Renal hydromineral metabolism in starry flounder, Platichthys stellatus

RENAL HYDROMINERAL METABOLISM IN STARRY FLOUNDER, PLATICHTHYS STELLATUS ROY C. FOSTER* Department

of Zoology, Cancer Research University of California,

Laboratory and Bodega Marine Berkeley. CA 94720. U.S.A.

Laboratory.

Studies are reported on urinary bladder modification of urine electrolyte composition in t?w and on renal Na and Mg excretion in SW and FW acclimated starry flounder. Plutidttlln otc/lrrtuF. 7. Kidney urine differs from bladder urine in electrolyte lcvcls and osmotic pressure in both SW and FW fish. SW flounder kidney urine has higher Na and lower Mg levels than bladder urine. whereas FW flounder kidney urine has higher levels of Na and Cl. and a grcatcr osmolarity than bladder urine. This suggests that the urinary bladder reabsorbs Na and possibly Cl in SW and FW fish. conccntratcs Mg through water reabsorption in SW fish. and restricts water influx during salt reabsorption in FW Bounder. 3. SW flounder urine is characterized by a significant inverse relationship between Na and Mg levels over a wide range of urine Na concentrations without alteration in urine osmolarity. FW flounder urine osmolarit> changes directly with urine Na content. but there is no correlation between Na and Mg concentration in bladder and kidney urines. These relationships can be used to assess renal ad.justmcnts during salinity transfers. 4. Rate of renal electrolyte and water excretion is infuenced by environmental salinity. Acclimation of SW flounder to fresh water results in increased urine flow. dccrcascd Mg excretion and increased Na excretion. Abstract---l.

of P. ,jlrsus (MacFarlane, 1974) unlike the urine produced by other sea water flounders such as P. lethosticpn (Hickman, 1968) and the starry flounder, Platichthys .stel/arus (Foster, 1972), where the magnesium content is approx 130 mM. In the present investigation renal function was studied in sea water- and m fresh water-acciimated starry flounder, P. strllatu.s. Reported are studies of kidney sodium and magnesium excretion and of urinary bladder modification of urine electrolyte levels

INTRODUCTION

Teleosts have been freed from osmotic and ionic conformity with their environment in part through the development of osmoregulatory epithelial surfaces located in the gills. intestme and kidney. The ability .^ ..I&_..s3zLLI”~ly “..I.._&:....,.. &L_ .._,I UIICl,LIUII ,I:___r:__ U1 ,.r clc~uulylc ^I..,.&.-,..+.. L” il‘l[;L u1T:.._*.. LdlC ill,U and water transfers at these surfaces has permitted many fish to osmoregulate when exposed to changed salinity. Gill and intestinal surfaces are principally involved m sodium, chloride and water movements, whereas the kidney is also active in regulating other electrolytes, principally magnesium, calcium, phosphate and sulfate. Kidneys of sea water fish usually elaborate urine cioseiy isosmotic to the biood but containing more magnesium. calcium, sulfate and phosphate and less sodium and chloride. Although fresh water fish have greater urine flow rates, the urine is markedly hypo-osmotic and composed largely of sodium and chloride (Hickman & Trump, 1969). It is becoming clear. however, that important exceptions may exist. Several fish in sea water or after transfer to sea water are reported capable of secreting hyperosmotic urine for a limited time, e.g. the killifish Fwldulus kmsue (Stanley & Fleming, 1964: Fleming & Stanley. 1965). the flounder Pluti~/~th~~~Jie~t~~(MacFarlane. 1974) and the southern flounder Purdichthys lethostignm (Hickman, 1968). Magnesium may not always be the dominant cation in the urine of sea water fish and may be as low as 12 mM in urine

