Pflfigers Archly
Pflfigers Arch. 380, 159-163 (1979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
A Micropuncture Study of the Renal Handling of Lithium John P. Hayslett and Michael Kashgarian Departments of Medicine, Pediatrics and Pathology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA
Abstract. Although clearance studies in man and experimental animals indicate that filtered lithium is reabsorbed primarily in the proximal tubule, it is unclear whether lithium is also reabsorbed in distal portions of the nephron. Micropuncture studies were, therefore, performed to determine the nephron sites involved in lithium transport during free flow. A method was established to estimate the concentration of lithium in nanoliter samples, using the Helium Glow photometer, which permitted the accurate measurement of lithium in tubular fluid samples over a range from 0 . 5 - 30.0 raM. Approximately 56 ~ of filtered lithium and tubular fluid was reabsorbed at the end of the proximal convolution, while at the early distal tubule 75 % of filtered lithium and water was reabsorbed. There was no change in net transepithelial movement of lithium beyond the loop of Henle. These data suggest that lithium transport is localized to the proximal tubule, including the pars recta. Lithium reabsorption does not occur in distal tubule or collecting duct. Beyond the early distal tubule net movement of lithium and sodium is dissociated. Key words: analysis.
Lithium transport
-
Micropuncture
this substance is important in the prevention and treatment of lithium toxicity. Clearance studies in man and experimental animals have shown that 70 - 80 ~ of the filtered load of lithium is reabsorbed predominantly in the proximal tubule [18, 19]. It is unclear, however, whether a small fraction of the filtered load is also reabsorbed in more distal portions of the nephron. For example, although clearance experiments have attempted to evaluate the handling of lithium in the distal nephron by using pharmacologic agents and physiological pertubations which are through to influence sodium transport in specific nephron segments, the results, as recently reviewed by Thomsen, have been contradictory [17]. Analysis of this question, therefore, necessitates a direct study of fractional transport rates in tubular segments at the level of the individual nephrons. To evaluate the transport sites for lithium reabsorption along the nephron, a method was developed to estimate its concentration in samples of tubular fluid and micropuncture studies were performed in normal, non-diuretic rats. The results of this study directly demonstrate that approximately 7 5 ~ of filtered lithium is reabsorbed in the proximal tubule and loop of Henle and that there is no evidence for further reabsorption at more distal nephron sites.
Methods Introduction Although lithium is not a major endogenous substance, recent interest in the renal handling of lithium and its effect on transport processes of the renal tubule has been generated by its use in clinical medicine to treat neuropsychiatric disorders. Since lithium has a low therapeutic index and serum lithium levels are largely influenced by the rate of renal lithium clearance, an understanding of the tubular sites of reabsorption of
Male Sprague-Dawley rats, weighing 200-300 g, were used in all experiments and were fed regular Purina Chow and allowed to drink tap water ad lib until the time of study. Following induction of anesthesia with Inactin (Promonta, Hamburg, Germany) in a dose of 80 - 100 mg per kg body weight, a tracheostomy was performed, two polyethylene catheters (PE 50) were placed into jugular veins for the administration of fluid and a catheter was placed into the urinary bladder for collection of urine. Following surgery 0.15 M NaC1, equal in volume to 1 ~ of body weight, was infused intravenously to replace surgical losses of body fluid. Animals were prepared for micropuncture as previously described in our laboratory [3].
0031-6768/79/0380/0159/$01.00
160 After administrating a priming dose of 100 Ixcof methoxy-inulinH a in 0.5 ml of 0.3 M LiC1, a sustaining infusion was administered to deliver 100 ~tcinulin-H 3 per hour in a volume of 1.2 ml of 0.3 M LiCI. Previous studies in our laboratory have shown that that priming and sustaining dose of LiC1 resulted in relatively constant plasma levels of lithium of 2 - 4 mEq per liter [2]. Following a 45 min equilibration period, three 30 rain urine collection periods were performed to determine the clearance of H3-inulin and the urinary excretion of lithium and sodium. A blood sample was obtained from the tail at the mid-point of each urine collection to determine plasma H3-inulin activity. During clearance periods timed collections of tubular fluid were obtained from end proximal and early distal tubular sites, using sharpended glass capillary micropipettes. The selection of puncture sites was made on the basis of the transit of 2 % FD & C green dye (Keystone Aniline and Chemical Co.). The terminal portion of the last accessible portion of the proximal tubule was determined during the rapid transit of dye. Early sites in the distal tubule were identified by the ratio of dye transit to the tubular segment selected for study to the dye transit to the earliest distal segment to fill with dye, after intravenous injection. A ratio of less than 1.3 was taken to identify the early portion of the distal tubule [21]. Dye was allowed to completely clear from tubular fluid before punctures were performed. Tubular fluid collections were analyzed for the concentration of lithium and activity of H3-inulin. Immediately following each tubular fluid collection a blood sample was obtained for determination of the plasma concentration of lithium and H3-inulin activity. Determination of lithium in samples of nanoliter size was performed on a Helium Glow Photometer (American Instrument Co., Inc.). Lithium emission at 6708 ~, was obtained with an interference filter (Baird-Atomics, Inc., Bedford, Mass.). Characteristics of the filter provided by manufacturer included the following: 90 A band width at ~/2 peak transmission; peak transmission 54% at t/2 band width. Emission was recorded by photomultiplier