer Smith in his famous treatise The Kidney Structure and Function in Health and Disease published in 1951 [3]; this was the `Bible' for the kidney physiologists of my generation. It may be worth recalling at this point that, when Smith wrote his book, the nature of the mechanisms responsible for tubular transport (either of reabsorption or of secretion) was still unknown. Net fluxes proceeding against the electro-chemical gradient were called active transports.

`Although we are still ignorant of how energy is made available or utilized in tubular excretion (such studies are just beginning), there is in our concept of it no implication of vitalism, and the term "secretion", stripped of its older ambiguity, may serve as a convenient synonym for it' [3; p. XVI].

Homer Smith's views on the sites and mechanisms of urine concentration and dilution within the mamalian kidney were deduced from micropuncture data in amphibians. They are summarized in Figure 2Go [from Ref. 3, Fig. 60 p. 327]. This highly schematic nephron includes a short `thin segment', but no loop of Henle. As a consequence of the marked reabsorption of salt occurring along the proximal tubule, the fluid delivered to the thin segment is assumed to be slightly hypotonic to plasma. According to Homer Smith [3; p. 243]:

`it may be presumed that, in so far as time permits, water diffuses from this hypotonic urine back into the blood in consequence of the resulting difference in osmotic pressure. Further diffusion occurs in the thin segment of the loop of Henle, the function of which is now considered to be the promotion of osmotic equilibrium between the tubular urine and the blood before the urine is delivered to the distal tubule'.



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Fig. 2. Mechanism of urine dilution and concentration, according to H W, Smith, 1951 [3].

 

In the absence of antidiuretic hormone (ADH), the distal nephron segments are impermeable to water, so that the active reabsorption of salt along those segments results in the formation of a large volume of diluted final urine. In the presence of ADH, the distal segments are permeable to water so that one should obtain a reduced volume of isotonic urine. In order to account for the formation of hypertonic urine in the presence of ADH, Homer Smith had to postulate the existence (in distal segments) of an additional active component of water reabsorption, TX(H2O) in Figure 2Go, i.e. a component working against the gradient of osmotic pressure.

As regards the correlation between the anatomical development of Henle's loops and the renal concentrating ability in various mammals noted by Peter [5] and confirmed by I. Sperber [10], H. Smith's comment is clear.

`This led to the view that the urine was concentrated chiefly in the thin segment, but the thinness of the tubular epithelium in this segment has always been a difficulty in this interpretation, and more recent work indicates that the function of this segment is to promote osmotic equilibrium before urine is delivered to the distal segment where the final operations on water and sodium are performed' [3, p. 245–246].

The physico-chemist Werner Kuhn (1899–1968) working in Basel University, hypothesized in 1942 [11] that the production of hypertonic urine by the kidney might result from a concentration mechanism by counter-current between descending and ascending limbs of Henle's loops, as later detailed in 1951 [12]. The first experimental evidence supporting this revolutionary hypothesis was obtained in the University of Basel by the Swiss physiologist Heinrich Wirz (1914–1993) (Figure 3Go) in collaboration with Kuhn. The results were presented in Copenhagen by Wirz [13] as early as 1950 at the International Congress of Physiology and published in 1951 [14]. The authors measured, under microscopic observation, the melting-point temperature of the urine crystals present in tubular structures of frozen kidney slices (from dehydrated rats) prepared at different levels along the cortico-papillary axis. In Figure 4Go which illustrates their data, it may be noted: (i) that, at a given level along the axis, the osmotic pressure of the tubular fluid was roughly similar in all structures, namely, the two limbs of the loops and the collecting tubules, and (ii) that its absolute value increased progressively from isotonicity at the cortico-medullary junction up to final urine osmolarity at the tip of the papilla. The authors interpreted their data as evidence for the presence of the cortico-medullary gradient of tissue osmotic pressure expected from the counter-current concentration multiplyer mechanism.



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Fig. 3. Heinrich Wirz (1914–1993).

 


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Fig. 4. The osmotic pressure of urine in collecting tubules and in the two limbs of loops increases along the medulla and papilla as measured by cryometry in frozen kidney slices from dehydrated rats. Reproduced from Wirz et al. [14].

 
Homer Smith, who attended this meeting, refused to discuss the new theory, arguing that the cryoscopic method used was not reliable. Anyhow, if a tissue hypertonicity existed in papilla, the wall of vasa recta would be facing an osmotic pressure difference with blood plasma much more difficult to consider than when this difference is present across the wall of collecting tubules, as in his own view of the concentration mechanism (Figure 2Go). Wirz (personal communication) refuted this argument by stating that, in the counter-current hypothesis, the blood circulating in vasa recta should participate in the osmotic gradient!

