TUBULAR CELL LESION, ALBUMINURIA, AND RENAL ALBUMIN HANDLING IN RATS TREATED WITH ADRIAMYCIN

Abstract

Several lines of evidence have suggested that renal handling of proteins in rats with several nephropathies may contribute to the tubulointerstitial damage observed in these animals. It has been suggested that proteins filtered by the glomeruli may be toxic for tubule cells. The aim of this study was to investigate the relationship between albuminuria and tubular lesions observed in rats during the first two weeks after treatment with adriamycin (AD). Thirty female Wistar rats were injected intravenously with adriamycin at the dose of 3.5 (17 rats) or 5 mg/kg body weight (13 rats), and 7 were injected with 0.15 M NaCl (control group). Seven days later, we replaced drinking water with a 0.10 M sodium bicarbonate solution for 6 of the animals injected with 5 mg/kg adriamycin (group AD-B). Urine samples were collected before and 7 and 15 days after treatment to quantify albumin. The rats were killed 7 and 18 days after the injections, and the kidneys removed for immunohistochemical study. We observed a significant increase in urinary albumin excretion 15 days after AD injection (3.5 mg/kg), but not 7 days after AD. However, in the animals injected with 5.0 mg/kg AD (group AD-5) the increase in albuminuria was observed as early as on day 7. The immunohistochemical studies showed increased vimentin and albumin immunoreaction in the tubular cells of the renal cortex from the kidneys of rats injected with 3.5 mg/kg (group AD-3) only 18 days after treatment (p < 0.05), whereas in the animals treated with 5 mg/kg AD these immunohistochemical alterations were more intense. However, treatment with sodium bicarbonate attenuated the tubular lesions and reduced albumin reabsorption in adriamycin-treated rats. In conclusion, these experiments showed a relationship between albuminuria and tubular lesions in adriamycin-treated rats.

• Adriamycin nephropathy
• Albuminuria
• Tubular lesions
• Proteinuria
• Nephrotoxicity

INTRODUCTION

Several studies support the hypothesis that filtered proteins may be toxic to tubule cells [1-2]. Higher proteinuria is frequently associated with more severe glomerular and tubular damage [3-4]. Interstitial fibrosis associated with proteinuria has been observed during the evolution of puromycin aminonucleoside nephritis [[5]], protein overload proteinuria [[6]] and adriamycin nephropathy [7-8]. Studies with tubule cell cultures have shown that some proteins, such as IgG and albumin, induced increased production of inflammatory and vasoactive factors by these cells [9-10].

A single injection of adriamycin (AD) can induce marked and persistent proteinuria in rats [7-8]. There are several lines of evidence showing that the renal handling of proteins may contribute to the development of tubulointerstitial damage observed in these animals [9-13]. A low-protein diet attenuates the proteinuria and tubulointerstitial lesions in rats injected with AD [13-15]. We have recently observed that treatment with sodium bicarbonate prevented renal protein reabsorption, the increase in renal TGF-β production, as well the tubulointerstitial damage in rats killed 105 days after AD-injection [[12]]. Treatment with sodium bicarbonate induces urine alkalinization and extracellular volume expansion that could reduce protein reabsorption by tubular cells [[2]], [[12]]. The modifications in protein electric charges caused by the change in urinary pH must interfere with protein binding to the luminal membrane of tubular cells, which is the first step of endocytosis [16-17]. Changes in protein concentration at endocytic sites due to extracellular volume expansion also influence protein reabsorption [[17]].

The aim of this study was to investigate in rats the relationship between albuminuria and tubular cells lesions in the first two weeks after the administration of AD, before the tubulointerstitial nephritis have been established.

MATERIALS AND METHODS

Animals and Experimental Design

Thirty-seven female Wistar rats weighing 180–220 g were used. Seventeen rats were injected with a single dose of adriamycin, 3.5 mg/kg body weight, through the tail vein (group AD-3), 13 with a single dose of adriamycin, 5 mg/kg body weight (AD-5), and 7 with 0.15 M NaCl solution. Seven days later we replaced the drinking water with a 0.10 M NaHCO₃ solution for 6 of the animals treated with 5 mg/kg adriamycin (group AD5-B). All animals had free access to chow of normal protein content (18%). The average amount of food consumed by the animals was not modified by sodium bicarbonate treatment. The range of urinary pH was 8.0 to 9.0 for rats treated with sodium bicarbonate and 6.0 to 7.0 for the animals from the other groups. Urine samples were collected to determine albumin excretion 7 and 15 days after the injections. The animals were killed with excess anesthesia at 7 days (7 from group AD-3) and 18 days (10 from group AD-3, 7 from group AD-5, 6 from group AD5-B and 7 from control group). Blood samples were collected to quantify creatinine and the organs were perfused with PBS solution (0.15 M NaCl plus 0.01 M phosphate solution, pH 7.4) and the kidneys were removed for immuno-histochemical study.