tube R446, supplied by American Instrument Co. for measurement of potassium. Standard solutions were made from reagent grade LiC1 dissolved in demineralized water or 0.15 M NaC1. The concentration range of lithium tested was 0.5 to 30 mM. With this system the sensitivity was found to be approximately 1 • 10-~2 Moles and the precision was reflected by the determination of a standard containing 2.50 mEq/1 of lithium. The mean _+ SD of 18 samples was 2.51 +0.07. Samples were diluted 1:4 in 5 mM CsNO3 and 5 mM NH4PO~ and the sample size measured by the photometer was 4 - 5 nl. The emission profile is shown in Fig. 1. To determine the accuracy of the micromethod in the measurement of lithium recovery studies were performed in which lithium concentration was estimated in solutions containing varying concentrations of LiC1 dissolved in Ringer's solution before and after the addition of a sample, to the wire of the photometer, containing LiC1 dissolved in water. The results of that study are shown in Fig. 2 and indicate that the recovery of the added lithium was 103 _+1%. Similar experiments were performed in which LiCI, dissolved in water, was added to samples of distal tubule fluid obtained from animals infused with LiC1. Recovery was 100 _+1 ~o- These data indicate that the constituents of tubular fluid do not interfer with the accurate estimation of lithium by the microanalytical method. Since changes in the concentration of other cations within tubular fluid may have influenced the emission of lithium, the effect of variations in the concentration of NaC1 and KC1 on the estimation of lithium concentration was examined. In this experiment solutions with the same concentration of LiC1 were prepared in which the concentration of NaC1 varied between 50--150 mM and KC1 varied between 5 50 raM, to simulate variations in Na and K that could be expected to occur in proximal and distal fluid. Neither the presence nor changes in concentration of NaC1 influenced the emission readings of lithium, compared to values obtained from a solution containing the same concentration of LiC1 dissolved in water.
Pflfigers Arch. 380 (1979)
Fig. 1. Lithium emission profile, LiC1, 6 mMoles, dissolved in 0.15 M NaC1. The period of integration is shown by the time square well
I000
f
800 stondords plus 600
400 stondard curve
/
L~Cl in d ilue nt - - " ~
~....~
200
~
a , 400 units
/ o
/
i i.o
2.o
I 3.0
LITHIUM CONCENTRATION, mEq / liter
Fig. 2. Experiment in which a known amount of LiCI was added to lithium standards to demonstrate quantitative recovery
The concentration of lithium in tubular fluid samples were determined by the Helium Glow photometer from the average value of quadriplicate determinations. Since the photometer was more sensitive for sodium [2] than for lithium, sodium concentration was not estimated in tubular fluid samples. Estimation of sodium would have required a higher dilution than used for measurement of lithium. Lithium concentration in plasma and urine was determined by a Perkin-Elmer Mass Spectrometer and sodium in plasma and urine by a flame photometer with an internal standard. The activity of H 3inulin (New England Nuclear) was determined with a liquid scintillation counter (Parkard Instruments). Results are presented as mean • SE and the Student's t-test was used for statistical comparison.
Results
S t u d i e s w e r e p e r f o r m e d i n 14 a n i m a l s d u r i n g t h e infusion of lithium, under non-diuretic conditions. P l a s m a l i t h i u m levels a v e r a g e d 3.90_+0.27 m E q p e r liter. T h e g l o m e r u l a r f i l t r a t i o n r a t e a v e r a g e d 442.3 + 58.8 ~tl/min/100 g B W p e r k i d n e y . D a t a r e l a t i n g t o
J. P. Hayslett and M. Kashgarian: Renal Handling of Lithium
161
Table 1. Summary ofmicropuncture data in normal animals. Values represent mean _+ SE. The SNGFR and urine values were averaged for each animal while the mean of separate determinations were computed for tubular fluid collections. The number of parenthesis indicates the number of observations
SNGFR, nl/min 1-P/TF], x 100 TF/PL~ I-TF/P Li/Inx 100 I-U/P Na/In x 100
Endproximal
Early distal
40 • 55.5 .+3.0 1.01• 56.7 .+3.6
36 + 2 (12) 79.3 +2.6 (10) 1.38_+0.12 (9) 74.6 • (10)
(14) (12) (12) (12)
the urinary excretion of sodium and lithium and the fractional reabsorption of lithium and water in different nephron segments is shown in Table 1 and Fig. 3. Mean SNGFR averaged 40 _+3 nl/min in proximal tubular collections. The somewhat lower value obtained from distal tubule collections was not statistically different. The fractional reabsorption of water, and presumably sodium, in the proximal convolution was 55.5+3%, under the conditions of this study. Fractional water reabsorption continued to rise in more distal segments of the nephron and averaged 79.3 _+2.6 ~ at the early distal tubule and 99.5 _+0.01% in urine. Tubular fluid/plasma lithium (1.01 • 0.06) was not statistically different from unity in proximal tubule fluid, indicating that net absorption of lithium and water occured at the same rates. At later nephron sites, however, the TF/PLi gradient increased to 1.38 • 0.12 in the early distal tubule and 48.80 • 5.16 in urine. Fractional reabsorption of lithium in proximal tubule was 56.7• a value that was not statistically different from that of tubular fluid. Approximately 18 % of the filtered load of lithium was reabsorbed in the loop of Henle. Since the ascending limb of Henle's loop is considered to be water impermeable and the fractional reabsorption of water and lithium were nearly identical through the early distal tubule, it seems likely that lithium absorption in the loop of Henle occurs in the pars recta. There was no evidence for further reabsorption of lithium beyond the early distal tubule, as shown in Table 1 and Fig. 3. The fraction of filtered lithium excreted was 24.8 • 1.3 %, a value that was statistically similar to the fraction of filtered lithium delivered to the early distal tubule, 25.4 • 3.4 % (P = NS). The overall fractional reabsorption of sodium, in contrast, was 99.3 •
Discussion
Previous studies have indicated that reabsorption of filtered lithium occurs primarily in the proximal tubule.
