The enigmatic genesis of the urinary concentrating mechanism.

Richard Dawkins Philosophical Reply

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This year marks the 50th anniversary of the ingenius and counterintuitive formulation of a new model of mammalian urinary concentration by Hargitay, Kuhn and Wirz. The compact, interdependent nature of the workings of this countercurrent exchange augmenting mechanism are examined as a special example of difficulty for formation by a gradual series of successive evolutionary modifications.

Key Words: Countercurrent, Concentration, Urine, Loop, Gradualism, Evolution.

The elegant intricacies of  the urinary concentrating mechanism are a subject of unending fascination, but no aspect more so than that of its origin and development. This year marks the 50th anniversary of the formulation of the countercurrent hypothesis by Hargitay, Kuhn and Wirz, a crucial milestone in our contemporary understanding [1]. However the hypothesis was by no means readily or universally accepted at first. On the contrary, that doyen of American renal physiologists, Homer Smith, was not reconciled to the idea until 8 years later. The evolution of the concentrating mechanism was of special interest to him, and he believed it to be foundational to elucidating renal function [2].  Ironically, as he colourfully recounts [3], it was an adherence to strict gradualism, which lead to his considerable resistance to the new theory. A recent examination of the evolution of renal function, marking the centenary of Homer Smith's birthday, judiciously bypasses this [4].

Darwin's first formal proposal of evolutionary mechanism relies on the need for each new modification of structure or function to be gradual and slight, and for each change to be justified by adaptive advantage. Adhering strictly to Carolus Linnaeus' ancient maxim 'Natura non facit saltum', he states, "If it could be demonstrated that any complex organ existed, which could not possibly have been formed by numerous, successive, slight modifications, my theory would absolutely break down." [5] He carefully qualifies this statement with three conditions under which relatively abrupt modifications might be observed. These are: firstly, the specialization of an organ possessing two functions into one function only, as for example with the Hydra's ability to respire and digest from the same surface; secondly, the modification of one of two organs both performing an identical function to a separate function, for instance the simultaneous  respiration of oxygen from water via the gills, and of oxygen from air via swimbladder, with its putative conversion to primitive lungs; and finally, the acceleration or retardation of the period of sexual reproduction in relation to ordinary maturation, subsequently coined heterochrony. One eminent, modern disciple rehearses this claim as a touchstone of Neo-Darwinian orthodoxy [6].

However, other writers, Niles Eldredge and Stephen Jay Gould of Harvard, especially, have advocated a more saltatory approach to adaptive changes, with respect to phenotype, though not to genotype [7]. The distinction between these schools is significant, as is demonstrated by the early discussion Gould cites between Huxley and Darwin [8].

Modern secular critics of gradualism argue that molecular sequencing data would only provide ammunition for Darwin's 19th Century typological critics by its failure to document clear transitional paths between similar genera, analagous to the gaps in the fossil record [9,10]. There is a rapidly enlarging wealth of data that may be examined for such evidence. The variations in protein structure and genomic sequence for the aquaporin or urea transporter families between genera and classes, which bridge important gaps in renal adaptation, exemplify just two contemporary opportunities [11-13]. As with comparative anatomical or physiological data, what is sought is not merely a confirmation of the classification of structurally homologous organisms, which amounts to near tautology, but mechanistic insights into and intermediate paths for a proposed course of evolution between related species. To date there is a notable paucity of such descriptions in multicellular organisms, reliant as they are on small, discrete changes. The homeotic gene mutations, such as their first description in man, polysyndactyly from mutations in HOXD13 [14], provide saltatory phenotypic change, perhaps a better substrate for 'punctuated equilibria'.

It seems impossible to account for the urinary concentrating mechanism by 'numerous, successive, slight modifications' of phenotype, even after taking each of Darwin's solicitous qualifications into account. According to countercurrent multiplier theory, urine concentration is necessarily dependent upon several, quite contrasting properties in different parts of the loop of Henle. Can anything other than a large and precise leap be conceived to account for its existence? These four main contrasting properties, each essential to any utility of the whole, are evident: its striking and biologically eccentric hairpin loop structure, a water permeable descending limb, apparently mediated by Aquaporin 1 in some species [15], a water impermeable ascending limb, combined with active salt reabsorption also localised in the ascending limb.

How could a structure derived from reptilian nephrons gradually progress towards a long  hairpin looped configuration, after a classical Darwinian manner, unless there was an adaptive advantage in doing so? What could this be if not to concentrate urine? Could there be any particular advantage over the function of the 'straight' reptilian 'convoluted tubule' otherwise? Could urine even begin to be concentrated until this process had progressed to very near similarity of shape to a mammalian nephron? How could the descending and ascending limbs progressively acquire contrasting water permeability characteristics despite the fact that such properties would be of no adaptive advantage until a medullary concentrating gradient had been established? What selection advantage could be gained if the ascending limb of the loop, as distinct from more distal portions of the nephron, progressively accumulated considerable potential for ionic transport until all the rest of the concentrating mechanism is in place? If the descending limb also shared this marked active ionic transport, then the necessity for a clear distinction between the two for both water and sodium permeability is only heightened. However multiple loops of Henle with all the other necessary properties but insubstantial active salt transport in the ascending limb would be completely futile for urine concentration. Nephrons with little difference in water permeability between the two limbs but every other necessary property would again serve no purpose, particular to the loop, other than to dissipate energy. A nephron of reptilian configuration with all the appropriate transport and characteristics, both active and passive, would achieve no special end other than generate valueless and transient ion fluxes, at the cost of its possessor.

