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¹ Department of Physiology and Centre for Nephrology, Royal Free & University College School of Medicine, London, UK

	Abstract

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Previous investigations into the functional responses of the surviving nephrons following reductions in renal mass have been performed largely in anaesthetized animals and have taken little account of how the compensatory changes develop with time. The present study has assessed a method for determining glomerular filtration rate (GFR) in unrestrained, uncatheterized, conscious rats (plasma disappearance of ^99mTc-diethylenetriamene pentaacetic acid (DTPA)) and has used this method to document the time course of the changes in GFR over a 32 day period following uninephrectomy or 5/6 nephrectomy. Concurrent measurements of excretion rates and of the clearance of lithium (the latter being an index of end-proximal fluid delivery) provided information on changes in overall tubular function and segmental reabsorption. After uninephrectomy, the GFR of the remaining kidney (compared with that of a single kidney of sham-operated animals) increased maximally (by 50%) within 8 days; after 5/6 nephrectomy, the increase in the GFR of the remnant kidney was maximal (at 300%) within 16 days. Overall excretion rates of sodium and potassium were well maintained in partially nephrectomized animals throughout the period of study, while the excretion of water increased (by 30% after uninephrectomy and by 120% after 5/6 nephrectomy), partly as a result of the compensatory increases in GFR but mainly as a consequence of moderate (after uninephrectomy) or marked (after 5/6 nephrectomy) reductions in fractional reabsorption. During the early period after 5/6 nephrectomy, potassium excretion sometimes exceeded the filtered load, indicating net secretion. Lithium clearance data indicated that the changes in tubular function after 5/6 nephrectomy include a reduction in fractional reabsorption in the proximal tubule, whereas after uninephrectomy any such effect on the proximal tubule is minor and transient.

(Received 15 June 2006; accepted after revision 30 October 2006; first published online 3 November 2006)
Corresponding author D. G. Shirley: Department of Physiology and Centre for Nephrology, Royal Free & University College School of Medicine, Hampstead Campus, Rowland Hill Street, London NW3 2PF, UK. Email: david.shirley{at}ucl.ac.uk

	Introduction

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Although considerable information is available concerning the functional response of the surviving nephrons in models of reduced renal mass such as uninephrectomy or 5/6 nephrectomy, most studies have been ‘snapshots’ of renal function at a particular point in time; little or no account has been taken of the development of the compensatory changes. Moreover, the overwhelming majority of such studies have been performed in anaesthetized animals, usually prepared for micropuncture. There is no doubt that anaesthesia and the relatively severe surgery necessary for micropuncture affect some aspects of renal function (Maddox et al. 1977; Shirley et al. 1990), and measurements made under these conditions may not reflect the real changes following partial nephrectomy.

A central requirement for assessing renal function, whether in conscious or anaesthetized animals, is the ability to measure glomerular filtration rate (GFR). Most studies in conscious rats have used standard renal clearance techniques, usually with inulin as the glomerular marker, necessitating cannulation of blood vessels and (usually) restraint of the animals (e.g. Burgess et al. 1993). However, implanted cannulae cannot be maintained in a functional state for the prolonged periods required for a full assessment of the response to partial nephrectomy. The need for cannulae and restraint can be avoided by the use of implanted osmotic minipumps for delivery of inulin (Shirley et al. 1989); however, the pumps will not function for more than 4 weeks. In order to circumvent some of the problems associated with the measurement of renal clearance, methods have been introduced in which GFR is calculated from the plasma disappearance of a glomerular marker, such as ⁵¹Cr-EDTA or ⁵⁷Co-EDTA, following a bolus intravenous injection (Seefeldt & Houghton, 1990; Ido et al. 1992). In a subsequent development, Nankivell et al. (1992) used ^99mTc-diethylenetriamene pentaacetic (DTPA) and injected the bolus dose intraperitoneally, thereby obviating the need for an intravenous cannula. A further potential advantage of this method is that the short half-life of ^99mTc (6 h) allows repeated measurements at frequent intervals. On the face of it, therefore, this method is ideally suited to assess changes in GFR in long-term studies where serial measurements are required in the same animal.

The initial purpose of the present study was to assess the plasma disappearance method described by Nankivell et al. (1992). The method was then used to document the time course of the changes in GFR in conscious rats following either uninephrectomy or 5/6 nephrectomy, while concurrent measurement of excretion rates allowed an assessment of changes in overall tubular handling of water and electrolytes.