in tlilso. >lATERIALS AND METHODS Starr! Rounder. P/~II~~/II/II~~ \r~d/~~tuv.of hoth sexes. uclghing between 600 and 1200 g. wc~c collected b! ottcl trawl from Bodega Bay. Sonoma Country. California. Fish were maintained at the Bodega Marinc Laborator! in running sea water (IO- I?‘C) for I week before transfer to sea water (SW) or fresh water (FW = tap water) at 15-18 C for at least 7 days prior to study. The data rcportcd in Fig. I. however, were randomly collected over a ?-year period from sea water flounder in captivity for O&21 daqs. In urine flow studies, urine was collected in a rubber balloon attached to a short length of PE 160 or 200 polyethylene tubing inserted into the urinary bladder via the urinary duct. The cannula was adequately secured by the natural constriction of the urinary papilla. During periodic (6-12 hr) urine collections, fish were removed from their tanks and the fluid-filled balloons and cannulae rinsed with distilled water and wiped dry. The balloons were then removed from their cannulae and replaced with empty, deflated balloons and the fish returned to their aquaria. The entire process took no more than 2 minIfish. Urine samples from the balloons were stored in sealed vials at -20-C until analyzed. Flounders were restrained from swimming by placing them between masonite sheets. Movable wooden blocks. positloned between these sheets and

*Present address: Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center. 5lst & Grove Streets. Oakland. California 94609. 135

I?6

Rev C FOSXR Srarisrics. Regression analysis was by the least squares method; significance was determined on the regression coefficients by Student’s t-test (Snedecor & Cochran. 1967).

RESL’LTS

I

I

100

200

f

No, mM /L Fig. 1. Sodium. magnesium and osmotic pressure relationships in bladder urine of sea water starry flounder. Coefficient of determination, rz = 0.651 (Na/Mg) (P < 0.001); r* = 0.160 (Najn) (not significant).

fastened with screws. were arranged for each Rounder so as to restrict vertical or lateral movements to less than I In. Urine flow measurements reported here were generally made during the first 3 days of cannulation. Sampling stored (bladder) urine of uncannulated flounder bladders involved insertion of a short length of polyethylene tubing into the Wadder via the urinary duct and allowing the fluid contents to drain freely into glass vials. Blood samples were collected by syringe from the caudal *vein and ‘LI,v*,.,” .,II,,I.,&Vl tr\ ,>t i<‘r. hd,,rv ~an.>lv
1. Electrolvte

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Table 1 presents results on the electrolyte and osmolar concentrations of serum, kidney urine and residual bladder urine from sea water- and fresh water-acclimated starry Hounder. Kidney urine, also termed free-flow urine (Lahlou. 1967), is the fluid obtained from fish with cannulated urinary bladders and represents urine exposed for a minimal period to the bladder mucosa. Bladder urine represents the residual fluid. collected from urinary bladders of uncannulated fish, which has been stored in the urinary bladder for an unknown length of time. Sea water and fresh water flounder urines and sera are distinct in electrolyte and osmotic concentrations. Sea water flounder urine is generally lower than serum in Nai, Cl and K’ concentration and in osmolarity and higher in Ca”+ and Mg*’ content. Fresh water flounder urine is also lower than serum in Na+, Cl- and K’ concentration and in osmolarity. although similar in Ca” and Mg” levels. Kidney urine tends to be higher in Na i and Cl levels than bladder urine (Table I) which suggests that the urinary bladder reabsorbs Na+ and Cl- both in sea water and fresh water fish. In sea water fish this is accompanied by an increase in the Mg” concentration without an alteration in the urine osmolarity. In fresh water flounder the reduction in urine Na’ concentration is not matched by a proportional increase in Mg’+ or any other ion and bladder urine osmolarity is consequently hype-osmotic to kidney urine. exists between Na’ and Mg*+ levels in bladder urine (Fig. I) and kidney urine (Fig. 2) of sea water flounder. ‘This relationship is sul?iciently exact over a wide range of urine Na’ concentrations that the urine osmotic pressure remains essentially constant. seen in Fig. The wider range of Na + concentrations 1 for bladder urine than in Fig. 2 for kidney urine may have resulted from greater variation in the

urine flow seawater

and

fresh seater

starrv

flounder.

SW: %X”ia

160.1+2.7

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(n=io) Bladder

(n=E)

13.p+_4.0

87.4+1.4

(n-13)

(n-71

44.425.0 ("-21)

94.529.4 (n-21)

serum

149.8+3.3 bi3)

121.W4.9 fn-14)

Bladder

UrinE

14.pt2.0 (n-13)

Kidney "r*tle

29.8f2.7 (n-13)

"I-ine Kid”ey

Uri".?