In order to substantiate this provocative aspect of the new theory, it appeared necessary to get a direct access to vasa recta so as to collect blood and measure plama osmolarity. In Copenhagen, Sperber (who like Peter had investigated the Henle loop in various species) mentioned to Wirz (personal communication) that this might be possible in golden hamsters, because, in this species, the kidney papilla penetrates deeply into pelvis, so that its tip should be accessible to micropuncture after opening of the pelvis. Wirz performed such experiments and measured the osmolarity of blood plasma and urine samples successively collected in vasa recta and collecting tubules at the papillary tip of golden hamsters. The results, published in 1953 [15] unequivocally established that vasa recta blood and collecting tubule urine are both hypertonic and of a similar osmotic pressure in the concentrating kidney (Figure 5Go), thereby confirming one unparalleled property of the counter-current theory. Again, this further evidence did not suffice to convince the scientific community, except German speaking kidney physiologists.



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Fig. 5. The osmotic pressure of blood plasma punctured in vasa recta at the papillary tip of golden hamsters is high and similar to that of the adjacent collecting tubule urine. From Wirz, 1953 [15].

 
In any case, two other main aspects in this mechanism remained to be established: (i) what kind of solutes accumulate in the cells of the kidney medulla during dehydration? And, (ii) what is the active process (`single effect') responsible for building up and maintaining the medullary gradient of osmotic pressure?

First, from 1955 onwards Karl Ullrich and associates measured the solutes and inorganic ions contained in the renal cortex and medulla of dogs undergoing various diuretic conditions [16]. They observed an increase in sodium, chloride, and urea content in the medulla of dogs during dehydration; they also noted the accumulation of a phosphorus-containing small organic molecule, which proved later to be glycerophosphorylcholine [17], i.e. a now well-established kidney osmolyte.

As regards the second question, Kuhn et al. had already hypothesized in 1951 [12,14] that an active transport of solutes (probably NaCl) out of water-tight thick ascending limbs of loops, associated with its back diffusion into thin descending limbs, should result in solute sequestration within medulla and might therefore represent the driving force required in the hairpin counter-current mechanism. If this active process existed along thick ascending limbs, these segments should deliver a hypotonic fluid to distal convoluted tubules, a characteristic that could be tested by performing micropuncture experiments. To this end, Wirz had to revive the technique developed by Walker et al. [8] for rodent kidneys in 1941 and never used thereafter. Figure 6Go reproduces the results reported by Wirz in 1956 [18]. The fluid was indeed markedly hypotonic to plasma in the early distal tubule, both in dehydrated rats (ADH present) and in rats undergoing water diuresis (ADH absent).



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Fig. 6. The osmotic pressure of the tubular fluid punctured in the early distal tubule is hypotonic to plasma in rats producing either diluted or concentrated urine. From Wirz, 1956, [16].

 
In spite of this progressive accumulation by Wirz of convincing evidence giving support to the counter-current mechanism, the new theory remained ignored by most kidney physiologists during this long period. Thus, Homer Smith, in his otherwise excellent small book entitled Principles of Renal Physiology published in 1956 [19], does not even mention the counter-current hypothesis. I personally met Heinrich Wirz on several occasions during these years, and I can certify how disappointed he was, in particular for not having been able to convince Homer Smith.

A couple of years later, in the US, Carl Gottchalk (1922–1997) and associates completed at Chapel Hill in 1959, a remarkable series of micropuncture experiments in the kidney papilla of rodents, which fully confirmed and extended the observations gathered by Wirz previously. Gottchalk told me that he had an opportunity to submit his data to Homer Smith at Chapel Hill before their full publication. Homer Smith was so impressed by the accumulated evidence supporting the counter-current mechanism, that he felt prompted to recognize its relevance and to make known his new opinion without any delay in a remarkable article, in which he explains and details the various aspects of the counter-current mechanism with both accuracy and humour! The publication of the results of Gottchalk et al. in the American Journal of Physiology [20] was postponed until the article by Homer Smith in the Proceedings of the New York Academy of Medicine [21] had appeared (Gottchalk, personal communication). What should be called the conversion of Homer Smith to the `new theory' was followed by most kidney physiologists, in particular those in the US.

The former view of Homer Smith on the urine concentrating mechanism included two weak points, namely: first, it omitted the loop of Henle, as already mentioned, and second, it postulated an active transport of water, what appears to be an irrelevant concept never confirmed in any living system. This is probably because an active transport of water is thermodynamically impossible, that tubular loops developed during evolution in terrestrial mammals in order to allow the production of hypertonic urine.

Finally, the counter-current theory completed and improved, rather than replaced the model of Homer Smith, since, in the new mechanism: (i) hyperosmotic urine is still formed in medullary collecting tubules by water abstraction (but by a passive instead of active transport); (ii) the larger part of the thin segment is indeed a segment of passive osmotic equilibration (but in a hypertonic instead of isotonic environment) and (iii) the effects and sites of action of ADH remain unchanged.

However, blood plasma hypertonicity in vasa recta, as required by the new theory, corresponded to a situation so unprecedented that it may have contributed to Homer Smith's initial reluctance to accept it.