An important aspect of the uncertainties over altered renal function after partial nephrectomy concerns the relative contributions of the proximal tubule and the distal nephron to the adaptive changes. Micropuncture studies in anaesthetized rats suggest that uninephrectomy is followed by a short-lived reduction in fractional proximal reabsorption, with most of the long-term compensation being attributable to the distal nephron (Shirley & Walter, 1991), whilst the results of micropuncture studies after more extensive renal ablation (mimicking severe renal failure) are conflicting, reporting either a fall in fractional proximal reabsorption or no change (Hayslett, 1979), with no information on the time course. Significantly, in neither model has any assessment been made of the segmental pattern of reabsorption in conscious animals. In the present study, we have therefore included determinations of the renal clearance of lithium, a semi-quantitative index of proximal tubular function (Boer et al. 1995; Thomsen & Shirley, 1997). In this way, we were able to assess, for the first time in conscious rats, the role of the proximal tubule in the maintenance of excretion rates in these two models of reduced renal mass.

	Methods

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All procedures were performed in accordance with UK legislation.

Assessment of the ^99mTc-DTPA plasma disappearance method for measurement of GFR

Male Sprague–Dawley rats were anaesthetized with Trapanal (thiopentone sodium, 110 mg kg^–1, I.P.; Byk Gulden, Konstanz, Germany) and prepared surgically for clearance studies (tracheotomy; cannulae inserted in a jugular vein, a femoral artery and the bladder). In order to obtain a wide range of values for glomerular filtration rate (GFR), rats of varying weight were used (160–780 g) and, in some, the left renal pedicle was ligated and the kidney excised. All rats were infused intravenously with isotonic saline (30 µl min^–1). Two types of experiment were performed, as follows.

(1) Comparison of the renal clearances of ^99mTc-DTPA and inulin.  In nine rats, 1 h after the completion of surgery, ^99mTc-DTPA and [³H]inulin (both from Amersham International, Aylesbury, UK) were included in the saline infusion (5 µCi bolus, 5 µCi h^–1 and 2 µCi bolus, 2 µCi h^–1, respectively), and, after a 1 h equilibration period, the renal clearances of the two markers were measured over a period of 3 h, arterial blood samples being taken every hour.

(2) Comparison of the plasma disappearance of ^99mTc-DTPA with the renal clearance of inulin.  One hour after the completion of surgery, [³H]inulin was infused intravenously into 26 rats as above. One hour later, a known dose (500 µCi) of ^99mTc-DTPA was injected intraperitoneally using an insulin syringe. In order to avoid injection into the bowel, a setback attachment was constructed by cutting off the end of the needle cap, allowing only 7 mm of the needle to protrude. Two accurately timed arterial blood samples were obtained approximately 50 and 100 min later. The clearance of ^99mTc-DTPA was calculated from its plasma disappearance (see Calculations subsection below) and compared with the simultaneously measured renal clearance of [³H]inulin.

At the end of each experiment, the rat was killed with an overdose of anaesthetic.

Assessment of renal function in conscious rats following partial nephrectomy

Male Sprague–Dawley rats, initially weighing 140–180 g, were placed in a room illuminated from 19.00 to 07.00 h in order to reverse their circadian rhythm and thus facilitate the assessment of renal function during the animals' active period. After an acclimation period of 1 week, the rats were placed in individual metabolism cages (Metabowl, Jencons Ltd, Leighton Buzzard, UK) which allowed free access to water and food (CRM diet, Labsure, Poole, UK; protein content 17%, sodium and potassium contents 140 and 180 mmol (kg dry weight)^–1, respectively). In order to allow the determination of lithium clearance, LiCl was mixed in with the food (15 mmol (kg dry weight)^–1 from 2 days before the first clearance measurement; this provided a plasma lithium concentration of 250–320 µmol l^–1. (In rats subsequently subjected to uninephrectomy or 5/6 nephrectomy, the LiCl content of the food was reduced to 12 and 6 mmol kg^–1, respectively, so that plasma lithium concentrations remained comparable to those of control animals.) Every 24 h the animals were weighed, the consumption of food and water registered, and the urine collected and analysed.

After 5 days in metabolism cages, measurements of urinary excretion rates and the clearances of ^99mTc-DTPA and lithium were made (see below) on two occasions separated by a 2 day interval. The animals were then divided into three groups, as follows.

5/6 nephrectomy (5/6NX; nine rats).  Under halothane anaesthesia, the left kidney was exposed via a mid-line incision and the renal capsule removed. The renal pedicle was temporarily clamped and approximately 7/8 of cortex was resected (equivalent to 2/3 of the single kidney mass). The remnant kidney was wrapped with part of the mesentery and the clamp on the renal pedicle was removed, bleeding from the cut surfaces being prevented by 5 min of digital pressure. The abdominal incision was then closed, analgesic (buprenorphine hydrochloride, 1.2 mg kg^–1, subcutaneously; Reckitt & Colman, Hull, UK) was administered and the animal was allowed to recover consciousness. After 2 h observation in an ordinary cage, the rat was returned to its metabolism cage. One week later, again under halothane anaesthesia, the right kidney was removed via a flank incision, and the same recovery procedure employed.