3.34$?.14

(n-6) 1.31 23.2 (n-8)

2.*7*0.*2

(n-6) 7.7El.24 fn=8)

0.8~0.13

(Il=lO) 138.556.4 (n-9)

332.1+4.8 b=W 290.0+_14.0 iLl=6)

_

105.7+6.3 (n-21)

291.f+4.3 (n=ZO)

*.6*.07 (n-7)

1.79+0.07 G7)

0.63?13.04 bF13)

228.3-1-4.0 in=i2)

0.58CO.U ("%I

[email protected] (*=9)

1.*fZ*0.11

FW:

2.28+(1.46 (n=13> 6.15+2.26 (n-6) -

Data are expressed as the MEAN f_ SEX, n = number of fish.

_

57.159.7

(“31)

(n=lO)

0.89fO.11 (n=14)

78.7+10.3 (n.13)

Renal hydromineral

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320

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300

$

280

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metabolism

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20

30

40

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50

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Not, mM /L

50

150

100

No, mM/L Fig. 2. Sodium. magnesium and osmotic pressure relationships in kidney urine of cannulated sea water starry flounder. Each datum represents average value for urine collections during 2-6 day cannulation period. rz = 0.419 (Na/Mg) (P < 0.001); rz = 0.210 (Na/n) (not significant), derrree --c)- --

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stress. In fresh water flounder, Na+, Mg’+ and osmotic pressure relationships in bladder urine (Fig. 3) and

in kidney urine (Fig. 4) are considerably different. Urine osmolarity of fresh water fish is positively correlated (P < 0.001) with urine Na’ content, whereas no significant correlation exists between Na+ and Mg” concentration in bladder and kidney urines. The rate of renal electrolyte and water excretion is markedly affected by environmental salinity. Na+ excretion by sea water flounder kidneys (Fig. 5) is neither constant nor dependent on the rate of urine excretion. In flounder acclimated to fresh water for >7 days, renal Nat excretion is significantly corre-

Fig. 4. Sodium magnesium and osmotic pressure relationships in kidney urine of cannulated fresh water starry flou*der. Each datum. rWV?Q=n+< S”PrYor nPr fish of ._r__~___.” _ .“._.~_ VllllP .I.__ r-. urine samples collection during the first 36 hr following urinary bladder cannulation. r’ = 0.365 (Na/Mg) (not significant): rz = 0.654 (Na/n) (P < 0.001).

lated with urine flow (Fig. 6). Acclimation to a hypoosmotic environment also involves a modification in renal water metabolism, resulting in a 4-fold increase in urine flow from ca. 0.38 ml hr-’ kg-’ (range 0,!5J V.79) (Fin 51 LA1 in UIU EPQ wa:er P,aur,&r +A s-n I Y, 1. ,6’ d, L” LU. 1.67 ml hr-’ kg-’ (range 1.05-2.83) (Fig. 6) in fresh water fish. Figures 7 and 8 suggest that renal magnesium excretion is positively correlated with urine flow in sea water flounder (P < 0.001) and possibly also in fresh water flounder (P < 0.01). Fresh water flounder (Fig. 8), however, have a substantially reduced rate of magnesium excretion when compared with sea water fish (Fig. 7). DISCUSSION

The euryhaline starry flounder, Platichthys strllatus, is a common and economically important demersal fish inhabiting the Pacific Coast from California to

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Not, mM /L Fig. 3. Sodium. magnesium and osmotic pressure relationships in bladder urine of fresh water starry flounder. r2 = 0.032 (Na/Mg) (not significant); r* = 0.735 (Na/n) (P i 0.001).