Quantification of Urinary Albumin and Plasma Creatinine

Urinary albumin was quantified by electroimmunoassay using a specific antibody against rat albumin [[2]], [[18]] and plasma creatinine was measured by the Jaffé reaction [[19]].

Antibodies

The primary antibodies used were: 1) a monoclonal mouse antivimentin antibody (Dako Corporation, Glostrup, Dennmark), 2) a monoclonal mouse anti-proliferating nuclear antigen (PCNA) antibody (Sigma Chemical Company, St. Louis, MO), and 3) a polyclonal anti-rat albumin antibody produced in our laboratory [[2]].

Immunohistochemical Studies

Thirty adriamycin-injected rats and 7 controls were killed 7 and 18 days after treatment. The rats were submitted to left intraventricular perfusion with PBS, pH 7.4, until the kidneys were blanched. The kidneys were also perfused with 4% formalin, fixed in 4% formalin for 2 h, and post-fixed in Bouin solution for an additional 4–6 h, rinsed with 70% ethanol to eliminate picric acid, dehydrated through a graded ethanol series, embedded in paraffin, sectioned into 3 mm slices, deparaffinized, and subjected to immunohistochemical staining [[20]].

The sections were incubated for 1 h at room temperature with 1/1500 monoclonal mouse antivimentin antibody, or for 1/2 hour with a 1/1000 monoclonal mouse anti-PCNA antibody, or overnight with a polyclonal rabbit anti-rat albumin antibody. The reaction product was detected with the avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA). The color reaction was developed with 3,3-diamino-benzidine (Sigma Chemical Company, St. Louis, MO) and the material was counter-stained with methyl green, dehydrated, and mounted. Nonspecific protein binding was blocked with 5% goat serum in PBS for 30 minutes. The controls consisted of replacing the primary antibody with normal rabbit (polyclonal antibody) or mouse IgG (monoclonal antibodies).

For evaluation of the immunoperoxidase stains for vimentin and albumin each tubulointerstitial grid field was graded semi-quantitatively, and the mean score per biopsy was calculated [20-21]. Each score reflects mainly changes in the extent, rather than the intensity, of staining and depended on the percentage of grid field showing positive staining: 0, absent or less than 5%; I, 5 to 25%; II, 25 to 50%; III, 50 to 75%, and IV, > 75%. To obtain total counts of PCNA-positive cells in the renal cortex, grid fields measuring 0.366 mm² each were analyzed, and again, mean counts per kidney were obtained.

Statistical Analysis

Urine albumin level and the immunohistochemical scores for vimentin obtained under control and experimental conditions were submitted to the Kruskal Wallis test and Dunn's post test. Data concerning the immunohistochemical scores for albumin, PCNA-positive cells and plasma creatinine concentration were submitted to analysis of variance with multiple comparisons by the Tukey test.

RESULTS

Urine Albumin Excretion and Plasma Creatinine Level

Urinary albumin level was increased in all groups of animals injected with adriamycin (p < 0.001). However, in the animals injected with 3.5 mg/kg adriamycin this increase was observed only on day 15 after injection (Table 1). The animals treated with 5 mg/kg adriamicin presented a progressive and intense increase in albuminuria which was significant 7 and 15 days after treatment and there was no difference between groups AD-5 and AD5-B (Figure 4). Plasma creatinine levels were not modified by these treatments, being 0.64 ± 0.06 mg% in the control rats and 0.69 ± 0.11 mg% in the rats killed 18 days after AD injection.

Figure 4. Urinary albumin excretion before and after the treatment with 5 mg/kg adriamycin (AD) or with 5 mg/kg adriamycin plus sodium bicarbonate (AD-B). Data are expressed as mean ± SD.

Table 1. Albuminuria and Immunohistochemical Data for the Renal Cortex from Control Rats and from Rats Killed 7 (AD 7 D) and 18 Days (AD 18 D) After Adriamycin Injection (3.5 mg/kg)

Rats	Albuminuria (mg/24H)	Albumin (Score)	Vimentin (Score)	PCNA (cells +/0.36 mm^2)
Control	0.31 ± 0.22	0.20 ± 0.14	0.04 ± 0.03	4.78 ± 1.22
AD 7 D	0.33 ± 0.03	0.18 ± 12	0.01 ± 0.01	4.19 ± 1.06
AD 18 D	71.4 ± 37.4^**	1.74 ± 0.29^**	0.36 ± 0.39^*	23.5 ± 17.1^*

Values are means ± SD,

^*P < 0.05;

^**P < 0.001 vs control.

