RE-ASSESSMENT OF THE RENAL HYDROSALINE DYSFUNCTION IN RATS BEARING THE WALKER-256 TUMOR

Abstract

Sodium retention is a frequent effect of cancer in humans and animals, but the mechanism involved is not yet understood. In the Walker-256 tumor, sodium retention has been considered to be a late effect, secondary to retention in the tumor mass, and/or to adrenal hypertrophy. Normally, (in rats receiving single tumor implants), the development of different tumor systemic effects (TSE) such as anorexia, sodium and fluid retention, anemia and immune depression in rats is synchronous within each individual but random among individuals of a given group in which they appear 6-47 days, or more, after inoculation. In present study, multifocal simultaneous inoculations of tumor cells resulted in a rapid and synchronous initiation of TSE (in 3-4 days) in all rats when no local effects of metastases could mask the results. Sodium retention is a special tumor effect on Na⁺ balance and a very sensitive indicator of TSE initiation. The results from multifocally inoculated rats were averaged in each (sub-clinical (SubC), moderate (mCP) and grave (gCP)) clinical phase and compared to food-restricted (FR) rats. There was a significant, early decrease in urinary Na⁺ excretion during mCP when compared to SubC and FR. The renal sites involved were studied in awake, unrestrained animals by measuring of sodium, creatinine and lithium clearances. There was an initial increase in the absolute proximal (mCP: 21.4 ± 1.7 vs FR: 16.0 1.1 mmol/min/100 g b.w., p< 0.05) and postproximal (mCP: 11.1 ± 0.4 vs FR: 6.6 ± 0.4 mmol min/100g b.w., p <0.001) Na⁺ reabsorption, which were partially compensated for by a rise in glomerular filtration rate (mCP: 213 ± 11.4 vs FR: 162 ± 10.2μ L/min/100 g b.w., p < 0.01) and by a fall in fractional proximal Na⁺ reabsorption (mCP: 62.8 ± 2.2% vs FR: 70.1 ± 1.7%, p < 0.05), despite significant Na⁺ and fluid retention. The terminal phase of the illness (gCP) culminated with a marked decrease in creatinine clearance, suggesting a significant fall in renal function. The multifocal model proved useful for studying the initial TSE, since the sites of action would, in principle, be easy to identify. These observations may be of physiological interest since TSE may result from the abnormal production of physiological modulators.

• Walker-256 tumor
• Urinary sodium excretion
• Diuresis
• Tumor systemic effects
• Multifocal tumor inoculation

INTRODUCTION

Water and sodium retention are frequent effects of cancer in humans and experimental animals [1-7], but the true pathophysiological mechanisms involved are not well understood. A need to assess these responses occurred during experiments designed to induce the rapid and synchronous initiation of systemic tumor effects (TSE), as part of a search for “the primary causative factors” of TSE [[8]]. A model involving simultaneous multifocal inoculations of Walker-256 tumor cells was allowed the rapid and synchronous initiation of the anorexia and fluid retention characteristic of this tumor at an early stage (3-4 days post-inoculation), when the total tumor mass was still very small [[9]].

Sodium and water retention by the Walker-256 tumor has been known for a long time [10-13], and was originally considered a late, secondary response, perhaps related to the tumor mass and/or to the concurrent adrenal hypertrophy. The present challenge is to find ways to experimentally detect the earlier effects of cancer in the chain of events initiated by the tumor. In the present study, we investigated the renal function, particularly the hydro-saline renal handling, in rats with multifocal inoculations of Walker-256 tumor cells. Sodium retention is not a critical effect, since neither an increase in its concentration in body fluids nor the magnitude of the associated water retention would lead to host death. Nevertheless, a study of sodium excretion in the multifocal model could facilitate the identification of systemic cancer involvement kidney function is qute easily assessed.

MATERIAL AND METHODS

Animals and Experimental Design

The experiments were performed on male Wistar-Hannover rats (200-250 g) housed individually in metabolic cages under controlled conditions of temperature and lighting. The rats had free access to tap water and standard laboratory chow and were assigned randomly to control pair-fed or tumor-bearering groups. The general guidelines established by the Declaration of Helsinki (1964) for laboratory animals were followed throughout the study.

Tumor-bearing Rats

The renal function study was performed in 50 rats divided into a control food restricted pair-fed group (FR, n=8) (see below) and two tumor bearing groups, with 30 rats in the moderate clinical phase (mCP) and 12 rats in the grave clinical phase (gCP).

