Renal Proximal Tubule Segment-Specific Nephrotoxicity: An Overview on Biomarkers and Histopathology

Abstract

The correspondence between histopathological findings and segment-specific biomarkers was investigated in rats treated with segment-specific nephrotoxicants. Male Wistar rats were treated with a single injection of K₂Cr₂O₇ (25 mg/kg sc in saline), cis-Pt (10 mg/kg ip in buffered MSO) or HCBD (100 mg/kg ip in corn oil). Twenty-four and 48 hours after treatment, the rats were sacrificed and the kidneys were drawn for histopathological and biochemical evaluation, i.e., GS activity in renal cortex and PAH uptake in renal cortical slices. Histopathological findings show that cis-Pt and HCBD cause diffuse necrosis of S₃ segment of proximal tubules in the outer stripe of outer medulla, respectively. On the contrary, K₂Cr₂O₇ damages exclusively S_1–S₂ segments, inducing vacuolization at 24 hr and diffuse necrosis at 48 hr after treatment. GS activity in renal tissue is significantly decreased after HCBD and cis-Pt, but not K₂Cr₂O₇ treatment. In contrast, PAH uptake is significantly reduced by K₂Cr₂O₇, but not by cis-Pt or HCBD treatment (even if HCBD causes a slight decrease 48 hr after treatment). The evidence of this study confirms the high specificity of GS activity as marker of S₃ segment injury, that PAH uptake is prevalently active in the S_1–S₂ segments, and that there is complete correspondence among segment-specific nephrotoxicants, biomarkers of segment-specific damage, and histopathological findings.

Introduction

Several xenobiotic substances damage renal proximal tubule, the portion of the nephron with the greater sensitivity to nephrotoxic effects. The proximal tubule is subdivided into three segments (S₁, S₂, and S₃) present in the cortical labyrinth and the medullar rays.

The proximal convoluted tubule (S₁ and S₂ segments) contains a very active endocytosis/lysosomal apparatus, thus representing the site of injury related to lysosomal overload, as well as protein-bound toxic moieties. The S₃ segment represents the most susceptible site of injury via metabolic activation, transporter-associated accumulation, and hypoxia/reperfusion (Khan and Alden, 2002).

Among chemicals damaging the proximal tubule are antibacterial drugs such as cephaloridine (Silverblatt et al., 1970) and aminoglycosides (Wellwood et al., 1976), which selectively affect S_1–S₂ segments, anticancer drugs such as cis-Pt (Dobyan et al., 1980), which affects S₃ segment, industrial chemicals (metals and solvents) such as cadmium (Squibb et al., 1979) and hexavalent chromium (Biber et al., 1968; Evan and Dail, 1974), which affects S_1–S₂ segments, or mercury (Eknoyan et al., 1982; Dobyan and Bulger, 1984), HCBD (Ishmael et al., 1982), and palladium (Trevisan et al., 2002), which affect S₃ segment.

As previously demonstrated, urinary markers may be used to evaluate segment-specific damage caused by chemicals (Trevisan et al., 1999) or to evaluate segment-specific distribution of renal biomarkers, treating animals with segment-specific nephrotoxicants (Trevisan et al., 1998). The classification of nephrotoxins by subtopographical target is an integral step in pathogenesis studies. Target site identification is also a prerequisite in understanding function impact, as well as in developing the most sensitive premonitory of injury from nephrotoxicants. However, simply identifying the target site for a xenobiotic does not identify the mechanism of injury.

The aim of this research is to study the effects of three xenobiotic chemicals, two (K₂Cr₂O₇ and HCBD) of industrial use and an antineoplastic drug (cis-Pt), producing renal proximal tubule segment-specific toxicity. Biomarkers of proximal tubule damage and histopathological findings are used to measure the segment-specific effects of the chemicals and to state the correspondence among chemicals, biomarkers, and histopathology.

Materials and Methods

Chemicals

HCBD (purity >97%), K₂Cr₂O₇, imidazole, 2-mercaptoethanol, hydroxylamine hydrochloride, ferric chloride anhydrous, PAH, and MSO were purchased by Fluka (Buchs, Switzerland); L-glutamic acid monosodium salt, adenosine 5′-triphosphate disodium salt, L-glutamic acid-γ-monohydroxamate, and cis-diammineplatinum(II) dichloride were supplied by Sigma Chemical Co. (St. Louis, USA).