Immunohistochemical Studies

The results of immunohistochemical analysis showed that the animals treated only with adriamycin (3.5 and 5 mg/kg) and killed on day 18 after adriamycin injection had higher immunohistochemical scores for vimentin and albumin in the tubular cells from the renal cortex than control animals (Table 1, Figures 1-2 and 5). Higher amount of PCNA-positive cells was also observed in the renal cortex from the animals injected with adriamycin on day 18 after injection (Table 1, Figure 3). Treatment with sodium bicarbonate reduced the immunostaining for vimentin (Figure 5) and for albumin in these cells. No difference in staining for vimentin, PCNA or albumin was observed between the animals killed 7 days after treatment with 3.5 mg/kg adriamycin and control animals (Table 1). Since vimentin and PCNA expression in tubular cells is an indicator of tubular cell regeneration suggesting recent injury [[22]], this damage may be related to an increase in protein reabsorption.

Figure 1. Immunolocalization of albumin in the renal cortex of a rat killed on day 18 after treatment with 3.5 mg/kg adriamycin (B) and of a control rat (A). Note that the immunostaining in the cytoplasm of the tubular cells is more intense in B. ×420.

Figure 2. Immunolocalization of vimentin in the renal cortex of a rat killed on day 18 after treatment with 3.5 mg/kg adriamycin (B) and of a control rat (A). Note that the immunostaining in tubular cells is more intense in B. ×420.

Figure 5. Immunostaining score for vimentin for control rats (C, N = 7), for rats killed on day 18 after treatment with 5 mg/kg adriamycin (AD, N = 7) and for rats killed on day 18 after treatment with 5 mg/kg adriamycin plus sodium bicarbonate (AD-B, N = 6). The median is shown as a horizontal line.

Figure 3. Immunolocalization of PCNA in the renal cortex of a rat killed on day 18 after treatment with 3.5 mg/kg adriamycin (B) and of a control rat (B). Observe that the amount of PCNA positive cells is higher in B. ×420.

DISCUSSION

Eighteen days after adriamycin treatment, the animals showed high levels of albuminuria and increased vimentin, PCNA and albumin expression in tubular cells from the renal cortex. Treatment with sodium bicarbonate reduced the immunostaining for albumin and vimentin in the renal cortex of these animals. Vimentin and PCNA expression is an indicator of tubular cell regeneration suggestive of recent injury [[22]], and therefore this damage may be related to an increase in the tubular reabsorption of albumin or other filtered plasma proteins. The fact that lower staining for vimentin, PCNA and albumin was observed in the kidneys from rats killed 7 days after injection of 3.5 mg/kg adriamycin, with no albuminuria, supports this hypothesis. Other studies have shown the presence of tubular cell damage in adriamycin-treated rats, and these alterations were attributed to proteinuria or to a direct toxic effect of this drug [[7]]. However, we did not detect such lesions in the first week after treatment in the animals without albuminuria, a fact suggesting that these lesions probably are not due a direct toxic effect of this drug.

Lower immunostaining for albumin and vimentin was also observed in rats killed 18 days after treatment with adriamycin plus sodium bicarbonate compared to the animals treated with adriamycin alone. Treatment with sodium bicarbonate induces urine alkalinization and extracellular volume expansion that can reduce the renal handling of proteins by tubular cells [[2]], [[12]], [[17]]. The decreased albumin expression in the cytoplasm of the tubular cells observed in the animals treated with adriamycin plus sodium bicarbonate despite a great rate of urinary albumin excretion confirms this hypothesis. Urine alkalinization can decrease positively charged groups of proteins, interfering with their accessibility to endocytic sites, with binding to the luminal tubular membrane, and consequently with the efficiency of their tubular handling [[2]], [[17]]. The extracellular volume expansion present in these animals can also contribute to a decrease in renal albumin by reducing the concentration of proteins at endocytic sites due to the reduced sodium reabsorption rate [[17]]. A higher urinary volume was observed in the animals treated with sodium bicarbonate. On the basis of these data, we can conclude that the renal reabsorption of other plasma proteins such as IgG or IgG fragments can also be reduced by this treatment.

In conclusion, the tubular lesions observed in adriamycin-treated rats seem to be related to the increase in urinary albumin excretion. These alterations were observed only in the animals killed 18 days after adriamycin injection (3.5 mg/kg) after the establishment of albuminuria. Treatment with sodium bicarbonate reduced the renal reabsorption of albumin and attenuated the lesions of tubular cells from the kidney cortex.

ACKNOWLEDGMENT

The authors thank Erika Dellaiogono for expert technical assistance.