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Fig. 5. Relationship flow in cannulated

0.4

0.6

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0.8

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flow, ml/hr/kg

of renal sodium sea water starry (not significant).

excretion flounder.

and urine r* = 0.124

Fig. 6. Relationship of renal sodium Iloa m cannulated fresh water starry (P < 0.01 I.

excretion flounder.

and urine r’ = 0.702

Alaska and across to northern Japan. At Bodega Bay. California. the sea water salinity remains relatively constant throughout the year at 33~35”,,,,. However, starry Rounder often invade the two estuaries which flow into Bodega Bay, the Estuaro Americana and the Estuaro San Antonio. Seasonal salinity fluctuations here can be extreme. ranging from low salinities, (cu. Y,,,,) during rhe spring rainy season to hypersaline conditions (L.u. 56”‘;,,,)during the late summer and fall when the mouths of the estuaries are closed by sand bars and evaporative water losses consequently become considerable. The present study of urinary bladder function in r>i~o in sea water- and fresh water-acclimated starry flounder provides further evidence that the urinary bladder aids in osmotic and ionic regulation. Modification of the electrolyte composition occurs during

1 ./ 1

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0.2

0.4

Urine

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0.6

flow, ml /hr

,

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/kg

Fig. 7. Relationship of renal magnesium excretion and urine Row in cannulated sea water starry flounder. r’ = 0.777 (P -c0.001).

storage of urine in the bladder of sea water and fresh water flounder. Although rates of electrolyte and water reabsorption were not determined. the differences in electrolyte content of urine excreted by- the kidney and of urine retained in the urinary bladder imply the reabsorption of Na and possibly also of Cl- and water and the concentration of Mg’+ in sea water flounder and the reabsorption of Na ’ and Cl from the urine in fresh water flounder. These findings agree with the observations of Lahlou (1967) that bladder urine and kidney urine (free-flow urine) of the flounder, Plurir~htl7ys ,flcaus. difrer in electrolyte content in sea water and fresh water fish and also in osmolarity in fresh water fish. Lahlou (1967) attributed this to a selective and slow reabsorption of Nat and Cl.- by the urinary bladder. Previous studies have demonstrated that preparations of isolated urinary bladders of sea water starry flounders when bathed in physiological saline show a greater rate of water movement (mucosa to serosa) than do bladders from fresh water flounder (Hirano et d., 1971 : Johnson c’f u/., 1970, 1972). Water reabsorption appears linked to active, ouabain-sensitive transport of Na’ in the urinary bladder of starry flounder (Johnson CTul.. 1972) and the winter flounder Psrunoplc~uronr~tus~~~~I.~c.IIII~Is. (Rcnfro. 1975). Isolatcd urinary bladders from sea water and fresh water starry ilounder (Foster. 1975) and the Japanese founder KIII.(?I(S hirn~o~~rr~.s(Hirano. 1974) reabsorb water, Nat and Cl- and appear almost impermeable to K+, Mg’+ and Ca’+. However. irl vitro the urinary bladder of sea water winter flounder, P. LIIU~I.;UU~LIS. is also capable of a small net secretion (serosa to mucosa) of Kf (Renfro, 1975). It follows that in the present study the greater Mg” + content of bladder urine than of kidney urine in sea water flounder could result from reduction in urine volume through the reabsorption of water from the stored urine, effecting the increased Mg2+ concentration. This condition would require the bladder to be relativeIy impermeable to Mg” in order to maintain the serosal-to-mucosal concentration gradient. Recent in rim studies on Mg’ ’ permeability P. stelluttr.surinary bladder support the view that no significant net transepithelial movement of Mg’+ occurs

I

I

I

I

2

Urine

I

3

4

flow, ml /hr/kg

Fig. 8. Relationship of renal magnesium excretion and urine flow in cannulated fresh water starry Hounder. r2 = 0.853 (P < 0.01).