Urine
99.5 • (14) 48.80_+5.I6 (14) 75.0 +1.3 (10) 99.3 • (8)
75
h 0
z
~
o
:E
r--~
50 n,. it_ ._1
__ rr z ILl
25
3 ill
IK
END PROXIMAL
EARLY DISTAL
URINE
Fig. 3. The fraction of filtered lithium remaining at the end proximal and early distal tubule and in the final urine is shown. Bars represent mean • SEM
Since lithium is not bound to plasma proteins [1] it passes freely through glomerular membranes. The fractional excretion of lithium of 2 0 - 30 % in man [18] and experimental animals [1, 10] corresponds to the fraction of filtered sodium reabsorbed in the proximal tubule. Moreover, osmotic diuresis and the administration of sodium bicarbonate and acetazolamide, maneuvers thought to inhibit proximal transport, increase fractional excretion of lithium [l 8]. In contrast, agents which predominantly impair sodium transport in ascending limb and more distal tubular sites, such as furosemide, bendofluomethiazide, ethacrynic acid and spironolactone, do not significantly influence the urinary excretion of lithium [18]. Corresponding changes in fractional excretion of lithium and phosphate, under several experimental conditions, also suggest a prominant role for the proximal tubule in the renal handling of lithium [12, 13] while volume concentration increases proximal reabsorption of sodium [20] and decreases fractional excretion of lithium [15]. Lastly, the correlation between the clearance of lithium and urine flow rate in rats with diabetes insipidus, due to hereditary lack of vasopressin, suggest that lithium and sodium are reabsorbed in parallel in the proximal tubule [16].
162 While these studies, taken together, indicate that filtered lithium is primarily reabsorbed in the proximal tubule, they do not exclude further reabsorption in the loop of Henle or in more distal nephron sites. Steel et al. infused furosemide to sodium loaded rats in which proximal reabsorption was blocked by acetazolamide [14]. The observed increase in lithium clearance after furosemide administration was interpreted to be the result of inhibition of lithium reabsorption in the ascending limb of Henle's loop. An inhibitory action of furosernide on proximal tubule, however, was not excluded and since furosemide treated animals received additional sodium it is possible that volume expansion may have also occurred. Further arguments for reabsorption of lithium at distal sites are based upon the action of lithium to impair distal transport functions [14]. The effect of lithium to enter cells in distal nephron sites, however, does not indicate transepithelial movement of lithium. As recently reviewed by Thomsen, therefore, the experimental data derived from clearance studies are unclear with respect to transport sites for lithium reabsorption beyond the proximal tubule [17]. To determine the direction and magnitude of net lithium movement in proximal and distal segments of the nephron free flow micropuncture studies were performed in the rat. The method developed in our laboratory to determine the concentration of lithium in nanoliter samples was shown to be sensitive, accurate and precise. These experiments indicate that net reabsorption of lithium is confined to the proximal convolution and loop of Henle and suggest that net transport is localized to the proximal tubule including the pars recta. Although the similarity between lithium and water transport in the loop of Henle strongly suggests, in our view, that lithium transport is localized to the pars recta, these data do not exclude possible net transport of lithium in thin portions of the loop or in the thick ascending limb. Further increases in the concentration of lithium in tubular fluid in distal convolution and collecting duct were due to the reabsorption of water. There was no evidence for net reabsorption or secretion of lithium in the distal tubule or collecting duct system. Although the present study was designed to study the renal handling of lithium and did not include an evaluation of the relationship between sodium and lithium reabsorption recent micropuncture experiments in our laboratory [4] determined the fractional reabsorption of sodium during the infusion of lithium, using conditions that were similar to those employed in the present study. Since the TF/PNa gradient in proximal tubule fluid in that study and the TF/PLi gradient in proximal tubule in the present study were not statistically different from one the fractional reabsorption of both cations in the proximal tubule are similar
Pfliigers Arch. 380 (1979) in these experiments. The rate of fractional reabsorption of sodium in the loop of Henle and in more distal tubular sites, however, exceeded the rates of lithium reabsorption observed in this study. Fractional delivery of sodium to early distal tubule was 12 ~ , compared to the delivery of 25 ~ of the filtered load of lithium. In contrast to the absence of net movement of lithium in distal tubule and collecting duct, approximately 11.5 of the filtered load of sodium was reabsorbed in those segments of the distal nephron. These data indicate, therefore, that net transport of lithium and sodium is dissociated beyond the proximal tubule. Previous studies concerned with the transport properties of lithium have resulted from its use an experimental device to evaluate transport characteristics of epithelium for alkali metals, as a general phenomena, and from a desire to obtain a better understanding of the renal handling of lithium because of its use in the treatment of patients with manic-depressive disorders. In vitro studies in urinary toad bladder have shown that N a § and Li § transport from mucosal to serosal surface occurs against electrical and concentration gradients, are stimulated by antidiuretic hormone and inhibited by diuretic agents [11]. Lithium competes with N a § at the luminal cell membrane, displaces intracellular K +, and may share N a § pathways for cellular extrusion at the basolateral cell membrane [5, 8]. Despite evidence for active transport of lithium it is pertinent that the electrochemical gradient against which movement occurs in toad bladder is approximately 10-fold greater for sodium than for lithium [5]. It is possible, therefore, that this factor may explain the absence of net movement of lithium in distal tubule and collecting duct, where N a § is absorbed against an electrochemical gradient o f nearly 80 mV [9].
