Renal regulation of plasma glucose in the freshwater rainbow trout
McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1
* Author for correspondence (e-mail: buckincp{at}mcmaster.ca)
Accepted 28 April 2005
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Summary |
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Key words: carbohydrate diet, glucose transport maximum, Na+ reabsorption, Oncorhynchus mykiss, phlorizin, renal function
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Introduction |
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Rainbow trout (Oncorhynchus mykiss Walbaum), like many other
species of fish, have difficulty peripherally utilizing glucose obtained from
their diets, which results in slow plasma clearance
(Legate et al., 2001). As a
consequence, carbohydrates are considered to be of limited value in the
nutrition of fish such as the rainbow trout
(Phillips and Brockway, 1959
;
Legate et al., 2001
). However,
due to recent findings that commercial fish feeds, manufactured with fish meal
and oils, may be contributing to the contamination of farmed fish stocks with
PCBs (polychlorinated biphenyls) and other toxins
(Hites et al., 2004
), there is
renewed interest to explore alternative sources of food energy such as
carbohydrates. Replacing traditionally used fish meal with cheaper
carbohydrates would also reduce feed production costs and demands on depleting
wild fish stocks, which are the current source of the feed
(Naylor et al., 2000
).
However, carbohydrates, such as glucose, may be lost in the urine of fish
due to their inability to clear the nutrient from the plasma
(Wright et al., 1998). In
mammals, prolonged hyperglycemia induces a cascade of three events in the
nephron, as summarized by Massry and Glassocks
(2001
). First, the filtered
glucose load exceeds the transport capabilities of the proximal tubule,
resulting in glucosuria. Thereafter, glucose gains access to more distal
portions of the nephron, creating a net suppression of water reabsorption, a
condition referred to clinically as osmotic diuresis. If osmotic diuresis
persists, the final event is the reduction in Na+ reabsorption by
the nephron, resulting in natriuresis. Whether these three events occur in the
kidney of a freshwater fish in response to hyperglycemia is unknown.
Work by Friere et al.
(1995) involving brush-border
membrane vesicles (BBMV) isolated and prepared from the proximal tubule has
aided in characterizing the glucose transporter in the rainbow trout kidney.
The BBMVs displayed a single Na+-dependent D-glucose
co-transport system that bound Na+ and glucose in a 1:1
stoichiometry and appeared to use the energy from Na+ and voltage
gradients to transport glucose in a manner similar to that in mammals.
Phlorizin (phloretin-2'-ß-glucoside) is a flavonoid present in
apples and derived from phloretin (phloretin-2'-O-glucose). In
mammals, phlorizin is a competitive inhibitor of glucose uptake via
Na+/D-glucose co-transporters (Alvarado et al., 1964;
Leung et al., 2000;
Crespy et al., 2002
), and the
physiological effect in vivo appears to be primarily the inhibition
of renal tubular reabsorption of filtered glucose. This is accomplished by
phlorizin binding to the transporter and inhibiting the conformational change
necessary for glucose transport (Horsburgh
et al., 1978
). In fish, phlorizin has been reported to inhibit
Na+-dependent glucose transport in BBMVs from the proximal tubule
of the kidney (Friere et al.,
1995
).
The goals of the present study were to examine the effects of prolonged hyperglycemia on the renal handling of glucose and to explore the in vivo pharmacological effects of phlorizin on glucose transport in the rainbow trout. The study was designed to increase the rate of renal tubular glucose reabsorption by exogenous glucose loading and thereby determine the plasma threshold and the transport maximum (TmG) of the freshwater trout kidney. We hypothesized that once these thresholds are surpassed, osmotic diuresis, and possibly natriuresis, would ensue. Also, in view of the likely coupling of glucose reabsorption to Na+ reabsorption, the effects of altering the glucose transporter's level of functioning on Na+ reabsorption was explored, as well as the effect of phlorizin on both processes.
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Materials and methods |
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Experimental protocol
While fish were anaesthetized with MS-222 (0.07 g l1;
Sigma, St Louis, MO, USA) and artificially ventilated on an operating table,
dorsal aortic (Soivio et al.,
1972) and internal urinary
(Curtis and Wood, 1991
;
Wood and Patrick, 1994
)
catheters were implanted. The dorsal aortic catheters (Clay-Adams PE-50,
Sparks, MA, USA) were filled with 0.3 ml of Cortland saline
(Na+=140 mmol l1; Cl=130 mmol
l1; K+=5 mmol l1;
Ca2+=1 mmol l1, Mg2+=2 mmol
l1, glucose= 5.5 mmol l1; pH 7.8;
Wolf, 1963
) containing 50 i.u.
ml1 of lithium heparin (Sigma) and sealed. The internal
urinary catheters (heat-molded Clay-Adams PE-60) were drained by gravity into
glass vials placed 3 cm below the water line. By placing the catheters inside
the bladder, any reabsorptive/secretory role of the bladder is prevented and
therefore the function of the kidney is solely examined.