Study Design

Albino, male Wistar rats (Harlan, Italy) weighing 200 ± 10 g (2 months old) were kept in steel cages, fed with standard diet (Nuova Zoofarm, Italy), drinking ad libitum, and with artificial dark-light cycle (12 hours). Rats (10 animals per group) were treated with single ip injection of 100 mg/kg bw (0.5 ml) of HCBD dissolved in corn oil, sc injection of 25 mg/kg bw (0.5 ml) of K₂Cr₂O₇ dissolved in saline (0.9%) or ip injection of 10 mg/kg bw (0.2 ml) of cis-Pt dissolved in buffered MSO. Three ancillary groups were prepared (10 animals per group) treated with corn oil (ip, 0.5 ml), saline (sc, 0.5 ml) or buffered MSO (ip, 0.2 ml), respectively. Five rats per group (controls and treated with chemicals) were sacrificed 24 and 48 hours after treatment under light ether anesthesia. Animal husbandry and treatment were performed according to Italian laws concerning animals for experimental use.

Renal Cortex GS Activity Assay

After the sacrifice, one kidney was quickly removed and placed in cold saline. The cortex and outer stripe of outer medulla were carefully separated from the medulla and a portion was homogenized in imidazole buffer 0.25 M, pH 7.2 (10% w/v) and centrifuged at 10,000g for 15 minutes at 4°C. The supernatant was then processed to determine GS activity according to Rowe et al. (1970) with slight modification (Trevisan et al., 1999). In brief, 100 μL of supernatant were incubated for 15 minutes at 37°C with 900 μL of a mixture composed by 0.25 M imidazole buffer pH 7.2 (200 μL), 0.4 M magnesium chloride (50 μL), 0.025 M 2-mercaptoethanol (100 μL), 0.25 M L-glutamic acid monosodium salt (substrate, 200 μL), 0.04 M adenosine 5′-triphosphate disodium salt (250 μL), and 1.0 M hydroxylamine hydrochloride (100 μL) buffered at pH 7.2 with NaOH pellets.

The reaction was stopped with 1.5 mL of a ferric chloride solution composed by 400 mL of 10% ferric chloride, 100 mL of 24% trichloroacetic acid, 50 mL of 6 N hydrochloric acid and 650 mL of distilled water. Ferric chloride solution was filtered 2–3 times with paper filter to clean one. Blank vials were prepared incubating for the same period 900 μL of the mixture and adding 100 μL of supernatant after ferric chloride solution at the end of the incubation. Vials were centrifuged 10 minutes at 1000g at room temperature and the samples were immediately read at 535 nm. Standardization was performed with L-glutamic acid-γ-monohydroxamate. Protein content was measured according to Miller (1959). Enzyme activity was expressed as nmoles of L-glutamic acid-γ-monohydroxamate formed per mg of protein (synthetase activity).

PAH Uptake Determination in Renal Cortical Slices

Renal cortical slices (100 ± 10 mg wet tissue, thickness ~300 μm) were prepared freehand with a scalpel according to Berndt (1976). The slices placed in a PAH-free medium composed by 97 mM NaCl, 40 mM KCl, and 0.74 mM CaCl₂, and sodium phosphate buffer 7.4 mM, pH 7.4, until all slices could be prepared. Slices were rinsed free of blood and enzymes released from damaged cells during the slicing process. After preparation, the slices were transferred into 25 mL Erlenmeyer flasks containing 4 mL of incubation medium enriched by the addition of 1 mM lactate and 75 μM PAH, and incubated at 25°C for 90 minutes in a Dubnoff metabolic shaker (100 cycles/min) under 100% oxygen to study organic anion accumulation.

After incubation, the slices were homogenized in 3% trichloroacetic acid (TCA, 10 mL/100 mg of tissue). A 1 mL aliquot of the incubation medium was treated with 4 mL of 3% TCA. After centrifugation (1000g per 10 minutes at room temperature), the supernatant was assayed for PAH according to Smith et al. (1945). The organic anion accumulation was expressed as the slice/medium (S/M) ratio, where the PAH concentration (μg/g tissue) of the slices was divided by the PAH concentration (μg/mL) of the medium.