Control pair-fed rats

Eight healthy rats, were housed in metabolic cages and, after 6 days with free access to food and water, underwent food-restriction baseded on a pattern similar to the average ingestion in tumor-bearing rats during the hypophagic phase of the disease.

The rats were weighed and housed individually in metabolic cages to measure of the food and fluid intakes, urinary flow rate, urine volume, and plasma osmolality and Na⁺ and K⁺ levels over a 24-h period. The clinical condition of the rats was evaluated daily. Somatic and visceral pain was explored by palpation and gentle compression of the axilary, inguinal and dorsal tumor sites, as well as the peritoneal and thoracic regions. The multifocal tumor model markedly reduces animal suffering since its evolution very rapid and death occurs before the development of significant metastases. Close to death, the rats became progressively unresponsive to the usual palpation.

Renal function tests were also performed. Fourteen hours before the renal test, 60 mmol LiCl/100 g body weight was given by gavage. The rats were subsequently housed individually in metabolic cages with free access to tap water but no food. The experiment was performed at the same time in each group of control pair-fed and tumor-bearing rats. At 8:00 a.m., each rat received a tap water load (5% of body weight) by gavage followed by a second load of the same volume 1h later. Twenty minutes after the second load, spontaneously voided urine was collected over a 2 h period. The voided urine passed through funnel in the bottom of cage into a graduated centrifuge tube. At the end of the experiment, blood samples were drawn by cardiac puncture and the kidney was immediately removed, decapsulated and weighed.

Tumor

The Walker-256 A line is maintained in our laboratory under liquid nitrogen and by consecutive subcutaneous (sc) or intraperitoneal (ip) passage in Wistar rats. Tumor cells were inoculated in the dorso-lumbar region, close to the midline. Each treated rat received four simultaneous inoculations (at least 1 cm apart) containing 4 × 10⁶ viable tumor cells diluted in 0.25 mL of Ringer-Lactate. The tumor cells were obtained from the ascitic fluid of a donor rat. To avoid mixing data from tumor bearers in different stages of the illness, a procedure similar to that used in discontinue parameter (single-fiber nerve or ECG analyses) was applied. Four points of synchronization were established, namely [[1]]the day of inoculation, [[2]] up to the last day free of systemic effects, defined here by the sudden onset of anorexia,[[3]] the last day of mild systemic effects, marked by aggravation of anorexia and by striking changes in body weight and or estimated water balance and [[4]] the last day of survival. By characterizing these aspects we established the limits of the sub-clinical (SubC), moderate (mCP) and grave (gCP) clinical phases. The average duration of each phase and the median duration point of SubC, mCP and gCP were defined and calculated by a computer program as described previously [[8]].

Biochemical Analysis

Plasma and urine sodium, potassium and lithium concentrations were measured by flame photometry. Urine osmolality and creatinine concentrations were determined by the cryoscopic point change and spectrophotometrically by the alkaline picrate method, respectively.

Statistics and Calculations

The results are reported as mean ± SEM per 100 g body weight. Renal clearance (C) was calculated by a standard formula (C = UV/ P) using the plasma creatinine and lithium levels for each period. Creatinine clearance was used to estimate the glomerular filtration rate (GFR) and lithium clearance (CLi⁺) was used to assess proximal tubule output. Fractional sodium (FENa⁺) and potassium (FEK⁺) excretions were calculated as CNa⁺/C^Cr and CK⁺/C_Cr, respectively, where CNa⁺ and CK⁺ are the ion clearances and C_Cr is the creatinine clearance. The fractional proximal (FEPNa⁺) and post-proximal (FEPPNa⁺) sodium excretions were calculated as CLi/C_Cr × 100 and CNa⁺/CLi⁺× 100, respectively [14-15]. Sodium and potassium fractional balances were calculated by a standard formula (fractional balance = (input − urine output) × (input)^–1, where input = food intake (g/day) × diet ions concentration (mEq/g) and urine output = urine volume (mL/day) × urine ion concentrations (mEq/mL). Statistical analysis of the data was done using one-way analysis of variance for repeated measurements and Student's paired t test as appropriate. When the results were significant, Bonferroni's contrast test was used to determine the extent of the differences. A p value <0.05 was considered to indicate significance.