Acknowledgement. This work was supported by USPHS grant 18061 and the American Heart Association. The authors wish to acknowledge the technical assistance of Ms. Barbara Lee and Mrs. Trudi Klein-Robbenhaar and the secretarial assistanceof Ms. Linda Frisco.
References 1. Foulkes,J., Mudge, G. H., Gilman, A. : Renal excretionof cation in the dog during infusion of isotonic solutions of lithium chloride. Am. J. Physiol. 168, 692 649 (1952) 2. Galla,J. H., Forrest, J. N., Hecht, B., Kashgarian, M., Hayslett, J. P. : Effect of lithium on water and electrolytemetabolism.Yale J. Biol. Med. 48, 305-315 (1975) 3. Hayslett, J. P., Kashgarian, M., Epstein, F. H.: Functional correlates of compensatoryrenal hypertrophy. J. Clin. Invest. 47, 774- 782 (1968) 4. Hecht, B., Kashgarian, M., Forrest, J. N., Jr., Hayslett, J. P.: Micropuncture study on the effects of lithium on proximal and distal tubule function in the rat kidney. PflfigersArch. 377, 69 74 (1978)
J. P. Hayslett and M. Kashgarian: Renal Handling of Lithium 5. Herrera, F. C., Egea, R., Herrera, A. M. : Movement of lithium across toad urinary bladder, Am. J. Physiol. 220, 1501-1508 (1971) 6. Hober, R. : Physical Chemistry of Ceils and Tissues. Philadelphia, Boston: Blakiston 1945 7. Latimer, W. M., Hildebrand, J. H. : Reference Book of Inorganic Chemistry. New York: MacMillan 1937 8. Leblanc, G. : The mechanism of lithium accumulation in the isolated frog skin epithelium. Pflfigers Arch. 337, l - 18 (1972) 9. MaInic, G., Klose, R. M., Biegisch, G. : Micropuncture study of distal tubular potassium and sodium transfer in the rat kidney. Am. J. Physiol. 211, 529-559 (1966) 10. Schou, M.: Lithium studies. 2. Renal elimination. Acta Pharmacol. 15, 8 5 - 9 8 (1958) 11. Singer, I., Rotenberg, D., Puschett, J. P.: Lithium-induced nephrogenic diabetes insipidus: in vivo and in vitro studies. J. Clin. Invest. 51, 1081-1091 (1972) 12. Steele, T. H., Dudgeon, K. L.: Reabsorption of lithium and phosphate by the rat kidney: Role of the parathyroids. Kidney Int. 5, 196-203 (1974) 13. Steele, T. H., Manuel, M. A., Newton, M., Bonder, S.: Renal lithium reabsorption in man: physiologic and pharmacologic determinants. Am. J. Med. Sci. 269, 349-363 (1975) 14_ Steele, T. H., Dudgeon, K. L., Tarmore, C. K. : Pharmacological characterization of lithium reabsorption in the rat. J. Pharmacol. Exp. Ther. 196, 184-193 (1976)
163 15. Thomsen, K. : The effect of sodium chloride on kidney function in rats with lithium intoxication, Acta Pharmacol. Toxicol. (Kbh.) 33, 9 2 - 1 0 2 (1973) 16. Thomsen, K. : The renal handling oflithium: Relation between lithium clearance sodium clearance and urine flow in rats with diabetes insipidus. Acta Pharmacol. 40, 491-496 (i977) 17. Thomsen, K. : Renal handling of lithium at non-toxic and toxic serum lithium levels. Dan. Med. Bull. 25, 106-115 (1978) 18. Thomsen, K., Shou, M. : Renal lithium excretion in man. Am. J. Physiol. 215, 823-827 (1968) 19. Thomsen, K., Shou, M., Steiners, I. : Lithium as an indicator of proximal sodium reabsorption. Pflfigers Arch. 308, 180-184 (1969) 20. Weiner, M. W., Weinman, E. J., Kashgarian, M., Hayslett, J. P. : Accelerated reabsorption in the proximal tubule produced by volume depletion. J. Clin. Invest. 50, 1379-1385 (1971) 21. Wright, F. S. : Increasing magnitude of electrical potential along the renal distal tubule. Am. J. Physiol. 220, 624-638 (1971) 22. Vurek, G. H., Bowman, R. L. : Helium-glow photometer for picomole analysis of alkali metals. Science 49, 448-450 (1965)
Received January 10, 1979
Pflfigers Arch. 380, 159-163 (1979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
A Micropuncture Study of the Renal Handling of Lithium John P. Hayslett and Michael Kashgarian Departments of Medicine, Pediatrics and Pathology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA
Abstract. Although clearance studies in man and experimental animals indicate that filtered lithium is reabsorbed primarily in the proximal tubule, it is unclear whether lithium is also reabsorbed in distal portions of the nephron. Micropuncture studies were, therefore, performed to determine the nephron sites involved in lithium transport during free flow. A method was established to estimate the concentration of lithium in nanoliter samples, using the Helium Glow photometer, which permitted the accurate measurement of lithium in tubular fluid samples over a range from 0 . 5 - 30.0 raM. Approximately 56 ~ of filtered lithium and tubular fluid was reabsorbed at the end of the proximal convolution, while at the early distal tubule 75 % of filtered lithium and water was reabsorbed. There was no change in net transepithelial movement of lithium beyond the loop of Henle. These data suggest that lithium transport is localized to the proximal tubule, including the pars recta. Lithium reabsorption does not occur in distal tubule or collecting duct. Beyond the early distal tubule net movement of lithium and sodium is dissociated. Key words: analysis.