The fish were then placed in individual darkened flux boxes and allowed to
recover for 24 h, during which time the functionality of the catheters was
assessed. Fish in which both catheters were operational were deemed to be
experimentally viable. Such fish were then injected, via the dorsal
aorta catheter, with 17 µCi (0.629 MBq) of [1,2-3H]polyethylene
glycol (PEG-4000; New England Nuclear, Boston, MA, USA) in 0.66 ml of Cortland
saline, followed by an additional wash of 0.3 ml of Cortland saline (Curits
and Wood, 1991; McDonald and Wood,
1998). The [3H]PEG-4000 was allowed to equilibrate for
12 h before starting the experiment. [3H]PEG-4000 is the glomerular
filtration rate (GFR) marker of choice in teleost fish because it
undergoes the least radioautolysis, metabolic breakdown or post-filtration
reabsorption relative to other GFR markers
(Beyenbach and Kirschner, 1976
;
Erickson and Gingrich, 1986
;
Curtis and Wood, 1991
).
The fish were separated into two treatment groups (N=7 each). The first group received a constant infusion of 140 mmol l1 glucose supplemented with an NaCl solution to achieve a typical plasma osmolality of 280 mOsmol kg1. The second treatment group received the same infusion solution with an additional 2 mmol l1 phlorizin added. The infusions, via a Gilson Minipulse peristaltic pump and at a rate of 500 µl kg1 h1 were begun after allowing the [3H]PEG-4000 to equilibrate for 12 h and were continued for 72 h. Blood samples (100 µl) were taken every 12 h and centrifuged at 13 000 g for 30 s to separate plasma and red blood cells, and urine samples were collected at the same time i.e. over 12 h periods. Plasma and urine samples were immediately frozen in liquid nitrogen and stored at 80°C for later analysis.
Analytical techniques
Glucose concentrations in the plasma and urine were measured enzymatically
(hexokinase, glucose-6-phosphate dehydrogenase) using a commercial kit (Sigma,
301A). The [3H]PEG-4000 radioactivities of the plasma (25 µl)
and urine (1 µl) were measured by diluting with double-distilled water to a
total volume of 5 ml, then adding 10 ml of ACS scintillation fluor (Amersham,
UK). The samples were then counted in an LKB Rackbeta 1217 Counter (Turku,
Finland). Tests demonstrated that quenching was uniform and therefore no
correction was necessary. Na+ concentrations in plasma and urine
were measured using a Varian 1275 Atomic Absorption Spectrophotometer (Walnut
Creek, CA, USA).
Calculations
Urinary excretion rates () of any
substance (X) were calculated as:
![]() | (1) |
![]() | (2) |
![]() | (3) |
The filtration rate (FR; also known as filtered load) of a
substance [X] at the glomeruli was calculated as:
![]() | (4) |
![]() | (5) |
![]() | (6) |
All rates were related to fish body mass, i.e. ml kg1 h1.
Statistics
Data have been generally reported as means ±
S.E.M. (N=number of fish), unless
otherwise stated. Regression analysis was used to determine if there was a
significant relationship between two variables. Regression lines were fitted
by the method of least squares, and the significance (P<0.05) of
the slope was assessed. Significant (P<0.05) differences between
two regression line slopes were calculated, according to Zar
(1974), by an analysis of
covariance (ANCOVA). Significant differences between treatment means were
evaluated using Student's paired and unpaired t-tests as appropriate
(P<0.05), with Bonferroni correction for multiple comparisons
(Nemenyi et al., 1977
).
Significant differences between time points were evaluated with an analysis of
variance (ANOVA) followed by a post-hoc test (LSD). All statistical
tests were run using SPSS, version 10.
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Results |
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UFR increased significantly over time to a similar degree within
each treatment (Table 1). As
with glucose excretion rates, the final UFRs (water excretion rates)
showed a greater elevation above background (1 ml kg1
h1) than the infusion rates (0.5 ml kg1
h1). GFR was also not significantly different
between the two treatment groups (Table
1), although GFR decreased over the course of the
experiment as UFR increased. However, for most time points of the
experiment, GFR was significantly higher than UFR,
indicating that reabsorption of water had occurred
(Table 1), with the exception
of the final experimental period (6072 h) in both treatments.
The filtration rate of glucose during the glucose-only infusion increased over time, peaking at 3648 h (Table 1). The same pattern was seen during the phlorizin infusion, with filtration rates peaking at 36-48 h, and for the course of the experiment there was no significant difference between the two groups. The final filtration rates were 60% higher than the initial filtration rates during 012 h.
