Redistribution of Epidermal Growth-factor Immunoreactivity in Renal Tissue After Nephrotoxin-induced Tubular Injury

Tubular necrosis elicits a process of renal tissue repair characterized by an increase of cell turnover in tubular epithelium. The present study was undertaken to examine the distribution of epidermal growth factor (EGF) and/or of its larger precursor proEGF in the kidney undergoing tubular regeneration. Sprague-Dawley rats were exposed to various drugs (aminoglycosides or platinum-based anticancer agents) known to induce tubular necrosis. The proliferative response resulting from renal tissue damage was measured by the incorporation of [H-3]thymidine into DNA of renal cells. EGF immunoreactivity was evidenced by immunocytochemical staining, using anti-EGF antibody and immunogold-silver staining. Concomitantly with the increase of cell proliferation resulting from tubular injury, a redistribution of EGF immunoreactivity was observed in renal tissue (from the inner stripe of outer medulla towards renal cortex). Amazingly, EGF was detected in proximal tubules of nephrotoxin-treated rats whereas, in the kidneys of control animals, it was almost exclusively found in distal tubules and collecting ducts. Insofar as the administration of exogenous EGF has recently been shown to enhance renal tubular regeneration after ischaemic injury [Humes et al: J Clin Invest 1989; 84:1757-1761], our observations lend further support to the concept that EGF might be involved in renal tissue repair

Mechanism of Protection Afforded By Polyaspartic Acid Against Gentamicin-induced Phospholipidosis .1. Polyaspartic Acid Binds Gentamicin and Displaces It From Negatively Charged Phospholipid Layers Invitro

Coadministration of polyaspartic acid protects rats against aminoglycoside-induced nephrotoxicity, with respect to functional and pathological changes as well as to early signs of renal alterations (lysosomal phospholipidosis of proximal tubular cells, increased proliferation of proximal tubular and peritubular cells), without reduction, but actually by increasing the drug cortical content (Williams et al., J. Pharmacol. Exp. Ther. 237: 919, 1986; Gilbert et al., J. Infect. Dis. 159: 945, 1989; Beauchamp et al., 1990). Because aminoglycoside accumulation in kidney cortex involves their segregation in lysosomes, we have examined the possibility of formation of intracellular aminoglycoside-polyaspartic acid complexes that would render the drug less toxic. We found that in vitro polyaspartic acid (MW 9-15,000) 1) binds gentamicin with an optimum at acidic pH (5.4), 2) displaces it from negatively charged liposomes and 3) restores the activity of gentamicin-inhibited lysosomal phospholipase A1 toward phosphatidylcholine included in negatively charged liposomes. In parallel, we also observed that at pH 7.0, polyaspartic acid binds and displaces gentamicin from purified brush-border membrane vesicles, causing an apparent decrease of affinity of gentamicin for these membranes, which was falsely interpreted by Williams et al., J. Pharmacol. Exp. Ther. 237: 919, 1986 as "competition for a common membrane binding site." Assuming that, after its administration in vivo, polyaspartic acid gains access to lysosomes of proximal tubular cells, as many low molecular weight proteins and polypeptides do, our results suggest that protection against gentamicin-induced nephrotoxicity is obtained by the binding of the aminoglycoside to the polyanion in lysosomes, preventing thereby the development of phospholipidosis and therefore interfering with the cascade of events leading from drug accumulation to nephrotoxicity

Mechanism of Protection Afforded By Polyaspartic Acid Against Gentamicin-induced Phospholipidosis .2. Comparative Invitro and Invivo Studies With Poly-l-aspartic, Poly-l-glutamic and Poly-d-glutamic Acids

Poly-L-aspartic acid (poly-L-Asp) protects rats against gentamicin (GM)-induced nephrotoxicity (functional and pathological changes) and early cortical alterations (phospholipidosis and increase in cell turnover) without decreasing, but actually increasing, the renal accumulation of the drug. We suggested that this protection occurs through the complexation of GM by poly-L-Asp, after their pinocytosis and accumulation in the lysosomes of the renal cortex (Kishore et al., J. Pharmacol. Exp. Ther. 867-874, 1990). Here we examine further our proposal by comparatively assessing poly-L-Asp (as provided by the Sigma Chemical Co., St. Louis, MO; MW 9-11,000), with two other polyanionic peptides, viz, poly-L-glutamic (poly-L-Glu; MW 14,300) and poly-D-glutamic (poly-D-Glu; MW 20,000) acids obtained from the same supplier. In vitro, all three polyanions showed a similar capacity to bind GM, to displace it from anionic phospholipids at acid pH and thereby to decrease the inhibitory potency of GM toward lysosomal phospholipase A1. In vivo, however, only poly-L-Asp and poly-D-Glu were able to prevent the development of GM-induced renal lysosomal phospholipidosis as assessed by key biochemical criteria (increase in lipid phosphorus and decrease of acid sphingomyelinase activity) and by examination of the lysosomal content in the electron microscope (accumulation of myeloid bodies). Based on these criteria, poly-L-Glu completely failed to protect. In vitro, poly-L-Glu was 13- to 17-fold more susceptible to hydrolysis by liver lysosomal extracts at pH 5.4 after 48 hr incubation, as compared to poly-L-Asp and poly-D-Glu, respectively. Assuming that all three polyanions tested are transported and accumulated in lysosomes of renal cortex to the same extent and that their respective rates of hydrolysis therein compare to that measured in vitro, these results suggest that stability of polyanions in lysosomes is an essential requisite for protection against GM-induced phospholipidosis and thus strengthens our earlier proposal that the site of action of poly-L-Asp must be in lysosomes. Although protecting from phospholipidosis, poly-D-Glu, however, caused a so far undescribed lysosomal storage disorder consisting of the accumulation of osmiophilic, nonlamellar material. This study, therefore, also demonstrates that not all polyanions resistant to lysosomal enzymes can be used as nephroprotectants, inasmuch as these, as is the case for poly-D-Glu, may cause renal alterations on their own

Comparative assessment of poly-L-aspartic and poly-L-glutamic acids as protectants against gentamicin-induced renal lysosomal phospholipidosis, phospholipiduria and cell proliferation in rats.