The real difficulty is that none of these quite heterogeneous and necessary properties appear to confer no distinguishing selective value unless all found together simultaneously, and found to be substantially present. Substantially enough that is to begin to subserve the concentration of urine, thus providing a selection advantage to its possessor. A slight tendency towards the demonstration of any or all of these properties by a reptilian nephron will not generate any medullary gradient, until a discrete state of quite advanced similarity in all four aspects to the mammalian nephron is attained. If one aspect lacks, urine concentration will fail.

It was just such a commitment to gradualism, that substantially underlay Homer Smith's considerable reluctance to adopt Kuhn and Hargitay's hypothesis. As he puts it, 'I still do no like it: it seems extravagant and physiologically complicated - though so is the whole glomerular filtration-tubular reabsorption pattern.... Least of all however, do I like to see the squamous epithelium of the thin segment freely permeable to water (if not to sodium also) in the descending limb, only to acquire water impermeability and active sodium transport at the tip of the loop* for no better reason, apparently, than the circumstance that it has turned a corner.' [2] (This author's emphasis).
Nor do these four principle characteristics constitute the only foundation of the mechanism. Henle's loop is associated with coadaptations of the medullary microcirculation, and of vasopressin or vasotocin regulation, the arrangements in some species realizing remarkable intricacy [15,16]. Whilst, for argument's sake, allowing scope to an trenchant advocate of gradualism to enable these mechanisms to refine with the passage of many generations, these concomitant processes must also be so configured as to allow for at least partial efficacy of the countercurrent exchanger, right from the outset. If the main blood supply failed to match the configuration of the nascent nephron closely, it would quickly disperse any medullary gradient otherwise established [18].

The nephron loop found in two species of lamprey, the anadromous Lampetra fluviatilis and Petromyzon marinus, have been cited as an example of a non-mammalian possessors of a looped nephron segment [19,20], which has been claimed as evidence of a vertebrate antecedent for the loop of Henle. The claim is dubious. At face value, some data is favourable: the loop marks a modest beginning for tubular dilution, is arranged in a parallel plane with its fellows and in close apposition to the urinary collecting duct. However micropuncture studies in the former showed no change in electrolyte concentration in the ascending limb of the loop, and although tubular fluid osmolarity falls by 13%, this appears mainly due to non-electrolytic osmolar transport [21], usually more characteristic of proximal tubules than the diluting segment in non-mammalian vertebrates [22]. Moreover the ascending limb, in contrast to its descending partner, reabsorbs water; the direction of flow in peritubular vessels supplying the ascending limb of the loop has not been confirmed as commensurate with countercurrent exchange [21]; the length of the loop, at 1.1 mm, appears too short compared to simple avian nephrons [23], and the renal perfusion rate too slow to enable countercurrent multiplication [24]. Comparative analysis of cytochrome C or hemoglobin sequencing corroborates traditional methods of classification and suggests that cyclostomata are not close ancestors of terrestrial vertebrates [9]. Therefore these loops whilst a curious anomaly, with uncertain significance, do not serve as a useful functional paradigm for Henle's loop, and are not observed in forms more closely related to the amniota.

Finally it seems to be no solution to this insurmountable obstacle for a gradualist to appeal to the smooth transition of forms between the reptilian and mammalian nephrons found in Gambel's quail [23], for example, as an indication, that 'however the avian nephron did attain that advanced state, it most likely did so by small, discrete alterations'. Is it not evident that the relatively weak concentrating ability of Lophortyx gambii, at 2 to 3 times plasma osmolarity is actually dependent not on transitional nephrons but on the longer looped mammalian nephrons ('longer' but not 'long' by mammalian standards)? Without denying a weak contribution from intermediate nephrons (choose the definition of these at whatever point one wishes short of full mammalian configuration, and yet attainable by a small modification from the reptilian), the initial, tentative contribution is wholly dependent on a pre-existent osmotic gradient, generated and maintained by the longer, 'more-advanced' nephrons, analogous to the heterogeneity of nephron length in mammals [16]. A kidney entirely composed of intermediate nephrons of an attainable kind could not concentrate. It is therefore no basis upon which to assert the gradual modification of structure, when adaptive utility to the whole organ or rather whole creature, is obligated for every new investment. Here unaided, strict gradualism appears too myopic and parsimonious a bank manager to justify its reputation as a 'watchmaker', an artificer capable of crafting improbable marvels.

Can any distinctive purpose for which Henle's loop exists be proposed, other than urinary concentration, which might obviate these difficulties? If not, here is another argument why the presuppositions of classical, gradualistic Darwinism require revision.

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Richard Dawkins Philosophical Reply