It may be relevant, at this point, to quote Homer Smith's views in 1951 [3; p. XXII] regarding controversies in renal physiology.

`It has been a history of rival theories, each based upon inconclusive evidence. Its errors have been compounded by oversimplification in the matter of theory and underexamination in the matter of critical investigation. Renal physiology has now passed into a quantitative phase where unsupported speculation and empirical description are no longer warranted'.

On his side, Heinrich Wirz followed Claude Bernard's advice [22].

`When the observed evidence is opposed to a theory prevailing at the moment, one must accept the data and give up the theory, even when it is supported by famous names and widely accepted.'

Beyond this controversy, the loop of Henle indirectly played another, major role in the recent history of kidney physiology. As a matter of fact, as mentioned, Wirz, then Gottchalk et al. in the late 1950s had to revive the micropuncture technique in order to measure tubular fluid osmolarity. Consequently, what was regarded as a rare technical achievement 20 years before, became, during the 1960s, a widely and routinely used approach for the experimental analysis of a many aspects of kidney functions. This opened the era of microtechniques that prevailed in kidney physiology during more than two decades. That is the reason why the loop on Henle may be regarded as a turning-point in the history of kidney physiology.

Notes

Editor's note

The present manuscript is based upon an article published in French in Nephrologie d'hier et d'aujourd'hui 1995: 6: 4–10

References

  1. Henle J. Handbuch der Eingeweidelehre des Menschen. F. Vieweg und Sohn, Braunschweig, 1866: 303
  2. Bernard C. Leçons sur les propriétés physiologiques et les altérations pathologiques des liquides de l'organisme. Vol. 2, Baillère, Paris: 1859; 157 (Author's translation)
  3. Smith H. The Kidney, Structure and Function in Health and Disease. Oxford University Press, New York: 1951; 1049
  4. Cushny AR. The Secretion of Urine. Longmans, Green and Co., London: (1st edn), 1916; (2nd edn.), 1926
  5. Peter K. Die Nierenkanälchen des Menschen und einiger Säugetierniere. In: Untersuchungen über Bau und Entwickelung der Niere. Vol. 1. Fisher Iena, 1909: 249–431
  6. Richards AN. Urine formation in the amphibian kidney. Harvey Lect, 1934/1935; 30: 93–118
  7. Richards AN. Processes of urine formation, Proc Roy Soc B 1938; 126: 398
  8. Walker AM, Bott A, Oliver J, McDowell MC. The collection and analysis of fluid from single nephrons of the mammalian kidney. Am J Physiol 1941; 134: 580–595[Free Full Text]
  9. Shannon JA, Smith HW. The excretion of inulin, xylose and urea by normal and phlorizined man. J Clin Invest 1935; 14: 393
  10. Sperber I. Studies on the mammalian kidney. Zoologiska Bidrag Uppsala 1944; 22: 249–431
  11. Kuhn W, Ryffel K. Herstellung konzentrierter Lösungen aus verdünnten durch blosse Membranewirkung. Ein Modellversuch zur Funktion der Niere. Hoppe Seyler's Z Physiol Chem 1942; 276: 145–178
  12. Hargitay B, Kuhn W. Das Multiplikationprinzip als Grundlage der Harnkonzentrierung in der Niere. Z Elektrochem 1951; 55: 539–558[ISI]
  13. Wirz H, Hargitay B, Kuhn W. The concentration process in mammalian kidney located by a microcryoscopic method. Proc XVIII Int Cong Physiol, Copenhagen, 1950:
  14. Wirz H, Hargitay B, Kuhn W. Lokalisation des Konzentrierung Prozesses in der Niere durch direkte Kryoscopie. Helv Physiol Acta 1951; 9: 196–207[ISI]
  15. Wirz H. Der osmotische Druck des Blutes in der Nierenpapille. Helv Physiol Acta 1953; 11: 20–29[ISI]
  16. Ullrich KJ, Drenckhahn FO, Jarausch KH. Untersuchungen zur Problem der Harn Konzentrierung und Verdünnung. Pflügers Arch 1955; 261: 62–77[Medline]
  17. Ullrich KJ. Glycerylphosphorylcholinumsatz und glycerylphosphorylcholinesterase in der Säugetierniere. Biochem Z 1959; 331: 98–102[ISI]
  18. Wirz H. Der osmotische Druck in den corticalen Tubuli der Ratteniere. Helv Physiol Acta 1956; 14: 353–362
  19. Smith HW. Principles of Renal Physiology. Oxford University Press, New York: 1956
  20. Gottschalk CW, Mylle M. Micropuncture study of the mammalian urinary concentrating mechanism: evidence for the counter-current hypothesis. Am J Physiol 1959; 196: 927–936[Abstract/Free Full Text]
  21. Smith HW. The fate of sodium and water in the renal tubules. Proc NY Acad Med 1959; 35: 293–316
  22. Bernard C. Introduction à l'étude de la Medecine Expérimentale J. B. Baillère, Paris: 1865; 288 (author's translation)




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