As with the urine concentrations discussed earlier, the effect of phlorizin was immediately apparent when examining the glucose excretion rates. During the first 12 h, excretion rates were 16 times higher in the phlorizin treatment than in the glucose-only treatment (Table 1). Both groups showed an increase in excretion over time, eventually becoming similar in value.
Glucose reabsorption for the glucose-only infusion group experienced a significant decrease at 48 h following an initial increase (Table 1), ending at 50% of the rate during the first 12 h. The glucose reabsorption rates for the phlorizin-exposed group were significantly lower than the glucose-only infusion group at all time points, ranging from 50 to 75% lower, and they underwent a similar decrease from 48 h onwards.
Glucose clearance ratios showed a similar pattern to glucose excretion
rates (Fig. 2A). During the
glucose-only infusions, the clearance ratio started out at 0.028±0.015,
which shows an almost complete reabsorption of glucose (approximately 97%). As
plasma glucose concentrations rose over time to reach a plateau, the clearance
ratios rose steadily as well, eventually reaching 0.75 (only 25%
reabsorption of glucose) by the final time period. During the phlorizin
treatment, the clearance ratio at 12 h had already reached 0.5 (50%
reabsorption of glucose), and by 48 h had reached a value (0.9) not
statistically different from 1.0, which would indicate that glucose was being
filtered with the same efficiency as the [3H]PEG 4000 and was not
being reabsorbed.
|
Glucose and Na+ reabsorption were well correlated during the glucose-only infusion (Fig. 3). Fig. 3 also shows that Na+ reabsorption was almost 10-fold higher than glucose reabsorption and that the phlorizin treatment effectively eliminated the relationship between Na+ and glucose reabsorption.
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Discussion |
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The infusion of glucose raised plasma glucose levels well beyond those
normally seen in rainbow trout (Wright et
al., 1989). At normal to moderately elevated plasma glucose
concentrations (520 mmol l1), glucose reabsorbed was
a linear function of glucose filtered (Figs
4,
5). However, as the
hyperglycemia worsened, the relationship between reabsorption and filtration
approached an asymptote as a result of the TmG of glucose being reached
(Fig. 4). Above a certain blood
glucose level (threshold value), excretion becomes a linear function of the
amount filtered (or plasma concentration), and reabsorption reaches a plateau.
Consequently, a plot of urinary excretion rate against plasma glucose
concentration yields a threshold value at which glucose is lost to the urine,
and a plot of urinary reabsorption rates against plasma glucose concentration
yields a sigmoidal curve, as shown in Fig.
5, from which a TmG can be calculated
(Massry and Glassocks,
2001
).
The TmG found in this study is similar to findings in another in
vivo study (Gray and Brown,
1985; Table 2).
However, it is approximately double compared with findings on in situ
perfused trout kidney studies (Amer and
Brown, 1995
; Brown et al.,
2000
; Table 2).
Part of these differences could be accounted for by variations in experimental
temperatures, as TmG varies with temperature, with higher temperatures
yielding higher TmGs (MacKay and Beatty,
1967
). However, it seems likely that part of the discrepancy could
reflect more efficient renal function in vivo than in perfused-organ
experiments.
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The plasma threshold value at which glucose started to be lost to the urine
was found to be 22 mmol l1. This is similar to other peak
plasma values found in the rainbow trout, after ingestion of glucose-rich
diets or injection of glucose-rich solutions, although different when compared
with other species (Table 2).
The minor differences seen within rainbow trout might be attributed to
experimental procedure, as the highest plasma values were seen with bolus
injections and infusions, while the lowest value was seen in a chronic dietary
exposure. This could be due to differential activation of physiological
processes, depending on the route of exposure. Indeed, Hemre et al.
(1995) observed that plasma
glucose levels in Atlantic salmon rose only marginally after oral ingestion,
while intraperitoneal administration resulted in prolonged hyperglycemia.
A possible explanation for the difference observed between species
(Table 2) could involve
variations in `glucose space', where glucose space is the space into which
glucose is dispersed and includes plasma and extracellular fluid
(Legate et al., 2001). Glucose
space has been shown to vary between species, ranging from 100 to 400 ml
kg1 (Garin et al., 1987), although only a limited number of
species have been examined to date. Another possible explanation for the
differences in glucose plasma peaks is a possible difference in the renal
glucose transport maximum between species. There are reported differences in
many renal transport processes between species, including those involved with
glucose, especially when comparing marine and freshwater fish
(Dantzler, 1989
). In addition,
there exists evidence of differential handling of dietary starches between
species. Perch have been shown to increase intestinal glucose absorption when
fed high starch diets, while rainbow trout remained unchanged
(Buddington, 1987
). Krogdahl et
al. (2004
) have also shown
that Atlantic salmon and rainbow trout exhibit differential handling of
dietary starches, wherein rainbow trout were observed to have a higher
capacity to digest and absorb starches compared with Atlantic salmon.