RESULTS

All inoculations with tumor cells gave rise to continuously growing tumors that were palpable within 2-3 days. TSE such as anorexia, body weight loss, sodium retention, and others that were not considered in this work (anemia, thymus atrophy, etc.), occurred rapidly in the multifocal tumor model. Thus, TSE initiated after 3-6 days and all deaths occurred 12-14 days after inoculation.

Figure 1 shows the changes in body weight and food intake in multifocally inoculated rats and their respective pair-fed controls. Anorexia was accompanied by body weight gain in all tumor bearers, while in the pair-fed rats, a similar degree of hypoalimentation produced a clear decrease in body weight.

Figure 1. Changes in food intake and body weight in rats that received multifocal simultaneous inoculations of the Walker-256 tumor (A), and in pair-fed controls (non-tumor bearers) (B). The points are the mean±SEM of x-y rats/group. Paired t-test on body weight data (day 7 vs day 4): tumor group = +5.5 ± 0.9%, p < 0.001; pair-fed group = −7.0 ± 1.4%,p < 0.01. (*) For pair-fed controls, day 5 was actually the first day in which food was restricted in this group of rats.

Sodium Retention

Figure 2 shows that renal sodium retention was significantly increased in the multifocally inoculated rats. Before the onset of TSE in tumor bearers, and of food restriction in pair-fed controls (days 1-4), about 75% of the ingested Na⁺ was excreted through the kidneys. Subsequently, under hypoalimentation, control rats increased their relative renal Na⁺ excretion to about 95% (days 6-7), while in tumor-bearing rats there was marked decreased to about 30% (days 7-8). The renal Na⁺ retention in tumor bearers resulted in a 10% rise in plasma (Na⁺), from 142±2.4 to 156±1.6 mM. The total amount of Na⁺ retained by the tumor bearers was estimated to be close to 30% of the total Na⁺ contained in the extracellular fluid of the control animals, i.e an ∼10% rise in concentration vs an ∼20% rise in fluid volume (Figure 4). The Na⁺ retention was coincident with the development of anorexia, and was a sensitive indicator for the onset of of TSE. Plasma potassium levels were not significantly different between control (4.8±0.3 mM) and tumor-bearing (5.1±0.2 mM) rats.

Figure 2. Average Na⁺ and K⁺ fractional balances in rats that received multifocal simultaneous inoculations of the Walker-256 tumor (A) and in the pair-fed controls (non-tumor bearers) (B). The points are mean±SEM of x-y rats/groups. Paired t-test (vs average of the control period), of the fractional Na⁺ balance data were highly significant after day 4, e.g., on day 7: tumor group = +0.42 ± 0.034, p < 0.001; control group = −0.20± 0.05, p < 0.01 Potassium fractional balances were not significantly different between groups. (^*) For pair-fed controls, day 5 was actually the first day in which food was restricted in these rats.

Figure 4. Actual changes in body weight (as%) in rats that received multifocal inoculations of the Walker-256 tumor relative to the pair-fed controls. The points are the mean±SEM of x-y rats per group. Paired t-test was significant after day 4. On days 5, 6, 7 and 8 the results were +2.8 ± 1.0% (p < 0.05), +6.5 ± 1.7% (p < 0.01), +11.3 ± 2.1% (p < 0.001) and +15.2 ±2.5% (p< 0.001) respectively. (^*)%Body weight = (body weight_{tumor rat} × body weight⁻¹_{pair-fed rats}−100.

Water Retention

The changes in the renal handling of water are shown in figure 3. In tumor bearers, the kidneys produced a lower volume of concentrated urine during the early stages of TSE, and this resulted in water retention; the pair-fed controls produced a high volume of diluted urine as a physiological response to hypoalimentation. The changes in fluid intake were not different between the two groups of rats: during days 5-8, water intake dropped by 8.7±1.5 mL/day and 5.5±2.0 mL/day in tumor-bearing and pair-fed control rats respectively; Student's paired ttest within groups was significant (p < 0.001 and p < 0.05, respectively), whereas the difference between groups was not. These alterations in urine output and water intake lead to the following changes in the renal involvement in water balance: before the initiation of TSE and hypoalimentation (days 1-4) in tumor-bearing and pair-fed control rats respectively, about 35% of the ingested water was excreted through the kidneys. Subsequently, under equivalent conditions of hypoalimentation, the PF rats increased their relative water excretion to about 50%, while in the tumor bearing rats this increase was abolished. An estimate of the resulting total amount of water retained was obtained by calculating the% increase in body weight of the tumor-bearing rats relative to the body weight of pair-fed controls. Figure 4 shows that this value was about 20%.