Lithium transport
-
Micropuncture
this substance is important in the prevention and treatment of lithium toxicity. Clearance studies in man and experimental animals have shown that 70 - 80 ~ of the filtered load of lithium is reabsorbed predominantly in the proximal tubule [18, 19]. It is unclear, however, whether a small fraction of the filtered load is also reabsorbed in more distal portions of the nephron. For example, although clearance experiments have attempted to evaluate the handling of lithium in the distal nephron by using pharmacologic agents and physiological pertubations which are through to influence sodium transport in specific nephron segments, the results, as recently reviewed by Thomsen, have been contradictory [17]. Analysis of this question, therefore, necessitates a direct study of fractional transport rates in tubular segments at the level of the individual nephrons. To evaluate the transport sites for lithium reabsorption along the nephron, a method was developed to estimate its concentration in samples of tubular fluid and micropuncture studies were performed in normal, non-diuretic rats. The results of this study directly demonstrate that approximately 7 5 ~ of filtered lithium is reabsorbed in the proximal tubule and loop of Henle and that there is no evidence for further reabsorption at more distal nephron sites.
Methods Introduction Although lithium is not a major endogenous substance, recent interest in the renal handling of lithium and its effect on transport processes of the renal tubule has been generated by its use in clinical medicine to treat neuropsychiatric disorders. Since lithium has a low therapeutic index and serum lithium levels are largely influenced by the rate of renal lithium clearance, an understanding of the tubular sites of reabsorption of
Male Sprague-Dawley rats, weighing 200-300 g, were used in all experiments and were fed regular Purina Chow and allowed to drink tap water ad lib until the time of study. Following induction of anesthesia with Inactin (Promonta, Hamburg, Germany) in a dose of 80 - 100 mg per kg body weight, a tracheostomy was performed, two polyethylene catheters (PE 50) were placed into jugular veins for the administration of fluid and a catheter was placed into the urinary bladder for collection of urine. Following surgery 0.15 M NaC1, equal in volume to 1 ~ of body weight, was infused intravenously to replace surgical losses of body fluid. Animals were prepared for micropuncture as previously described in our laboratory [3].
0031-6768/79/0380/0159/$01.00
160 After administrating a priming dose of 100 Ixcof methoxy-inulinH a in 0.5 ml of 0.3 M LiC1, a sustaining infusion was administered to deliver 100 ~tcinulin-H 3 per hour in a volume of 1.2 ml of 0.3 M LiCI. Previous studies in our laboratory have shown that that priming and sustaining dose of LiC1 resulted in relatively constant plasma levels of lithium of 2 - 4 mEq per liter [2]. Following a 45 min equilibration period, three 30 rain urine collection periods were performed to determine the clearance of H3-inulin and the urinary excretion of lithium and sodium. A blood sample was obtained from the tail at the mid-point of each urine collection to determine plasma H3-inulin activity. During clearance periods timed collections of tubular fluid were obtained from end proximal and early distal tubular sites, using sharpended glass capillary micropipettes. The selection of puncture sites was made on the basis of the transit of 2 % FD & C green dye (Keystone Aniline and Chemical Co.). The terminal portion of the last accessible portion of the proximal tubule was determined during the rapid transit of dye. Early sites in the distal tubule were identified by the ratio of dye transit to the tubular segment selected for study to the dye transit to the earliest distal segment to fill with dye, after intravenous injection. A ratio of less than 1.3 was taken to identify the early portion of the distal tubule [21]. Dye was allowed to completely clear from tubular fluid before punctures were performed. Tubular fluid collections were analyzed for the concentration of lithium and activity of H3-inulin. Immediately following each tubular fluid collection a blood sample was obtained for determination of the plasma concentration of lithium and H3-inulin activity. Determination of lithium in samples of nanoliter size was performed on a Helium Glow Photometer (American Instrument Co., Inc.). Lithium emission at 6708 ~, was obtained with an interference filter (Baird-Atomics, Inc., Bedford, Mass.). Characteristics of the filter provided by manufacturer included the following: 90 A band width at ~/2 peak transmission; peak transmission 54% at t/2 band width. Emission was recorded by photomultiplier tube R446, supplied by American Instrument Co. for measurement of potassium. Standard solutions were made from reagent grade LiC1 dissolved in demineralized water or 0.15 M NaC1. The concentration range of lithium tested was 0.5 to 30 mM. With this system the sensitivity was found to be approximately 1 • 10-~2 Moles and the precision was reflected by the determination of a standard containing 2.50 mEq/1 of lithium. The mean _+ SD of 18 samples was 2.51 +0.07. Samples were diluted 1:4 in 5 mM CsNO3 and 5 mM NH4PO~ and the sample size measured by the photometer was 4 - 5 nl. The emission profile is shown in Fig. 1. To determine the accuracy of the micromethod in the measurement of lithium recovery studies were performed