As the hyperglycemia created from the infusions increased, glucose was lost
to the urine as a consequence of the transport maximum being reached,
resulting in glucosuria as expected. As mentioned in the Introduction, this is
the first event of three that occurs in the kidney during hyperglycemia in
mammals. The second event, the decrease in reabsorption of water over time,
was also present in the rainbow trout kidney, as evidenced by the increasing
clearance ratio for water (Fig.
2C). This is probably due to the increasing osmotic pressure in
the distal portion of the nephron due to the increasing concentration of
glucose in the urine (Massry and
Glassocks, 2001). This increase in osmotic pressure raises the
apparent permeability of the distal nephron to water, as mentioned earlier
(Nishimura et al., 1983
).
The third event that occurs in mammalian kidneys, the loss of
Na+ to the urine, became clearly apparent in the last 1224 h
of the study (Table 1;
Fig. 2B). Natriuresis caused by
osmotic diuresis is the final occurrence in the kidney in response to
hyperglycemia and tends to show a delayed response
(Massry and Glassocks, 2001).
This is supported by the phlorizin treatment group, which exhibited a decrease
in Na+ reabsorption 12 h before the glucose-only treatment. The
former group was experiencing elevated glucosuria compared with the
glucose-only infusion group over a longer period of time
(Fig. 2A). Overall, the
freshwater rainbow trout kidney displays similar trends in response to
hyperglycemia when compared with the mammalian kidney.
Despite the desire to include carbohydrates in fish food, there exists
evidence that carbohydrate levels must be closely controlled when incorporated
into the feed. Reduced growth rates have been observed in some fish species
fed carbohydrate-free diets (Anderson et
al., 1984; Degani et al., 1986). Conversely, feeding excessive
dietary carbohydrates to fish has been shown to adversely affect an array of
physiological parameters, such as growth and liver size and function
(Hilton and Atkinson, 1982
;
Dixon and Hilton, 1985
). These
deleterious effects have been attributed to the poor peripheral utilization,
or poor peripheral uptake, of glucose and other carbohydrates by the tissues
or to insulin production and binding difficulties
(Hilton and Atkinson, 1982
;
Legate et al., 2001
).
The elevated levels of glucose found in the plasma of the rainbow trout
after a carbohydrate-rich meal or injection are close to the plasma
concentration at which the transport maximum is reached in this study
(Fig. 5;
Table 2; Philips et al., 1948;
Palmer and Ryman, 1972; Bergot,
1979; Legate et al.,
2001
). This indicates that fish fed high starch diets may lose
some of the glucose to the environment via urinary excretion. For
aquaculture, this would affect the amount of starch or glucose incorporated
into diets because, above a certain concentration of glucose, it would be of
no use to, and possibly detrimental to the health of, the fish. The present
study indicates that disturbances in ionoregulation and osmotic regulation
associated with glucosuria may contribute to these deleterious effects.
Although the phlorizin infusion at 1 µmol kg1 h1 was effective in reducing the reabsorption of glucose, it did not immediately block it entirely (Table 1). Glucose excretion rates only became equal to filtration rates after 48 h of phlorizin infusion, as shown by the clearance ratios in Fig. 2A and Table 1.
Administration of 110 µmol kg1
h1 of phlorizin caused complete inhibition of glucose
transport in several in vitro studies conducted in fish
(Pritchard and Kleinzeller,
1976), while previous studies on mammals have encountered complete
blockade of glucose reabsorption at rates of <1 µmol phlorizin
kg1 h1
(Horsburgh et al., 1978
;
Silverman et al., 1970
). The
slow onset of blockade in the present study was probably due to competition.
Indeed, Kimmich (1990
) found
that at glucose concentrations much greater than phlorizin concentrations,
glucose was able to out-compete phlorizin for access to the transporter
despite having a much lower binding affinity for the transporter. This was
probable in the present experiment as the glucose concentration was 70 times
greater than the phlorizin concentration in the infusion solution.
In summary, constant infusion of glucose into the plasma of the rainbow trout results in persistent hyperglycemia. As a result, the kidney filters glucose at rates that exceed the transport maximum of the nephrons, resulting in glucosuria. This causes osmotic diuresis and natriuresis. As freshwater fish are hyperosmotic to their environment, this poses a detrimental risk to their osmotic and ionic regulation. With regard to the desire to incorporate carbohydrates into the diets of farmed fish, this should be approached with caution as, above a certain concentration, glucose obtained from the diet will result in glucosuria.
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Acknowledgments |
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