Uninephrectomy (UNX; nine rats).  Under halothane anaesthesia, a mid-line incision was made as for 5/6NX, but the left kidney was merely gently manipulated before closure of the incision and recovery. One week later, under halothane anaesthesia, the right kidney was removed via a flank incision.

Sham operations (SO; nine rats).  The same operations were performed as for 5/6NX and UNX except that on each occasion the kidney was gently manipulated rather than being partially resected or removed.

Clearance measurements.  Day 0 was defined as the day of the second operation. Clearance measurements were performed on days –12 and –10 (preoperative values), and 2, 4, 8, 16 and 32 days after the second operation. The procedure was as follows.

Urinary excretion rates and lithium clearance were measured during the period 11.00–16.00 h; the plasma disappearance of ^99mTc-DTPA was measured during the final 2 h. At 10.45 h, a blood sample (120 µl) for measurement of lithium was obtained from a small cut made in the tip of the tail (Shirley et al. 1989) after first cleaning the tail with warm water. At 11.00 h, bladder emptying was provoked by placing the rat on a sloping glass plate and holding its tail upright (Shirley et al. 1989). The rat was then returned to its metabolism cage and urine collection was begun. At 14.00 h, a known dose of ^99mTc-DTPA was injected intraperitoneally as described above, and further blood samples were taken at 15.00 h (150 µl), for measurement of ^99mTc-DTPA, and 16.00 h (250 µl), for measurement of ^99mTc-DTPA and lithium. Immediately before the final blood sample, bladder emptying was again provoked as described above. Split urine collections (11.00–14.00 and 14.00–16.00 h) indicated no systematic differences in excretion rates before and after ^99mTc-DTPA injection.

Immediately after the final clearance measurement (day 32), a larger blood sample was taken from each rat for measurement of haematocrit, plasma urea nitrogen, and the plasma concentrations of sodium and potassium. The following day, each rat was anaesthetized with Trapanal (110 mg kg^–1, I.P.) for clearance experiments (not reported here) and killed 6 h later with an overdose of anaesthetic.

Glomerular counting in 5/6NX rats

In order to evaluate the extent of the changes in GFR in conscious rats after partial nephrectomy, it is necessary to know the number of glomeruli remaining. In the case of UNX, this can be assumed to be half the original number. However, in the case of 5/6NX, the situation is not so straightforward. Although the operative procedures involve removal of one kidney plus resection of two-thirds of the other, the precise number of glomeruli removed from the resected kidney is not known; it is likely to be greater than two-thirds, since a proportionally greater amount of cortex (containing the glomeruli) is excised than medulla. Therefore, in the present study, the number of glomeruli remaining after two-thirds resection of the left kidney was determined in anaesthetized rats by staining the glomeruli with Alcian Blue and counting them after digesting the kidney with acid, using a modification of the method of Bankir & Hollenberg (1983). Male Sprague–Dawley rats (n = 6) were anaesthetized with Trapanal (110 mg kg^–1, I.P.) and the right jugular vein cannulated and infused with isotonic saline (100 µl min^–1). The left kidney was exposed via a mid-line incision, the renal pedicle clamped and 2/3 resection of the left kidney performed as described above. Twenty minutes after the completion of surgery, the intravenous infusate was changed to 5% Alcian Blue 8GX dye (Sigma, Poole, UK) for 10 min, after which the animal was killed with an overdose of anaesthetic. The entire right kidney and the remnant left kidney were removed, cut into small pieces and immersed in 1% ammonia solution. After 6–12 h, the kidney sections were digested in 15% hydrochloric acid for 12–24 h.

The digest of the right kidney was diluted with distilled water to a total volume of 75 ml, and that of the remnant kidney to a total volume of 20 ml. Each suspension was mixed thoroughly, and a 50 µl aliquot pipetted onto a single-cavity microscope slide in which a grid had been etched in order to facilitate counting. For each suspension, the number of stained glomeruli in 20 aliquots was counted (20–30 glomeruli per aliquot), the total in each aliquot tabulated, and the sum of the counts multiplied by the appropriate dilution factor to obtain the total number of stained glomeruli.

In another seven rats, the number of glomeruli in the intact left kidney (in the absence of resection) was compared with that in the right kidney.