Renal hydromineral metabolism when the bladder wall separates equal or 70: 1 (mucosa-serosa) Mg’ ’ concentration differences under isomotic conditions (Hirano, 1974; Foster, 1975 and unpublished observations). In sea water flounder Na+ and water reabsorption appear to match in osmotic equivalents the increased Mg’+ content. creating the similar osmolarity of kidney and bladder urines. In fresh water flounder the extraction of Na’ from bladder urine without a net reabsorption of water is probably a key factor making bladder urine hypo-osmotic to kidney urine. It is within the kidney that urine is initially formed as an ultra-filtrate of the blood and is subsequently modified by reabsorption and sccrction of essential nutrients. water and electrolytes (Hickman & Trump. 1969). The kidneys of sea water P. .sfcllatus. in common with other marine fish, secrete considerable quantities of Mg’+ and reabsorb Na+ from the ultrafiltrate before passing this fluid to the urinary bladder. Functionally, the sea water flounder kidney operates as though the transport of Na+ and Mg2’ are coupled. Urinary MgZ+ content is significantly and inversely proportlonal to the Na- level over the wide range of Na’ concentrations that can exist in bladder and kidney urines of sea water flounder without altering urine osmotic pressure. This coordinated adjustment of Na’ and Mg” resulting in urine isosmotic to the blood does not necessarily suggest a coupled transport process of Na’ reabsorption of Mg’+ secretion. Bayenbach & Kirschner (1974) find no clear correlation between the rates of Mg’+ excretion and Na+ reabsorption in sea water-adapted rainbow trout as might be expected from a coupled system. However, an interaction of Mg’+ and Na’ could exist in sea water flounder kidneys as in rat proximal kidney tubules where increased pla.srncc Mg’* reduces net Na’ transport by dccrcasing lumen-to-plasma unidirectional Nat flux without change in plasma-to-lumen unidirectional Na* flux (DiBona. 1974). Whatever coordinated interactions, if any. betwren Mg’+ and Na * occur in kidneys of sea water founder. the situation in fresh water flounder appears different. In fresh water fish, urinary Mg2+ content is not correlated with the Na+ level and any variation in urinary Nat proportionally changes urinary osmotic pressure. Differences in renal Na’ and Mg’- excretion in relation to urine Row in sea water starry flounder support the long-held idea (Forster & Berglund. 1956) that urine production. although meager in sea water fish. is directed toward eliminating excess Mg” entering the blood directly from the external environment and from ingested sea water. Although increased urine flow parallels increased Mg’+ excretion. it is clear that Na+ excretion is not altered. Increased urine flow. however. would result in depletion of body water (dehydration) unless recovered. The sea water flounder urinary bladder accomplishes this through an apparently continuous, Na+-linked reabsorption of water from urine stored in the urinary bladder (Johnson er al.. 1972). The highly diuretic and natriuretic condition of fresh water flounder could lead to significant depletion of blood electrolyte levels if renally eliminated salts are not recovered. Foster (1975) has shown that Nat excretion in fresh water flounder. although 4-fold greater than Na’ excretion in sea water

139

flounder, arises more as a result of increased urine flow (a 4-fold increase), than from decreased Na+ reabsorption. Increased urine flow implies a decreased period of residency of urine in the kidney tubule. A compensatory increase in tubular Na+ reabsorption may therefore account for the similarity in Na+ concentration in free-flow (kidney) urine of sea water and fresh water flounder. The Na+ reabsorptive capacity of the urinary bladder must play an important adaptive role in restricting Na+ losses in the low sodium, fresh water environment. There is considerable evidence for an active absorption of Na+ across the relatively waterimpermeable urinary bladder wall of fresh water acclimated fish (Johnson et ctl., 1970. 1972: Lahlou & Fossat. 1971 ; Hirano er ~(1..1971) and an indication that the rate of Na+ uptake is increased during fresh water acclimation (Foster. 1975). Such events are clearly consonant with the idea that the urinary bladder is operative in ion and osmotic homeostasis. A~,kr~o~~lrl!clc,,~~rnrs~~ I thank the personnel of the Bode&a Marine Laboratory for their considerable assistance and hospitality during my stay and Malcolm Erskin and Thomas Gross for continuous and cncrgetic technical assistance. I am indebted to Professor Howard A. Bern for his support and keen interest. This work was aided by N.I.H. grants CA-05388 and CA-O.5035 and I N.I.H. postdoctoral fellowship. REFERENCES BLI~NBACH K. W. & KfKSc’f~NfR L. B. (1474) Renal interactions in the sea water adapted rainNat-Mg”’ bow trout. F&l. Proc,. Fc1tl11ilnl. &KS c’\-p. hio[. 33. 305. DIIKINA G. F. (1974) Effect of magnesium on unidirectional and net sodium fluxes in microperfused rat proximal tubules. .41n. J. Ph>xiol. 226. 470 474. FLLMIYG W. R. & STANLr:y J. G. (1965) Effects of rapid changes in salinity on renal function of a curyhaline teleost. Afn. J. Ph~siol. 200. 1025 1030. FOKSTEK R. P. & B~KGL~IND F. (1956) Osmotic diuresis and its effect on total electrolyte distribution in plasma and urine of the aglomerular tcleost. Lophilrs rrn~r,ric~r/~us. J. grr~. Physiol. 39. 349-359. Fosrf-K R. C. (1972) Prolactin modification of renal electrolyte excretion in the starry flounder. AI,I. Zoo\. 12. 677. FOSTIX R. C. (1974) Comparative biochemical studies of osmoregulation in Sipuncula I. Steady-state characterlstics of two sipunculids in full-strength sea water. Corlt/~. Bio&,,~. Phj~.siol. 47A. 855 X66. FOSTI:R R. C. (1975) Changes in urinary bladder and kidncb function in the starry flounder (Pluticltth~~~ S/I,//CI[IU) in