Figure 3. Changes in urine volume and osmolality in rats that received multifocal simultaneous inoculations of the Walker-256 tumor as compared with the pair-fed controls. The points are the mean±SEM of x-y rats per group. Urine volume, paired t-test: a) tumor group, significant decrease of during days 5-8, −2.5 ±0.9 mL day, p< 0.05; b) control group, significant increase of during days 6-7, +1.8± 0.7 mL/ day,p < 0.05. Urine osmolality, paired t-test : a) tumor group, significant rise of from day 3, 220±70 mOsm, p < 0.05, b) control group, significant decrease of 309±79 mOsm on days 6-8, sp < 0.01. (^*) For pair-fed controls, day 5 was actually the first day in which food was restricted in these rats.

Renal Sodium Handling

The endogenous creatinine clearance showed a significant increase during the moderate clinical phase compared to the sub-clinical and food-restricted groups (p < 0.01). Nevertheless, no significant changes in creatinine clearance were observed in the food-restricted group (Table 1) compared to the sub-clinical phase. A striking decrease in the glomerular filtration rate estimated by creatinine clearance occurred in the grave clinical phase in tumor bearers.

Table 1. Comparison of the creatinine clearance (C_Cr) and renal tubular sodium handling in food-restricted (FR) rats and tumor-bearing rats, during the sub-clinical (subC) and moderate clinical (mCP) phases of the latter. FENa⁺, FEPNa⁺ and FEPPNa⁺ are the fractional, fractional proximal and fractional post-proximal sodium excretion, respectively. The data are expressed as the mean±sem of x-y rats/group

	CCr	FENa^+	FEPNa^+	FEPPNa^+
	(μl/min/100 g b.w.)	(%)	(%)	(%)
FR	162.5±15.2	0.39±0.07	70.1±1.7	29.5±1.7
subC	140.6±12.0	0.76±0.111^*	75.6±2.31^*	23.7±2.21^*
mCP	213.0±11.41^*	2.20±0.471^*	62.8±2.21^*	36.7±2.21^*
gCP	62.8±16.41^*	–	–	–

^* p< 0.05 vs FR.

The approximate tubule sodium handling was assessed by lithium clearance at the median duration point of the sub-clinical (third day) and moderate clinical (sixth day) phases after Walker-256 tumor inoculation. No analysis was done during the grave clinical phase when the creatinine clearance was significantly altered.

There was a significant decrease in the renal fractional sodium excretion of food-restricted rats with no change in fractional water excretion (21.6±1.6%) when compared to the sub-clinical phase (20.9±0.9%). The decreased renal sodium excretion in food-restricted rats was accompanied by a fractional decrease in the proximal tubule followed by a striking enhancement of post-proximal tubule sodium reabsorption (Table 1).

During the moderate clinical phase, six days after tumor inoculation, the tumor bearers showed a decrease in the fractional renal sodium excretion when compared with food-restricted and sub-clinical phases (Table 1). A similar drop in proximal tubule absorption in food-restricted and Walker-256 tumor rats was followed by a significant rise in fractional post-proximal tubule sodium reabsorption (mCP: 9.6 ±1.3% vs SubC: 3.5± 2.2%, p < 0.05), which was significantly higher in the Walker-256 tumor group.

DISCUSSION

In human cancer and unifocal tumor models, the onset of systemic effects is usually sudden although the timing varies among individuals making it almost impossible to obtain a group of animals in which the systemic effects are synchronized [1-7]. In the present study, some synchronization of the symptoms was achieved by multifocal inoculation. The short duration of the clinical phases (3-4 days after inoculation) allowed the renal function tests to be done before the development of cancer metastases, which would otherwise have interfered with adequate function of this organ.

The short duration and high reproducibility of the experiments with minimal apparent animal suffering allowed the observation of the sharp transition from the normal physiological condition (sub-clinical phase) to that in which fundamental homeostatic mechanisms were quickly and irreversibly altered.