Analyses

Activity of ^99mTc-DTPA in urine and plasma samples was counted in a sodium iodide crystal well counter (Cobra 5003, Canberra Packard, Pangbourne, UK), with automatic correction for background and radioactive decay from a common time point. In experiments in which the isotope was injected intraperitoneally, the residual activity in the syringe and needle was counted similarly. [³H]Inulin activity in urine and plasma samples was measured 3–4 days after the ^99mTc-DTPA measurements (to allow the ^99mTc activity to decay to background) by ß-emission spectroscopy (model 2000 CA, Canberra Packard) after dispersal in Aquasol 2 scintillation cocktail (Perkin Elmer, Cambridge, UK). Lithium concentrations in urine and plasma were measured by atomic absorption spectrometry (model 151, Instrumentation Laboratory, Warrington, UK), and sodium and potassium concentrations by flame photometry (model 543, Instrumentation Laboratory). Urine osmolality was measured by freezing point depression (Roebling, Camlab, Cambridge, UK); haematocrit was determined using microhaematocrit tubes (Fisons, Loughborough, UK); and plasma urea nitrogen was measured colorimetrically using a commercially available kit (Sigma).

Calculations

The renal clearances of [³H]inulin, ^99mTc-DTPA, lithium, sodium and potassium were calculated using the standard clearance formula. Appropriate plasma values for [³H]inulin, ^99mTc-DTPA and lithium were interpolated from those measured. For sodium and potassium, the value measured on day 32 was used.

The clearance of intraperitoneally injected ^99mTc-DTPA was calculated from the plasma disappearance according to the method of Nankivell et al. (1992), using the formula:

(1)

where I is the injected dose (counts min^–1), C₀ the time zero intercept (counts min^–1 ml^–1) and k the exponential slope of the plasma disappearance curve (min^–1). In calculating I, care was taken to subtract the residual activity in the syringe and needle; k was calculated as ln(P₁/P₂)/t, where P₁ and P₂ represent the ^99mTc activity in the first and second plasma samples, respectively, after injection, and t is the time interval between the two samples; C₀ was then determined by substituting C₀ for P₁.

As a consequence of the comparison of the renal clearance of inulin with the clearance of intraperitoneally injected ^99mTc-DTPA (as determined from its plasma disappearance), GFR in conscious rats was calculated from the plasma disappearance of ^99mTc-DTPA with an appropriate correction factor (see Results). Fractional excretions of lithium (FE_Li), sodium (FE_Na), potassium (FE_K) and water (FE_H2O) were calculated as C_Li/GFR, C_Na/GFR, C_K/GFR and V/GFR, where C_Li, C_Na and C_K are the renal clearances of lithium, sodium and potassium, respectively, and V is the urine flow rate.

Statistics

Results are presented as means ± S.E.M. Comparison of the ^99mTc-DTPA and ³[H]inulin methods for measurement of GFR was performed using regression analysis and the Bland–Altman test (Bland & Altman, 1986). Comparisons of haematocrit and plasma data between SO, UNX and 5/6NX rats were made using one-way ANOVA. Differences in renal function data between conscious SO, UNX and 5/6NX rats were assessed using two-way ANOVA with one within-group repeated measures factor (the effect of time). When overall differences were found to be significant, post hoc comparisons were made using the test of least significant difference.

	Results

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Measurement of GFR with ^99mTc-DTPA

The renal clearance of intravenously infused ^99mTc-DTPA was virtually identical to that of [³H]inulin over a wide range of clearance values (Fig. 1A), indicating that the ^99mTc-DTPA employed in this study was a valid glomerular marker. However, when the plasma clearance of ^99mTc-DTPA, calculated from the plasma disappearance of intraperitoneally injected tracer, was compared with the renal clearance of inulin, the latter was found to be consistently higher (Fig. 1B), although the two methods were highly correlated. On average, the renal clearance of inulin was 20% greater than the plasma clearance of ^99mTc-DTPA. Assuming that the renal clearance of inulin is an accurate measure of GFR, the regression equation was used to calculate the ^99mTc-DTPA value for each GFR value. The actual, measured ^99mTc-DTPA value was then compared with the calculated value, and the measure of agreement was assessed using the formula of Bland & Altman (1986) in which the difference between each pair of values obtained by two methods is plotted against the average of the two values. In 25 out of 26 cases, the difference between the two values fell within two standard deviations of the mean difference, with 95% confidence intervals of 0.08 ml min^–1. Therefore, in subsequent experiments, GFR was calculated from the plasma clearance of ^99mTc-DTPA, using this regression equation to provide the appropriate correction factor.

