Localisation of Renal Function

An article based on a Dissertation read before the R.M.S. on 17th January, 1964. Classical renal physiology as taught to the undergraduate during his medical course, regards the kidney as an entity with little attempt to relate function to the basic unit of the nephron. In most instances this is permissible as it gives a functional understanding of renal processes enabling the clinician to diagnose and treat conditions where this function is impaired, either from intrinsic or extrinsic causes. However, it is not sufficient today to regard complicated organs solely in this fashion. Thus the functions of the kidney, particularly that of ‘acid-base balance' are briefly discussed at a more fundamental level. Copyright Royal Medical Society. All rights reserved. The copyright is retained by the author and the Royal Medical Society, except where explicitly otherwise stated. Scans have been produced by the Digital Imaging Unit at Edinburgh University Library. Res Medica is supported by the University of Edinburgh’s Journal Hosting Service: http://journals.ed.ac.uk ISSN: 2051-7580 (Online) ISSN: 0482-3206 (Print) Res Medica is published by the Royal Medical Society, 5/5 Bristo Square, Edinburgh, EH8 9AL Res Medica, Autumn 1964, 4(3): 25-27 doi: 10.2218/resmedica.v4i3.430


D. I. NEWBLE, B.Sc.
An article based on a Dissertation read before the R.M .S. on 17th January, 1964.Classical renal physiology as taught to the undergraduate during his m edical course, regards the kidney as an entity with little attem pt to relate function to the basic unit of the nephron.In m ost instances this is per m issible as it gives a functional understanding of renal processes enabling the clinician to diagnose and treat conditions where this function is im paired, either from intrinsic or extrinsic causes.H owever, it is not sufficient today to regard com plicated organs solely in this fashion.T h u s the functions of the kidney, particularly that o f 'acid-basc balance' are briefly discussed at a m ore fundam ental level T h e first m ajor advances in the localisation of renal function stem from the classical work o f A .N .Richards in the 19 20 's.H e was the first to develop m icropuncture techniques into renal physiology.H e related the acidification process, in the frog, to the distal tubule.W alker et al (1946) later developed sim ilar m ethods applicable to m am m alian kidneys.Basically, the technique was to insert a micropipette into the renal substance and w ithdraw samples of the tubular fluid.T h e site o f the puncture was marked by the injection o f India ink and being accurately located by maceration and m icroscopic exam ination.
Furth er progress was m ade in 19 5 7 when M alvin, Sullivan and W ild e described their " stop flow" analysis m ethod.Pitts et al (1958) used this m ethod to investigate tubular func tion in dogs.T h e ureters were catheterised and prim ing doses o f creatinine P A H and phosphate were given.Infusions were m ain tained until stabilisation occurred.
T h e catheters were then clam ped fo r varying times ranging from 2 to 8 m inutes.20 to 40 one m illilitre sam ple were collected autom atically in vials fixed into a m oving bar.T heoretically, clam ping the ureter produces a rapid pressure build up in the tubules until the back pressure equals that of the glom erular filtration pressure.A stationary colum n of fluid is then in contact with the tubular epithelium w hich perform s, in an exaggerated fashion, its norm al functions.W h en the clam p is released, the fluid is for cibly ejected.T h e first samples obtained are those from the distal tubules and the later ones from the proxim al tubules.T h e results showed that acidification, am m onia production and potassium-sodium exchange all reached peak values in the same samples, these being those from the distal tubules.Phosphate reabsorption occurred in the proxim al tubule sam ples and was in no way related to the acidification pro cess.A lthough this m ight appear conclusive there arc m any criticism s o f the m ethod.As samples were collected in air sm all p H changes could be missed.A lso, the pelvis o f the kidney acts as a m ixing cham ber, this effecting the later samples in particular.T h e m ethod therefore provides valuable qualitative infor m ation but care m ust be taken in interpretation of the results.
O ne further technique has been used.U lrich and E igler (1958) managed to insert a polyethylene catheter into the collecting ducts o f hamsters.T h e y confirmed the long suspected fact that there is a large p H fall at this site.