Apparatus

Spectrophotometric analyses were performed with a UV-Vis spectrophotometer Perkin-Elmer Lambda 5 model.

Histopathological Examination (Light Microscopy)

A portion of the kidney from each rat was removed and fixed in 10% neutral phosphate-buffered formalin. The fixed tissue was processed in paraffin wax, cut (5 μ thickness) and stained with haematoxylin and eosin or PAS stains.

Statistical Analysis

Statistical evaluation of the results was by means of variance analysis (ANOVA) and Student’s t-test; significance was stated from p < 0.05.

Results

Renal Cortex GS Activity

GS activity in the kidney cortex appears significantly decreased (p < 0.001) 24 hours after treatment with a single ip injection of HCBD or cis-Pt. Further significant decrease (p < 0.002) occurred 48 hours after treatment with HCBD, but not with cis-Pt. Conversely, K₂Cr₂O₇ does not affect GS activity, even if a slight not significant decrease was observed at 24 hours, but not at 48 hours after treatment. Results are summarized in Figure 4.

PAH Acid Uptake in Renal Cortical Slices

PAH uptake is significantly reduced (p < 0.002) by K₂Cr₂O₇ 24 hours after a single sc injection, with a further significant decrease (p < 0.02) 48 hours after treatment. Conversely, HCBD and cis-Pt do not affect PAH uptake 24 hours after treatment, whereas a slight but significant (p < 0.02) decrease was observed 48 hours after treatment with HCBD only (Figure 4).

Histopathological Findings

Light microscopy of the kidney of rats treated with HCBD or cis-Pt shows acute necrosis of S₃ segment of the proximal tubules in the outer stripe of outer medulla, evident 24 (Figure 1) and 48 (data not shown) hours after single ip injection. The extent of the lesions was scored as diffuse in HCBD and cis-Pt-treated animals. No morphological changes were seen in S_1–S₂ segments. Conversely, the damage caused by treatment with K₂Cr₂O₇ involves S_1–S₂ segments of the proximal tubules only, with vacuolar degeneration 24 hours (Figure 2) and diffuse necrosis of epithelial tubular cells 48 hours after single sc injection (Figure 3). No morphological change was observed in the renal tubular structures from control rats treated with the used vehicles.

Discussion

The investigated xenobiotic substances exert renal toxic effects with different mechanisms, even if HCBD and cis-Pt show some similarity. According to Dekant and Vamvakas (1996), there are three main mechanisms of nephrotoxicity: (1) accumulation of xenobiotics and xenobiotic-induced accumulation of endogenous macromolecules in renal tissue, (2) renal accumulation of toxic metabolites biosynthesized in other organs or tissues, and (3) intrarenal bioactivation of xenobiotics to reactive metabolites.

Classically, HCBD, a halogenated alkene, undergoes GSH conjugation and further transformation to S-(pentachlorobutadienyl)-L-cysteine and is cleaved by cysteine conjugate β-lyase to a reactive, nephrotoxic thioketene (Dekant et al., 1991). Similarly, cis-Pt is metabolized to a nephrotoxin through a GSH-conjugate intermediate and excreted into the bile. The GGT-dependent pathway plays a key role (Hanigan et al., 2001), as GGT expression is required for the nephrotoxic effects, but diminishes the tumor toxicity of the drug, indicating that nephrotoxicity and tumor toxicity are via two distinct pathways (Hanigan et al., 1999). Finally, hexavalent chromium can react with hydrogen peroxide to hydroxyl radical, inducing DNA damage (Aiyar et al., 1991). The metal is accumulated in the renal cell by an anion transport system and subjected to intracellular reduction (Standeven and Wetterhahn, 1989).

Independently of the mechanism of nephrotoxicity, the end point is always cell death; therefore, it is important to identify the mechanism in addition to the site of action, in order to formulate a strategy of damage prevention.

To measure the entity of damage, biochemical and histopathological findings were evaluated in male Wistar rats treated with segment-specific nephrotoxicants such as K₂Cr₂O₇, HCBD, or cis-Pt.