View larger version (12K): [in this window] [in a new window]   Figure 1.  Comparison of 99mTc-DTPA clearance with the simultaneously measured renal clearance of [3H]inulin in anaesthetized rats A, individual values for the renal clearance of intravenously infused [3H]inulin versus the renal clearance of intravenously infused 99mTc-DTPA (n = 9 rats); the line of identity is shown. B, individual values for the renal clearance of intravenously infused [3H]inulin versus the plasma clearance of intraperitoneally injected 99mTc-DTPA (n = 26 rats). The line of identity and the regression line are shown. The equation for the regression line is y = 0.03 + 0.83x; r = 0.98.

Glomerular counting.  Confirming previous observations (Kaufman et al. 1974; Larsson et al. 1980), the number of glomeruli present in the left kidney was not significantly different from that in the right kidney (36 750 ± 1820 versus 37 609 ± 2493, n = 7). In rats in which two-thirds of the left kidney mass had been resected, the total number of glomeruli in the remnant kidney was 9417 ± 1022 (n = 6), compared with 42 014 ± 1812 (n = 6) in the intact right kidney. Assuming that the two-kidney glomerular count was twice the figure found in the right kidney, the number of functioning glomeruli in the remnant kidney was therefore reduced to 11.4 ± 1.4% of the normal two-kidney complement.

Excretion rates.  Figure 2 shows data from the daily measurements in the three groups of rats. Rats in the SO group grew steadily throughout the study, apart from a transient slight fall in body weight after the first operation (Fig. 2A). At no time in the study was the body weight of UNX rats significantly different from that of SO animals. In 5/6NX rats, however, body weight was slightly lower than that of SO rats after the first operation; this difference became more obvious after the second operation: 5/6NX rats failed to gain weight on days 1–8 (coincident with a significantly reduced food intake). Thereafter, however, the rate of growth of 5/6NX rats was indistinguishable from those of the other two groups.

View larger version (38K): [in this window] [in a new window]   Figure 2.  Twenty-four hour measurements (means and S.E.M.) of body weight and excretion rates in conscious rats before and after sham operations (SO), uninephrectomy (UNX) or 5/6 nephrectomy (5/6NX) A, body weight; B, urine flow rate; C, sodium excretion; and D, potassium excretion. The operations were performed on day –7 and day 0 (arrowed). There were nine rats in each group.

Overall sodium excretion (Fig. 2C) changed little in SO and UNX rats throughout the study, except for reductions on the days of operations. In 5/6NX rats, sodium excretion followed the same pattern as in the other groups except for a small reduction during the first week after the second operation (P < 0.05, days 1–4, 6 and 7). Potassium excretion (Fig. 2D) followed a similar pattern to that described for sodium.

Clearance data.  Similarities and differences in absolute excretion rates between the three groups of rats during the 5 h clearance periods (data not shown) followed the same pattern as those described for 24 h collections. However, absolute values for rates of sodium, potassium and water excretion during the clearance periods were 20–25% higher than the corresponding 24 h excretion rates. This can be explained as a consequence of diurnal variations in excretion rates (Roelfsema et al. 1980; Shirley et al. 1989), since the clearance period was in the middle of the rats' active period.

No systematic differences in clearance values were observed between the two preoperative days in any group of rats. Therefore, the two values were averaged for each rat and a single mean value presented for each group. For none of the variables measured was the mean preoperative value significantly different between the groups.

Values for GFR in the three groups of animals are shown in Fig. 3A. In SO rats, GFR (per 100 g body weight) gradually decreased as the animals aged. In UNX rats, GFR on day 2 was 57% of the corresponding value in the SO group. It then increased rapidly, reaching a maximum of 77% of that in SO rats by day 8. In 5/6NX rats, GFR on day 2 was 23% of that in SO rats. Given that the number of functioning glomeruli had been reduced to 11%, this represents a doubling of GFR in the remnant kidney. During the next 2 weeks, the GFR increased further, to a maximal value of 36% of that in SO rats.

View larger version (15K): [in this window] [in a new window]   Figure 3.  Glomerular filtration rate (GFR) and fractional lithium excretion in conscious rats before and after sham operations (SO), uninephrectomy (UNX) or 5/6 nephrectomy (5/6NX) Values are means ± S.E.M.A, GFR (per 100 g body weight); and B, fractional lithium excretion. Preoperative values for each group were calculated from the average of two determinations per rat (on days –12 and –10). There were nine rats in each group.

Figure 4 shows values for the fractional excretions of water, sodium and potassium. In the light of the changes in absolute excretion rates and in GFR described above, changes in fractional excretion rates followed a predictable pattern. In SO rats, FE_H2O remained at slightly below 1% throughout the study (Fig. 4A), while in UNX rats it was somewhat higher, although this difference was only statistically significant (P < 0.05) on day 2. In 5/6NX rats, the increase in absolute urine flow rate, together with the reduced GFR, meant that FE_H2O was increased eightfold on day 2. As the GFR increased, FE_H2O fell somewhat, but was still fourfold higher than that in SO rats on days 8–32.