in which lithium concentration was estimated in solutions containing varying concentrations of LiC1 dissolved in Ringer's solution before and after the addition of a sample, to the wire of the photometer, containing LiC1 dissolved in water. The results of that study are shown in Fig. 2 and indicate that the recovery of the added lithium was 103 _+1%. Similar experiments were performed in which LiCI, dissolved in water, was added to samples of distal tubule fluid obtained from animals infused with LiC1. Recovery was 100 _+1 ~o- These data indicate that the constituents of tubular fluid do not interfer with the accurate estimation of lithium by the microanalytical method. Since changes in the concentration of other cations within tubular fluid may have influenced the emission of lithium, the effect of variations in the concentration of NaC1 and KC1 on the estimation of lithium concentration was examined. In this experiment solutions with the same concentration of LiC1 were prepared in which the concentration of NaC1 varied between 50--150 mM and KC1 varied between 5 50 raM, to simulate variations in Na and K that could be expected to occur in proximal and distal fluid. Neither the presence nor changes in concentration of NaC1 influenced the emission readings of lithium, compared to values obtained from a solution containing the same concentration of LiC1 dissolved in water.
Pflfigers Arch. 380 (1979)
Fig. 1. Lithium emission profile, LiC1, 6 mMoles, dissolved in 0.15 M NaC1. The period of integration is shown by the time square well
I000
f
800 stondords plus 600
400 stondard curve
/
L~Cl in d ilue nt - - " ~
~....~
200
~
a , 400 units
/ o
/
i i.o
2.o
I 3.0
LITHIUM CONCENTRATION, mEq / liter
Fig. 2. Experiment in which a known amount of LiCI was added to lithium standards to demonstrate quantitative recovery
The concentration of lithium in tubular fluid samples were determined by the Helium Glow photometer from the average value of quadriplicate determinations. Since the photometer was more sensitive for sodium [2] than for lithium, sodium concentration was not estimated in tubular fluid samples. Estimation of sodium would have required a higher dilution than used for measurement of lithium. Lithium concentration in plasma and urine was determined by a Perkin-Elmer Mass Spectrometer and sodium in plasma and urine by a flame photometer with an internal standard. The activity of H 3inulin (New England Nuclear) was determined with a liquid scintillation counter (Parkard Instruments). Results are presented as mean • SE and the Student's t-test was used for statistical comparison.
Results
S t u d i e s w e r e p e r f o r m e d i n 14 a n i m a l s d u r i n g t h e infusion of lithium, under non-diuretic conditions. P l a s m a l i t h i u m levels a v e r a g e d 3.90_+0.27 m E q p e r liter. T h e g l o m e r u l a r f i l t r a t i o n r a t e a v e r a g e d 442.3 + 58.8 ~tl/min/100 g B W p e r k i d n e y . D a t a r e l a t i n g t o
J. P. Hayslett and M. Kashgarian: Renal Handling of Lithium
161
Table 1. Summary ofmicropuncture data in normal animals. Values represent mean _+ SE. The SNGFR and urine values were averaged for each animal while the mean of separate determinations were computed for tubular fluid collections. The number of parenthesis indicates the number of observations
SNGFR, nl/min 1-P/TF], x 100 TF/PL~ I-TF/P Li/Inx 100 I-U/P Na/In x 100
Endproximal
Early distal
40 • 55.5 .+3.0 1.01• 56.7 .+3.6
36 + 2 (12) 79.3 +2.6 (10) 1.38_+0.12 (9) 74.6 • (10)
(14) (12) (12) (12)
the urinary excretion of sodium and lithium and the fractional reabsorption of lithium and water in different nephron segments is shown in Table 1 and Fig. 3. Mean SNGFR averaged 40 _+3 nl/min in proximal tubular collections. The somewhat lower value obtained from distal tubule collections was not statistically different. The fractional reabsorption of water, and presumably sodium, in the proximal convolution was 55.5+3%, under the conditions of this study. Fractional water reabsorption continued to rise in more distal segments of the nephron and averaged 79.3 _+2.6 ~ at the early distal tubule and 99.5 _+0.01% in urine. Tubular fluid/plasma lithium (1.01 • 0.06) was not statistically different from unity in proximal tubule fluid, indicating that net absorption of lithium and water occured at the same rates. At later nephron sites, however, the TF/PLi gradient increased to 1.38 • 0.12 in the early distal tubule and 48.80 • 5.16 in urine. Fractional reabsorption of lithium in proximal tubule was 56.7• a value that was not statistically different from that of tubular fluid. Approximately 18 % of the filtered load of lithium was reabsorbed in the loop of Henle. Since the ascending limb of Henle's loop is considered to be water impermeable and the fractional reabsorption of water and lithium were nearly identical through the early distal tubule, it seems likely that lithium absorption in the loop of Henle occurs in the pars recta. There was no evidence for further reabsorption of lithium beyond the early distal tubule, as shown in Table 1 and Fig. 3. The fraction of filtered lithium excreted was 24.8 • 1.3 %, a value that was statistically similar to the fraction of filtered lithium delivered to the early distal tubule, 25.4 • 3.4 % (P = NS). The overall fractional reabsorption of sodium, in contrast, was 99.3 •
Discussion
Previous studies have indicated that reabsorption of filtered lithium occurs primarily in the proximal tubule.