The results show that 24 and 48 hours after treatment, HCBD and cis-Pt, chemicals that selectively affect the S₃ segment of the proximal tubule (Ishmael et al., 1982 and Dobyan et al., 1980, respectively), cause diffuse necrosis of this segment with a correspondent large decrease of GS activity in the kidney cortex. No impairment of PAH uptake was observed, even if slight but significant decrease was caused by HCBD 48 hours after treatment.

This confirms previous observations (Trevisan et al., 2001), and the possible explanation may be a slight involvement of the S₂ segment, which is difficult to disclose at light microscopy. Moreover, the last portion of convoluted tubule, comprised in the straight tubule, may be affected by HCBD but not by cis-Pt. These findings could explain the greater sensitivity of biochemical markers than found in histology. Conversely, K₂Cr₂O₇, known to damage S_1–S₂ segments (Biber et al., 1968; Evan and Dail, 1974), causes vacuolar degeneration of these segments 24 hours and diffuse necrosis 48 hours after treatment. Simultaneously, the metal salt produces high impairment of PAH uptake 24 hours and further decreased 48 hours after treatment, whereas GS activity was not affected.

These findings are relevant because they clearly illustrate a correspondence between segment-specific nephrotoxicant, biomarkers of segment-specific involvement, and histopathology. Currently, there are a number of different views concerning the localization of PAH and GS in kidney, especially for the former. Indeed, several authors have studied segment-specific localization of PAH transport, which does not appear to be concordantly localized in one specific tubule segment. The PAH transport, or organic anion transporter 1 (OAT1), is one of at least three basolateral membrane organic anion transport systems (named OAT2 and OAT3, other than apical membrane OAT4) in rat proximal tubule cells. In rabbit (McKinney, 1982) and mouse (Srimaroeng et al., 2005) it appeared localized in the S₂ segment, in contrast to Tune et al. (1969) who reported active secretion in both segments, the secretion rate being 3 times higher in the S₃ segment.

In rats, results are conflicting: Roch-Ramel and Weiner (1980) and Roch-Ramel et al. (1980) localized PAH transport in the S₂ and S₃ segments; Hook et al. (1982), by means of a segment-specific nephrotoxicants as HCBD suggested localization in S₃ segment. Recently, using the same techniques, localization was defined prevalently in S_1–S₂ segments (Trevisan et al., 2001). Finally, Kwak et al. (2005) reported that rat OAT1 mRNA is expressed in both the cortex and the outer medulla. This study further supports the segment-specific localization of PAH uptake

On the other hand, enzyme distribution along the nephron and, especially in the proximal tubule segments is well defined for a large number of tubular enzymes (Guder and Ross, 1984). Among tubular enzymes, GS is a huge mitochondrial enzyme (M.W. 620 kDa) playing a pivotal role in nitrogen metabolism, and it is responsible for the production of glutamine (Lund, 1970). The enzyme is generally accepted to be a marker of astroglial cells (Schousboe, 1982) where 65 is largely expressed. It is also present in a variety of tissues such as liver, kidney, spleen, adipose, testes, skeletal muscle, and stomach (Cooper, 1988). In the kidney, GS is localized in the early and late portion of the S₃ segment (Burch et al., 1978), as recently confirmed (Trevisan et al., 1999; Levillain et al., 2005). Cis-Pt uptake is known to occur in the proximal tubules of the kidney, via an organic cationic transporter in humans (Ciarimboli et al., 2005). In our study, via the GS activity evaluation, we clearly demonstrated that Cis-Pt is selectively toxic to the S₃ segment of the proximal tubules in rat.

In conclusion, the results clearly illustrate that (1) GS activity in the kidney cortex confirms its high specificity as marker of S₃ segment injury, (2) PAH uptake is prevalently active in S_1–S₂ segments, and it is a good ex vivo biomarker of nephrotoxicity caused by chemicals that affect these portions, (3) there is high relationship between segment-specific nephrotoxicants, histopathological findings and segment-specific biomarkers, and (4) cis-Pt toxicity is highly specific for the S₃ segment of the proximal convoluted tubule. This definitive evidence allows us to conclude a research program initiated 10 years ago, to determine the possibility of studying segment-specific nephrotoxicity by means of simple and inexpensive methods.