View larger version (14K): [in this window] [in a new window]   Figure 4.  Fractional excretion of water and electrolytes in conscious rats before and after sham operations (SO), uninephrectomy (UNX) or 5/6 nephrectomy (5/6NX) Values are means ± S.E.M.A, fractional excretion of water; B, fractional excretion of sodium; and C, fractional excretion of potassium. Preoperative values for each group were calculated from the average of two determinations per rat (on days –12 and –10). There were nine rats in each group.

Haematological data.  Table 1 shows values for haematocrit, plasma urea and plasma electrolyte concentrations on day 32. Although the mean value for haematocrit in UNX rats was slightly lower than that in SO animals, the difference did not achieve statistical significance; however, there was a marked reduction in the 5/6NX group. Plasma urea in UNX rats was similar to that in the SO group, but in 5/6NX rats it was approximately doubled. There were no significant differences in plasma sodium concentration between the three groups, but plasma potassium was significantly elevated in the 5/6NX rats.

	Discussion

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Plasma clearance of ^99mTc-DTPA

If the time course of the changes in GFR is to be assessed, a method that allows serial measurements in conscious rats over the full period of compensation is required. The slope-intercept method developed by Nankivell et al. (1992), in which the plasma clearance of a single intraperitoneal injection of ^99mTc-DTPA is measured using two small blood samples, seemed suited to this purpose, but before using it we assessed its precision by comparing the plasma clearance of ^99mTc-DTPA with the ‘gold standard’, namely the renal clearance of inulin. Over a wide range of renal function, the latter was found consistently to exceed the plasma clearance of ^99mTc-DTPA, the slope of the regression line relating the two variables being 0.83.

The reason for the discrepancy between our data and those of Nankivell et al. (1992), who found substantial agreement between the two methods, is not obvious. Both the present study and that of Nankivell et al. (1992) assessed the plasma clearance of ^99mTc-DTPA in anaesthetized animals. However, in an earlier study we found that in conscious rats the plasma clearance of ^99mTc-DTPA was also consistently lower (on average, by 20%) than the renal clearance of inulin, the latter delivered by osmotic minipump (Chamberlain & Shirley, 1993). The integrity of the ^99mTc-DTPA used in the present study was not an issue, since the conventional renal clearance of ^99mTc-DTPA was identical to that of inulin. (We also confirmed routinely the integrity of the [³H]inulin by observing a single peak after Sephadex separation, although this could not explain the difference between our results and those of Nankivell and colleagues who used the relatively stable [¹⁴C]inulin.)

Potentially, two factors might contribute to the observed discrepancy between the plasma clearance of ^99mTc-DTPA and the renal clearance of inulin. Firstly, the possibility cannot be excluded that a (consistent) proportion of the dose of ^99mTc-DTPA was sequestered at the injection site, although we were careful to check the absence of a subcutaneous swelling after each injection. Secondly, although current preparations of ^99mTc-DTPA are subject to very little binding by plasma proteins (as witnessed by the virtual identity of the renal clearances of ^99mTc-DTPA and inulin), a small proportion of the administered dose is bound (< 1%), and there is evidence that the absolute amount bound remains constant, so that as unbound activity is cleared by the kidneys the bound proportion increases (Rehling, 1988). This would reduce the slope of the disappearance curve. However, it is difficult to see this constituting more than a minor error.

Whatever the reason for the discrepancy found in the present study, a very high correlation existed between the two methods. Importantly, when the Bland–Altman test was applied to the plasma clearance of ^99mTc-DTPA versus the value calculated from the regression line relating inulin clearance (i.e. true GFR) and plasma clearance of ^99mTc-DTPA, the difference between the two values was found to be small and consistent over the whole range of GFR values encountered. This led us to conclude that the use of the regression equation for calculating true GFR from the plasma clearance of ^99mTc-DTPA was justified. In earlier studies, in a group of conscious rats, we had established that the coefficient of variation of measurements of the plasma clearance of ^99mTc-DTPA, calculated from five determinations on alternate days, was 11 ± 3% (Chamberlain & Shirley, 1993). Thus, the method is at least as reproducible as other methods of GFR assessment and is particularly suited to the serial measurement of GFR in the same animal, without the need for catheterization, anaesthesia or restraint, over prolonged periods of time. It was therefore adopted in the present study.