T h e first indication that these do not provide the com plete answer cam e from E llin ger as early as 1940.H e observed colour changes of an indicator passing along the tubule.In both the frog and the rat he found that acidification occurred specifically in the distal tubule only during a m ild acidosis.If the urine was strongly acid then he found colour changes along the length o f the nephron.
O ne o f the m ost com plete scries o f micropuncture studies was perform ed by G ottsch alk, Lassiter and M ylle in 1 960.T h e fluid collect ed was scaled in the m icropipe ttes which also acted as m icroelectodes.T h e pH was deter m ined by potential changes in the fluid.A ll their equipm ent was equilibrated w ith air con taining C O 2 at 27 m m H g. C o llection s were obtained from non-diuretic anim als and from those in a state o f osm otic diuresis, both nor m ally and during an am m onium chloride acidosis.In all cases there was a progressive acidification along both sections o f the tubule.(F ig 1.) T h is is conclusive proof that, in rats at least, proxim al tubules can acid ify urine.B io chem ical analysis has shown that equal am ounts of carbonic anhydrase , the enzym e necessary for hydrogren ion exchange, arc present in both sites.O bviously this state o f know ledge is tar from satisfactory bu t it m ight be profitable to attem pt to sum m arise the m echanism s propos ed at this tim e.A b o u t 8 0 % o f the glom erular filtrate is reabsorbed in the proxim al tubules under w hat Sm ith called " obligatory reabsorp tion" .T h e evidence suggests that the bulk of the hydrogen ion exchange also occurs here.
T h e distal tubules are capable o f the sam e pro cesses and probably act as the fine adjustors of p H in the same way as they regulate the 'facultative reabsorption" o f water.T h e collect ing ducts can m ake very little contribution to overall sodium and bicarbonate reabsorption as the load presented is very sm all.L arge pH changes could occur with a relatively low hydrogen ion secretion rate.
H aving thus attem pted to localise the pro cesses in the occurring kidney the actual m ode of transport o f ions by the tubular cells m ust be discussed.T h e first recorded experim ents on active transport in the kidney cam e from W ilb ran d t in 1 9 38.T h ese he perform ed on N ecturus which is an anim al having conveni ently large nephrons w ith long straight proxi m al tubules.
H e measured potential differ ences between the surface o f the kidney and the lum en o f the tubules using for elcctrodes m icropipettes sim ilar to those used by Richards.H e obtained " transtubular p otential" values of up to -12 m V , negative inside the lum en.T h is he interpreted as being due to different ion perm eabilities on the two sides of the cell.
U ssing et al ( 19 5 1) dem onstrated a potential difference across frogs' skin arising as a conse quence o f active transport.H e defined this as ion transport against an electro-chemical giad ient.Perhaps m ore w ell known arc the experim ents o f H odglin et al (1952) where the electrical activity o f nerves was shown to arise from the passage o f N a and K ions across the cell m em brane.N o t unnatur ally, workers turned to the kidney to study these processes as it is an organ where it is relatively easy to m ake electrical recordings and to determ ine ionic concentrations w ithout sub stantially altering the physiological conditions.
Solom on (19 5 7), utilising the specialised electrodes developed in nerve and m uscle studies by L in g and G erard, observed a bim odal distribution o f potentials on random insertions into the tubules o f rats.D urin g the puncture, transient higher potentials were recorded indicating that the electrodes were passing through cells with a greater negativity than the lum en.T h e low er range was related to the proxim al and the higher to the distal tubules.
G iebisch in a m uch fuller investigation using the proxim al tubules o f N ecturus found a m ean value o f -72m V for the peritubular m em brane potential (i.e. the P .D .between the peritubular fluid and the inside of the cell) and of -20m V for the transtubular potential.B y difference, In investigations o f the transtubular poten tia l, d irect microanalysis showed no concentra tio n gradient between the p e ritu b u la r and tu b u la r fluids fo r Na, H and C l ions.T h e po te n tia l difference is not, therefore, m a in tained by io n ic concentration gradients.There m ust be one or m ore active mechanisms in volved.M a n y workers believe th a t Na is the o n ly ion actively transported w h ile the other ions fo llo w passively dow n the electro-chem ical gradients set up.I n N e c tu ru s , at least, there is probably some active K transport.