Urine
99.5 • (14) 48.80_+5.I6 (14) 75.0 +1.3 (10) 99.3 • (8)
75
h 0
z
~
o
:E
r--~
50 n,. it_ ._1
__ rr z ILl
25
3 ill
IK
END PROXIMAL
EARLY DISTAL
URINE
Fig. 3. The fraction of filtered lithium remaining at the end proximal and early distal tubule and in the final urine is shown. Bars represent mean • SEM
Since lithium is not bound to plasma proteins [1] it passes freely through glomerular membranes. The fractional excretion of lithium of 2 0 - 30 % in man [18] and experimental animals [1, 10] corresponds to the fraction of filtered sodium reabsorbed in the proximal tubule. Moreover, osmotic diuresis and the administration of sodium bicarbonate and acetazolamide, maneuvers thought to inhibit proximal transport, increase fractional excretion of lithium [l 8]. In contrast, agents which predominantly impair sodium transport in ascending limb and more distal tubular sites, such as furosemide, bendofluomethiazide, ethacrynic acid and spironolactone, do not significantly influence the urinary excretion of lithium [18]. Corresponding changes in fractional excretion of lithium and phosphate, under several experimental conditions, also suggest a prominant role for the proximal tubule in the renal handling of lithium [12, 13] while volume concentration increases proximal reabsorption of sodium [20] and decreases fractional excretion of lithium [15]. Lastly, the correlation between the clearance of lithium and urine flow rate in rats with diabetes insipidus, due to hereditary lack of vasopressin, suggest that lithium and sodium are reabsorbed in parallel in the proximal tubule [16].
162 While these studies, taken together, indicate that filtered lithium is primarily reabsorbed in the proximal tubule, they do not exclude further reabsorption in the loop of Henle or in more distal nephron sites. Steel et al. infused furosemide to sodium loaded rats in which proximal reabsorption was blocked by acetazolamide [14]. The observed increase in lithium clearance after furosemide administration was interpreted to be the result of inhibition of lithium reabsorption in the ascending limb of Henle's loop. An inhibitory action of furosernide on proximal tubule, however, was not excluded and since furosemide treated animals received additional sodium it is possible that volume expansion may have also occurred. Further arguments for reabsorption of lithium at distal sites are based upon the action of lithium to impair distal transport functions [14]. The effect of lithium to enter cells in distal nephron sites, however, does not indicate transepithelial movement of lithium. As recently reviewed by Thomsen, therefore, the experimental data derived from clearance studies are unclear with respect to transport sites for lithium reabsorption beyond the proximal tubule [17]. To determine the direction and magnitude of net lithium movement in proximal and distal segments of the nephron free flow micropuncture studies were performed in the rat. The method developed in our laboratory to determine the concentration of lithium in nanoliter samples was shown to be sensitive, accurate and precise. These experiments indicate that net reabsorption of lithium is confined to the proximal convolution and loop of Henle and suggest that net transport is localized to the proximal tubule including the pars recta. Although the similarity between lithium and water transport in the loop of Henle strongly suggests, in our view, that lithium transport is localized to the pars recta, these data do not exclude possible net transport of lithium in thin portions of the loop or in the thick ascending limb. Further increases in the concentration of lithium in tubular fluid in distal convolution and collecting duct were due to the reabsorption of water. There was no evidence for net reabsorption or secretion of lithium in the distal tubule or collecting duct system. Although the present study was designed to study the renal handling of lithium and did not include an evaluation of the relationship between sodium and lithium reabsorption recent micropuncture experiments in our laboratory [4] determined the fractional reabsorption of sodium during the infusion of lithium, using conditions that were similar to those employed in the present study. Since the TF/PNa gradient in proximal tubule fluid in that study and the TF/PLi gradient in proximal tubule in the present study were not statistically different from one the fractional reabsorption of both cations in the proximal tubule are similar
Pfliigers Arch. 380 (1979) in these experiments. The rate of fractional reabsorption of sodium in the loop of Henle and in more distal tubular sites, however, exceeded the rates of lithium reabsorption observed in this study. Fractional delivery of sodium to early distal tubule was 12 ~ , compared to the delivery of 25 ~ of the filtered load of lithium. In contrast to the absence of net movement of lithium in distal tubule and collecting duct, approximately 11.5 of the filtered load of sodium was reabsorbed in those segments of the distal nephron. These data indicate, therefore, that net transport of lithium and sodium is dissociated beyond the proximal tubule. Previous studies concerned with the transport properties of lithium have resulted from its use an experimental device to evaluate transport characteristics of epithelium for alkali metals, as a general phenomena, and from a desire to obtain a better understanding of the renal handling of lithium because of its use in the treatment of patients with manic-depressive disorders. In vitro studies in urinary toad bladder have shown that N a § and Li § transport from mucosal to serosal surface occurs against electrical and concentration gradients, are stimulated by antidiuretic hormone and inhibited by diuretic agents [11]. Lithium competes with N a § at the luminal cell membrane, displaces intracellular K +, and may share N a § pathways for cellular extrusion at the basolateral cell membrane [5, 8]. Despite evidence for active transport of lithium it is pertinent that the electrochemical gradient against which movement occurs in toad bladder is approximately 10-fold greater for sodium than for lithium [5]. It is possible, therefore, that this factor may explain the absence of net movement of lithium in distal tubule and collecting duct, where N a § is absorbed against an electrochemical gradient o f nearly 80 mV [9].