Critical Importance of the Multifocal Model

The onset of TSE in rats receiving a single inoculation of Walker 256 tumor cells occurs 6-47 days, or more, after inoculation [[9]]. In all models, the onset of TSE is sudden and is followed by a continuous and rapid rise in intensity until death, about 6-11 days later. As a result, the daily (or every 3 days) cross- sectional averaging of data, as usually done in unifocally inoculated animals, leads to the loss of critical information because of the mixing of values from animals with markedly different intensities of TSE. In contrast, multifocal simultaneous inoculations initiated TSE in all animals 3-4 days after inoculations. Another advantage of the multifocal model in renal function studies lies in the short duration of the experiments which are usually completed before the local effects of metastases alter the urinary apparatus or thoracic reflexes (respiratory and volume receptors reflexes influence renal hydroelectrolyte excretion).

Sodium and Fluid Retention and Tubule Sodium Handling

The initiation of renal sodium and water retention was an early phenomenon in this experiment, and was concomitant with the onset of anorexia (body weight increase despite hypoalimentation) (Figure 1). When these effects were started, the total tumor mass was very small, since by the end of the third day, each one of the four tumors had a mass of little more than 100 mg. In contrast, in rats receiving single inoculations, TSE initiated when the tumor masses weighed 20 g on average. As shown here, Walker-256 tumor caused a significant and continuous time-dependent decrease in renal sodium excretion, which was associated with an increase in body weight and creatinine clearance during the moderate clinical phase. This effect on sodium homeostasis occurred before a pronounced increase in tumor mass i.e., sodium retention started on the fifth day, when each one of the four tumors weighed about 100 mg, compared to an average of 19.5 g when fluid retention was initiated in the single inoculation model [[8]]. These results contrast with previous data for single inoculation tumor development in which renal sodium retention was seen only when the tumor weight was more than 10% of the body weight [[11]]. The present data do not support an adrenal involvement in renal ion handling [[9]] since there was no change in the renal K⁺ balance and plasma K⁺ levels in tumor-bearing rats[[8]].

Sodium retention seemed to be the driving force for fluid retention suggested by the elevated plasma sodium level, which indicated that more sodium than water was retained and by the data on sodium and water balances (pair-fed controls increased their relative Na⁺ and water excretions, while tumor bearers markedly decreased their relative Na⁺ excretion without a parallel decrease in relative water excretion) (Figure 2). Under this pathophysiological condition, the hydroelectrolytic regulatory centers would receive conflicting peripheral signals from the elevated extracellular sodium level and the elevated extracellular volume, both of which require opposite compensatory responses (fluid retention and fluid excretion, respectively). Compensation of the elevated extracellular Na⁺ concentration was prefered here, and an antidiuretic response was elicited with the production of a lower volume of more concentrated urine (Figure 3), this effect disappeared around day 10, 1-2 days before death, when there was a rapid decrease in creatinine clearance [[14]]. The antidiuretic response was obviously important in the ensuing fluid retention. This retention is better appraised by the changes in water fractional balance: control rats excreted a larger fraction of the ingested water (∼50%) than the tumor bearers (∼35%), and by the resulting percentage increase in the body weight of tumor bearers relative to that of pair-fed controls (Figure 4).

Thus, a plasma sodium concentration increase of about 10%, together with a body fluid increase of about 20% would result in a total Na⁺ accumulation of about 30%, relative to the controls.

These observations on sodium and water retention suggest a more specific link between tumor and Na⁺ retention than the suggesting that Na⁺ retention is secondary to its sequestration in the tumor mass [[13]]. The latter conclusion was based on experiments using unifocal models of tumor growth in which the effect was detected when tumors were large (>10% of body weight). Our conclusion that the sodium retained is not associated with the tumor, is reinforced by the fact that the signal for Na⁺ retention in this case should have been a low plasma Na⁺ concentration. Instead, the rats in our experiments had eleved plasma Na⁺. Furthermore, the tumor mass should also have sequestered K⁺ which would have led to a reduction in plasma K⁺ concentration and to its renal retention, events not observed in this study (Table 1 and Figure 2). The decrease in renal sodium excretion was not accompanied by changes in creatinine clearance in food-restricted rats. This finding suggests a physiological response to the daily decrease in sodium intake involving a rise in post-proximal tubule sodium reabsorption. No renal fluid retention was observed in these rats. Previous studies have shown that in the early phase of weight reduction after food restriction, the composition of the material lost varies widely [[16]]. Water and sodium are the main elements responsible for this variability, since they are incorporated into the tissues as a complex with organic material, i.e., protein and glycogen [17-18].

In tumor-bearing rats, there was significant renal sodium and water retention despite an increased creatinine clearance (Figure 1). This enhancement in the glomerular filtration rate probabily reflects a compensatory attempt to raise renal hydro-electrolyte excretion.