T h e p rim ary process can be regarded as a s h ift o f positive ions from the lum en leaving i t at a negative pote ntia l.T h is is a process requiring energy.
M icroanalysis also shows th a t the Na con centration inside the cell is less than th a t in the tu b u la r fluid .As shown above, the inside o f the cell is at a m ore negative p o te n tia l than the lum en so Na ions can passively enter the cell along an electrical and a chem ical concentia tio n gradient.H owever the opposite is true o f the pe ritu b u la r border so i t is logical to assume th a t the active mechanism is situated here.T h is appears to be substantiated by electronm icroscopy where the m ito ch o n d ria are shown to be alm ost exclusively situated on this border.T here is some evidence to suggest th a t K uptake in to the cell is lin ke d to this " sodium p u m p " as the concentration o f K in side is greater than w ould be expected if only passive forces were involved.(Fig. 3 T h is in te rp re ta tio n is obviously oversim pli fied.Refined techniques in vo lvin g single nephron perfusion, measurement o f io n ic fluxes u tilis in g radioactive isotopes have all been used, b u t the calculations involved in these methods are com plex, and cannot be ade quately discussed here.I t w ould also be unwise to a tte m p t clear interpretations and explanations at this stage o f research.
M edicine is no longer an em pirical art.Soon i t w ill be inadequate to know sim p ly w hat alterations in blood and urine biochem istry indicate.T h e basic changes occurring at cellular and sub-cellular levels m ust be under stood.T h is article illustrates the lim ite d advances made in o n e small field b u t perhaps indicates also the trend o f research in the future.

Fig. 1 .
Fig. 1. M ic u ro -p u n ctu re stu d y illu stra tin g progessive acidification along th e n e phron, (after G o tts chalk e t al. 1960) T he pH of tu b u la r fluid relative to plsm a pH was m easured a t different p a rts of the tub ule.
Fig. 2 Diagrammatic representation of Proximal Tubular Potentials (Values afterSolomon, 1957)    the lu m in a l mem brane p o te n tia l was about 52m V negative inside the cell.Reductions in these potentials were produced by oxygen lack and by m ercurial diuretics w hich have b o th been shown to reduce active transport pro cesses.(Fig.2.)In investigations o f the transtubular poten tia l, d irect microanalysis showed no concentra tio n gradient between the p e ritu b u la r and tu b u la r fluids fo r Na, H and C l ions.T h e po te n tia l difference is not, therefore, m a in tained by io n ic concentration gradients.There m ust be one or m ore active mechanisms in volved.M a n y workers believe th a t Na is the o n ly ion actively transported w h ile the other ions fo llo w passively dow n the electro-chem ical gradients set up.I n N e c tu ru s , at least, there is probably some active K transport.T h e p rim ary process can be regarded as a s h ift o f positive ions from the lum en leaving i t at a negative pote ntia l.T h is is a process requiring energy.M icroanalysis also shows th a t the Na con centration inside the cell is less than th a t in the tu b u la r fluid .As shown above, the inside o f the cell is at a m ore negative p o te n tia l than the lum en so Na ions can passively enter the cell along an electrical and a chem ical concentia tio n gradient.H owever the opposite is true o f the pe ritu b u la r border so i t is logical to assume th a t the active mechanism is situated here.T h is appears to be substantiated by electronm icroscopy where the m ito ch o n d ria are shown to be alm ost exclusively situated on this border.T here is some evidence to suggest th a t K uptake in to the cell is lin ke d to this " sodium p u m p " as the concentration o f K in side is greater than w ould be expected if only passive forces were involved.(Fig.3.)