Disturbances of homeostasis after partial nephrectomy

In the UNX rats, used as a model of donor nephrectomy, there were no measurable disturbances of homeostasis, whereas in 5/6NX rats, used as a model of chronic uraemia, there was clear evidence of azotaemia and anaemia. The latter observations accord with those found by others in 5/6 nephrectomized animals (Sterner & Wennberg, 1988; Priyadarshi et al. 2002; Michimata et al. 2003). Although, in all animals, there were transient reductions in the rate of gain of body weight on the days of the two operations, food intake and growth rates in SO and UNX rats were indistinguishable throughout the study. After the second operation in 5/6NX rats, however, body weight failed to increase for 1 week, coincident with a reduced food intake, but normal growth resumed thereafter. Kleinknecht et al. (1988), who also observed this temporary dysphagia, noted that in ‘pair-fed’ intact rats, in which food intake was matched to that of their uraemic partners, growth was similarly halted.

The 24 h excretion data confirmed that, following reductions in functional renal mass, sodium and potassium balance is largely maintained by means of increased excretion rates by the remaining nephrons. The only time when overall sodium and potassium excretion rates were reduced was during the first week after 5/6NX, when food (and therefore sodium and potassium) intake was also reduced. Thus, even at this stage there was little evidence of an inability to maintain excretion rates. Nevertheless, some disturbance of potassium homeostasis was present, as indicated by the moderate hyperkalaemia at the end of the study period.

Compensatory changes after partial nephrectomy

Glomerular filtration rate.  As would be anticipated, in SO rats absolute GFR increased steadily throughout the 6 week study period. However, the gain in body weight during this period exceeded that in GFR, so there was a gradual reduction in GFR when expressed per 100 g body weight. This emphasizes the need to take into account changes in SO rats when assessing compensatory changes in GFR after partial nephrectomy. In UNX rats, there was little evidence for a substantial change in the GFR of the remaining kidney after 2 days (total GFR being 57% of the SO value). This contrasts with previous studies in conscious rats, in which an increase in the GFR of the remaining kidney (of 20–40%) within 24 h was described (Dicker & Shirley, 1971; Potter et al. 1974), although it is notable that in the earlier studies the animals were volume expanded, which may have modified the response. After day 2, we found clear signs of a compensatory increase in GFR, which was maximal by 8 days; during the period 8–32 days after UNX, GFR remained at 75% of the corresponding value in SO rats, representing a compensatory increase of 50%. The magnitude of the maximal increase is in reasonable agreement with that previously reported in conscious (Dicker & Shirley, 1971) and anaesthetized (Shirley & Walter, 1991; Pollock et al. 1992) rats, but little previous information was available on the time course of the compensatory changes after UNX.

After more extensive loss of renal mass, no information at all was previously available on either the time course or the extent of the compensatory changes occurring in conscious animals. The resection procedure used in the present study resulted in a reduction in the total functional glomerular count of the remnant kidney to 11% of the full two-kidney complement. Therefore, the observation that, 2 days after removal of the partner kidney, GFR was 23% of that of SO rats represents a doubling of GFR per glomerulus. Although this seems a remarkably rapid compensatory increase, it is likely that some of it occurred during the 7 day period between partial resection and contralateral nephrectomy. Moreover, given the fact that GFR was expressed per 100 g body weight, the transient reduction in body weight immediately after the second stage of 5/6 nephrectomy will have exaggerated the apparent increase in GFR. During the subsequent 2 weeks, GFR increased further, to reach a maximal value by 16 days of 36% of that of SO rats, representing a threefold compensatory increase. This figure is in broad agreement with values found in anaesthetized rats 2–6 weeks after 5/6 nephrectomy (Lafferty et al. 1989; Bidani et al. 1990), although in the studies cited the exact proportion of functioning glomeruli in the remnant kidney was not determined.

Tubular function.  Using this previously undocumented knowledge of the compensatory changes in GFR in the two groups of partially nephrectomized animals, we were able to determine the changes in fractional excretion rates. These generally followed a predictable pattern. During the early period after UNX, FE_Na, FE_K and FE_H2O all increased significantly, but subsided as the compensatory increase in GFR became evident, so that in the long term fractional excretion rates were only moderately (and non-significantly) elevated. In 5/6NX rats, the increases in FE_Na and FE_K were much more marked, even after the compensatory increase in GFR was complete. Thus, maintenance of overall sodium excretion rates after extensive loss of renal mass requires a substantial reduction in fractional reabsorption by the remaining nephrons. In the case of potassium, although the maintenance of normal excretion rates might result partly from reduced reabsorption, a much more important factor is likely to be enhanced secretion in the distal nephron, as underlined by the observation that during the early period after 5/6NX, FE_K sometimes exceeded 100%. Evidence has been provided in conscious rats for enhanced amiloride-sensitive potassium secretion during the early period after UNX (Aizman et al. 1996); and, although not previously studied in conscious 5/6NX animals, experiments in anaesthetized rats and in isolated cortical collecting tubule segments indicate enhanced potassium secretion in the distal tubule/collecting tubule following 3/4 nephrectomy (Bank & Aynedjian, 1973; Kunau & Whinnery, 1978; Fine, 1982).