Acknowledgement. This work was supported by USPHS grant 18061 and the American Heart Association. The authors wish to acknowledge the technical assistance of Ms. Barbara Lee and Mrs. Trudi Klein-Robbenhaar and the secretarial assistanceof Ms. Linda Frisco.
References 1. Foulkes,J., Mudge, G. H., Gilman, A. : Renal excretionof cation in the dog during infusion of isotonic solutions of lithium chloride. Am. J. Physiol. 168, 692 649 (1952) 2. Galla,J. H., Forrest, J. N., Hecht, B., Kashgarian, M., Hayslett, J. P. : Effect of lithium on water and electrolytemetabolism.Yale J. Biol. Med. 48, 305-315 (1975) 3. Hayslett, J. P., Kashgarian, M., Epstein, F. H.: Functional correlates of compensatoryrenal hypertrophy. J. Clin. Invest. 47, 774- 782 (1968) 4. Hecht, B., Kashgarian, M., Forrest, J. N., Jr., Hayslett, J. P.: Micropuncture study on the effects of lithium on proximal and distal tubule function in the rat kidney. PflfigersArch. 377, 69 74 (1978)
J. P. Hayslett and M. Kashgarian: Renal Handling of Lithium 5. Herrera, F. C., Egea, R., Herrera, A. M. : Movement of lithium across toad urinary bladder, Am. J. Physiol. 220, 1501-1508 (1971) 6. Hober, R. : Physical Chemistry of Ceils and Tissues. Philadelphia, Boston: Blakiston 1945 7. Latimer, W. M., Hildebrand, J. H. : Reference Book of Inorganic Chemistry. New York: MacMillan 1937 8. Leblanc, G. : The mechanism of lithium accumulation in the isolated frog skin epithelium. Pflfigers Arch. 337, l - 18 (1972) 9. MaInic, G., Klose, R. M., Biegisch, G. : Micropuncture study of distal tubular potassium and sodium transfer in the rat kidney. Am. J. Physiol. 211, 529-559 (1966) 10. Schou, M.: Lithium studies. 2. Renal elimination. Acta Pharmacol. 15, 8 5 - 9 8 (1958) 11. Singer, I., Rotenberg, D., Puschett, J. P.: Lithium-induced nephrogenic diabetes insipidus: in vivo and in vitro studies. J. Clin. Invest. 51, 1081-1091 (1972) 12. Steele, T. H., Dudgeon, K. L.: Reabsorption of lithium and phosphate by the rat kidney: Role of the parathyroids. Kidney Int. 5, 196-203 (1974) 13. Steele, T. H., Manuel, M. A., Newton, M., Bonder, S.: Renal lithium reabsorption in man: physiologic and pharmacologic determinants. Am. J. Med. Sci. 269, 349-363 (1975) 14_ Steele, T. H., Dudgeon, K. L., Tarmore, C. K. : Pharmacological characterization of lithium reabsorption in the rat. J. Pharmacol. Exp. Ther. 196, 184-193 (1976)
163 15. Thomsen, K. : The effect of sodium chloride on kidney function in rats with lithium intoxication, Acta Pharmacol. Toxicol. (Kbh.) 33, 9 2 - 1 0 2 (1973) 16. Thomsen, K. : The renal handling oflithium: Relation between lithium clearance sodium clearance and urine flow in rats with diabetes insipidus. Acta Pharmacol. 40, 491-496 (i977) 17. Thomsen, K. : Renal handling of lithium at non-toxic and toxic serum lithium levels. Dan. Med. Bull. 25, 106-115 (1978) 18. Thomsen, K., Shou, M. : Renal lithium excretion in man. Am. J. Physiol. 215, 823-827 (1968) 19. Thomsen, K., Shou, M., Steiners, I. : Lithium as an indicator of proximal sodium reabsorption. Pflfigers Arch. 308, 180-184 (1969) 20. Weiner, M. W., Weinman, E. J., Kashgarian, M., Hayslett, J. P. : Accelerated reabsorption in the proximal tubule produced by volume depletion. J. Clin. Invest. 50, 1379-1385 (1971) 21. Wright, F. S. : Increasing magnitude of electrical potential along the renal distal tubule. Am. J. Physiol. 220, 624-638 (1971) 22. Vurek, G. H., Bowman, R. L. : Helium-glow photometer for picomole analysis of alkali metals. Science 49, 448-450 (1965)
Received January 10, 1979