response to prolactin and fresh water tr:msfct-. G~vI. conlp. Entiocr. 27, 153-l 61. HI~~~MAY C. P. (1968) Urine composition

and kidnq tubular function in southern Rounder. Ptrrtr/ichr/tr.\ /(‘~/IOsriqjtlu. in sea water. Ctr,~. J. Zoo/. 46. 439. Hf(.KMAN C. P. JR. & TRUMP B. F. (1969) The kidney. In Fish Ph~~.sio/oq~~ (Edited by HOAR W. S. & RANIIALL D. J.). Vol. 1. pp. 91-139. Academic Press. New York. HIRAM T. (1971) Effect of prolactin on water and electrolyte movements in the isolated urinary bladder of the flounder, Karrius hicolorrrtus. Grn. cornp. Eudocr. 22. 382. HIRANO T.. JOHNSON D. W. & BERN H. A. (1971) Control of water movement in flounder urinary bladder by prolactin. Naturr. Lmd. 230. 46947 I. JO~~NSOND. W., HIRANO T. & BERK H. A. (1970) Water and sodium movements in flounder (Plarichrh~ .str~//arlrs) urinary bladder and their hormonal control. .4,x Zoo/. IO. 497.

I40

Rou C. FOSTER

JOHNSOUD. W.. HIRANO T.. B~KN H. A. & C‘ONTEF. P. (1972) Hormonal control of water and sodium movements in the urinary bladder of the starry flounder. P/utichrllcs vtdlutrr,~. Cm. co~~~p. Emhcr. 19. 1I5 128. LAHLOLB. (1967) Excretion renale che7 un poisson euryhalin. le Ret (P/~rtic~ht/~~~.s,fustrs L.): Caractkristiques de I’m-inc normalc en eau deuce et en eau de mer et effets de changements de milieu. Cofttp. Biochcr~t. Ph,vsio/. 20. Q?j ‘,.7X. L,\HLOI B. & FOSSAT B. (1971) Mt:caniamc du transport dc I’cau et du scl ;I travcr\ Ia v&c urinaire d’un Poisson tklkostten en eau deuce. la tuite arc-en-ciel. C.r. hrhd. .%tnc. Actr[/. Sci.. Ptrris 273, 21 O&21 IO. MA< ~-\I
on osmoregulation in the euryhahnc Hounder. Pltrricl~rh?s flcsus (L). in sea water and in fresh water. C~I,II/J. Biod~m. Physiol. 47A. 201 3 17. RITVFK~J. L. (1975) Water and ion transport by the urinary bladder of the teleost P.sc~utlop/ru~ort~~~~r.\ mwic~rruus. .It~r. J. P k~~tiol. 228. S2 6 I. RFNFKO J. L. (1974) Ion transport processes in the urinary bladder of the winter Ilounder. Pst,~rr/op/cu~or~~,~/~,,\ tr~~i~‘111111.5. Fd,l. Ptw. Fdll .A,rr .SOC~\“I:,‘, Hid 33. 305, SN~DECOR G. W. & COCHRAN W. C;. (1967) Sturi.\rko/ mrrhods. 6th Edn. Iowa State University Press, Ames. Iowa. SIAW:Y J. G. & FLI.MIK W. R. (1964) Excretion of hypcrtonic urine by ;I teleort. .Scr~v~c~<~. .\‘.I’ 144. 63 64.