The well-known impairment of urinary concentrating ability in renal insufficiency was reflected in the present study by large increases in urine flow rate and FE_H2O in 5/6NX rats. (Values of FE_H2O increased more than 10-fold during the early period, subsiding to six- to sevenfold in the long term.) Urine osmolality was reduced correspondingly. The pathophysiology of the reduced concentrating ability is likely to be multifactorial. Plasma vasopressin levels are increased in renal failure patients (Jawadi et al. 1986) and in 5/6NX rats (Bouby et al. 1990), but there is in vitro evidence for a blunted response of collecting duct segments or cultures to vasopressin-stimulated accumulation of cAMP (Fine et al. 1978; Teitelbaum & McGuinness, 1995) and for downregulation of V₂ receptors in the inner medullary collecting duct (Teitelbaum & McGuinness, 1995), while decreased expression of aquaporins 2 and 3 has been reported (Kwon et al. 1998). In addition, however, we have shown that the osmolality of papillary interstitial fluid is severely reduced in 5/6NX rats (Chamberlain et al. 1997). In this context, the large increase in GFR per nephron, together with reduced fractional reabsorption in the proximal tubule (see below), will result in a greatly increased delivery of fluid to the loops of Henle (mainly juxtamedullary) of the remnant kidney; this increase may be at least partly responsible for the ‘washout’ of the medullary osmotic gradient.

In order to obtain information on segmental changes in tubular function after partial nephrectomy, we used the relatively non-invasive technique of lithium clearance. Although lithium reabsorption in the proximal tubule lags slightly behind that of water and a small proportion of filtered lithium is reabsorbed in the loop of Henle (Boer et al. 1995), lithium clearance remains a useful qualitative index of proximal tubular function (Boer et al. 1995; Thomsen & Shirley, 1997).

In UNX rats, FE_Li increased significantly during the first few days and then returned to preoperative values. Although no previous information is available in conscious animals, a transient increase in FE_Li after UNX has been reported in anaesthetized rats (Atherton et al. 1990), and this is consistent with micropuncture data showing reduced fractional proximal reabsorption acutely, but not chronically, after UNX (Shirley & Walter, 1991). The present results are also consistent with lithium clearance data in human renal transplant donors (Kamper et al. 1994). It should be noted, however, that the changes in FE_Li were small and, moreover, only on the second postoperative day was the value in UNX animals significantly different from that in SO rats. Thus, distal nephron segments appear to be of overriding importance in maintaining excretion rates after UNX.

More substantial increases in FE_Li were observed after 5/6 nephrectomy, and these persisted throughout the study period. On days 2 and 4, FE_Li increased by more than 15% of the filtered load. Increases of this magnitude could not result solely from a reduction in lithium reabsorption in the loop of Henle, and it therefore seems clear that fractional fluid reabsorption in the proximal tubules must have been reduced. This observation in conscious rats is consistent with the clinical findings of Kamper et al. (1989), who reported raised values for FE_Li in patients with severe, but not mild, renal failure. It also accords with a report that the quantities per nephron of the proximal tubule transporters NHE3, NaPi2 and Na⁺,K⁺-ATPase do not increase in proportion to nephron enlargement after 5/6 nephrectomy (Kwon et al. 1999). Our conclusion, therefore, is that in severe renal insufficiency, both proximal and distal nephron segments contribute towards maintaining homeostasis.

Conclusions

In summary, the present study has documented both the time course and the extent of the compensatory changes in renal function, measured serially in uncatheterized, unrestrained conscious animals, following UNX or 5/6NX. After UNX, the increase in the GFR of the remaining kidney was maximal (at 50%) within 8 days; after 5/6NX, the increase in the GFR of the remnant kidney was maximal (at 300%) within 16 days. Overall excretion rates were maintained (or, in the case of water, increased) partly by the compensatory increases in GFR and partly by moderate (after UNX) or marked (after 5/6NX) reductions in fractional reabsorption. Lithium clearance data indicate that the changes in tubular function after extensive loss of renal mass include a reduction in fractional proximal reabsorption; after UNX, this effect is minor and transient.

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	Acknowledgements

 
We are grateful to Kidney Research UK for financial support and to Mr J. Skinner for technical assistance.

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