Coadministration of poly-L-aspartic acid (poly-L-Asp) protects rats against all measured signs of aminoglycoside nephrotoxicity. Based on in vitro and acute in vivo models, previously we hypothesized that poly-L-Asp protects by forming complexes with the drug in lysomes of proximal tubular cells. However, another closely related peptide, poly-L-glutamic acid (poly-L-Glu), could not protect against gentamicin-induced phospholipidosis and nephrotoxicity, presumably because it is susceptible to rapid hydrolysis in sysosomes in vivo. The present study expands the in vivo comparison between these two polyanions to a subacute model of rats and examines in detail the influence of these polymers on the qualitative and quantitative morphological alterations of lysosomes, phospholipiduria and proliferation of cortical cells induced by gentamicin. Our results not only demonstrated that despite a significantly higher drug cortical accumulation, the coadministration of poly-L-Asp almost completely protects against the development of all these early renal alteration but also pointed to the possibility of a mild, albeit apparently nonlethal, lysosomal thesaurismosis to develop under these conditions. In contrast, poly-L-Glu could not protect against these early renal alterations, though cortical drug accumulation was not significantly higher; however, it induced a conspicuous proliferation of peritubular interstitial cells. Therefore, the present work, taken together with the earlier results of ours as well as that of others, tends to strengthen the hypothesis that the site of action of poly-L-Asp must be in lysosomes, which are also the organelles that sequester and accumulate the drug

Inhibition of aminoglycoside-induced nephrotoxicity in rats by polyanionic peptides.

In the present study, we compared poly-L-Asp with poly-L-Glu and poly-D-Glu in vitro and in vivo for their ability to inhibit the GM-induced nephrotoxicity. In vitro, all three polyanions (i) bound GM over a wide range of pH; (ii) displaced GM previously bound to negatively charged phospholipid bilayers at acid pH (i.e. under the conditions prevailing in lysosomes in vivo), and thereby (iii) decreased the inhibitory potency of GM towards lysosomal phospholipase A1. Thus, one was tempted to predict that all three polyanions would have the potential of protecting against AG-induced nephrotoxicity. However, when co-administered to rats with GM, poly-L-Asp and poly-D-Glu completely suppressed the development of lysosomal phospholipidosis, as assessed by biochemical criteria and increased drug accumulation, whereas poly-L-Glu did not offer such protection despite a relatively lower increase in drug accumulation levels. Histoautoradiography also confirmed that poly-L-Asp, but not poly-L-Glu, was a nephroprotectant against the tissue proliferative response induced by GM. Morphologically, poly-L-Asp almost completely and poly-D-Glu totally prevented the accumulation of myeloid bodies in lysosomes. In vitro incubation in the presence of purified lysosomal extracts revealed marked differences in the hydrolysis rate of these peptides (poly-D-Glu:poly-L-Asp:poly-L-Glu = 1:1.2:16.9). Assuming that all three polyanionic peptides are transported and accumulated in lysosomes to the same extent, these results not only suggest that their stability in lysosomes is an essential requisite for protection against lysosomal phospholipidosis, but also strengthen our hypothesis that the site of action of poly-L-Asp is inside the lysosomes but not at the level of the renal membranes. In addition, poly-D-Glu alone or combined with GM induced another type of morphological lesion, not related to AG-induced nephrotoxicity which, to our knowledge, has not yet been described

Distribution of Epidermal Growth-factor in the Kidneys of Rats Exposed To Amikacin

The distribution of epidermal growth factor (EGF) was examined by immunocytochemistry in the kidneys of rats exposed to amikacin, an aminoglycoside antibiotic causing tubular necrosis at high dose. Five-animal groups were treated for 4 or 10 days with amikacin at daily doses of 15, 40, 80 or 200 mg/kg. The drug was delivered i.p. twice a day. One hour before termination, each rat received an i.p. injection of [H-3] thymidine to evaluate DNA synthesis in renal tissue. After sacrifice, the kidneys were processed for morphological (semithin and paraffin sections) and biochemical analysis (measurement of DNA synthesis by [H-3] thymidine incorporation in vivo). Amikacin induced in proximal tubules a dose-related lysosomal phospholipidosis, which was assessed by the morphometric evaluation of altered lysosomes ("myeloid bodies") on semithin section. However, frank evidence of acute tubular necrosis was only observed in rats receiving amikacin at a daily dose of 200 mg/kg. Concomitantly with the development of tubular necrosis, there was a rise in the rate of cell turnover, reflected by an increase of DNA synthesis in renal tissue. This sign of tubular regeneration was accompanied by a redistribution of EGF immunoreactivity, as revealed by immunocytochemical staining. Within renal cortex of control rats, EGF immunoreactivity predominantly appeared in distal tubules and collecting ducts (97% of examined tubular sections). In contrast, in treated animals where the renal cortex displayed evidence of tubular necrosis/regeneration, EGF immunoreactivity was frequently associated with proximal tubules (more than 30% of examined tubular sections, as compared to 3% in controls). This change in the topography of EGF immunoreactivity suggests that the growth factor might be involved in the process of tissue repair consecutive to drug-induced tubular necrosis