Although there was an increase in proximal sodium reabsorption in mCP (Table 1), when expressed relative to the filtrated sodium load, there was actually a decrease in fractional proximal tubule absorption. However, the significant drop in this reabsorption was not enough to increase the renal sodium excretion (Table 1). The rise in fractional post-proximal tubule Na⁺ reabsorption probably reduced the urinary sodium excretion seen here. Together, these data suggest the presence of marked renal tonic antinatriuretic activity which is not altered by a decrease in food intake or body fluid volume expansion (indicated by increased body weight) or by an increased filtration rate. Additionally, this study showed that the terminal phase of the illness (gCP) culminated with a marked decrease in creatinine clearance, suggesting a significant fall in renal function.

Nature and Importance of the Factors Linking the Tumor with Sodium Retention

Sodium retention may be a useful phenomenon for identifying factors of tumor origin involved in the production of TSE. In principle, should be much less complex than the study of the mechanisms leading to anorexia, immune depression, protein catabolism, etc. Interest about the molecular mediators of TSE has been directed towards peptide-like molecules either produced by the tumor, or whose production may be induced by the tumor [16-20]. Tumor-related molecules may induce renal Na⁺ retention by central signals reaching the kidney through neural pathways [[21]], although in kidney denervated animals there were no tumor-induced changes in Na⁺ retention (unpublished data). If the molecular mechanism does not involve central signals, then its main action on the kidney should be through the blood stream. Substance such as prostaglandins, renin, ADH, may also be involved in this Na⁺ retention. Indomethacin and captopril do not alter the effect of the tumor on Na⁺ and fluid retention (unpublished data). ADH is known to induce some Na⁺ retention, [[22]], and ADH levels may have risen in the present experiments (decreased urine volumes with elevated osmolality; Figure 3), although this hormone retains more water than Na⁺ and should thus not raise plasma sodium levels. More recently [[19]], renal Na⁺ retention was reported to be stimulated directly by cytokine-like peptides. The divergent results obtained in the present work when compared with studies performed several years ago, cannot be attributed to eventual changes in the tumor line, since the results obtained with unifocal inoculations of this tumor [[9]] are remarkably similar to those reported by others several years ago [13-14].

Importance of the Multifocal Tumor Approach and of Increased Sodium Reabsorption in Studies on the Effects of Cancer and Other Pathophysiological Conditions

Multifocal tumor inoculation allows the real time study of the sudden transition from an essentially normal physiological condition to one in which fundamental homeostatic mechanisms are rapid and irreversibly altered. Sodium retention in itself is not a critical effect since neither the increase in its concentration nor the extent of associated fluid retention would lead to host death. However, the extraordinary coincidence between such retention and the initiation of the disruption of fundamental mechanisms that regulate sodium levels, feeding, immune responses and protein catabolism, which culminate in death, suggests that the molecules involved in TSE are probably closely linked or may even be the same. Identification of the mediators involved would be of interest not only for cancer research, but also for physiological and pathophysiological studies.

Bias in the Action of Multifocal Tumor Inoculations

The explanation as to why several small tumors may be more effective in inducing TSE and death than a single large tumor must have to do with the kinetics of the total population of proliferative tumor cells (PTC) residing in any tumor bearing individual. Bassukas and Maurer-Schultze [[23]], demonstrated experimentally that the proportion of PTC in small tumors and metastases is large and grows rapidly while in large tumors this proportion is small and its size increases very slowly. These observations agree with our general knowledge of cell proliferation [[24]]. Thus, a single relatively large tumor would represent a temporarily stable condition that would be broken when its metastatic dissemination occurred, a condition now characterized by multifocal growth, a rapid rise in the total population of PTC, and the appearance and rapid increase in the intensity of TSE until death.

In conclusion, the present study has demonstrated that renal Na⁺ retention is an early and sensitive marker for the onset of the systemic effects of of Walker-256 tumor. Sodium retention was the driving force in fluid retention, leading to a paradoxical increase in body weight despite anorexia. The results also support the contribution of post-proximal nephron segments to this sodium retention. The terminal phase of the illness (gCP) culminated with a marked decrease in creatinine clearance suggesting a significant decline in renal function. Further studies are being required to investigate the neurohumoral pathways involved in functional kidney-tumor interactions.

ACKNOWLEDGMENT

Financial support by FAPESP, CNPq and FAEP-Unicamp.