Polyanionic compounds as protectants against aminoglycoside-induced nephrotoxicity : biochemical and morphological studies

The doctoral work presented here, in our view, contributed not only to decipher the mechanism of protection afforded by polyaspartic acid but also to the in depth understanding of the basic mechanism of aminoglycoside-induced nephrotoxicity itself. It also resulted in certain incidental discoveries and opened new vistas for further research. These are summarised below.
1 Contribution to the understanding of the mechanism of protection afforded by polyaspartic acid
1.1. Points proved beyond doubt :
(a) Apart from polyaspartic acid, in vitro a variety of other polyanions could bind gentamicin and displace it from negatively-charged phospholipid layers. This displacement, at least for the polyanionic peptide tested, was associated with a restoration of the activity of gentamicin-inhibited lysosomal phospholipase A1.
(b) We have also disproved the hypothesis of Williams on the nature of the interference seen with polyaspartic acid in the membrane binding of gentamicin and questioned her theory that membrane binding is the key event in the toxicity of these drugs.
(c) We demonstrated that the protection afforded by different polyanionic peptides varied enormously depending upon the nature of the peptides? Thus, poly-L-Gly could not protect despite the fact that its in vitro behaviour was very similar to poly-L-Asp. The reason(s) for this could be many, including the sensitivity or resistance of the polyanions for lysosomal hydrolases (which we could demonstrate in vitro) or the different rates of transport and accumulation of these polyanions in lysosomes of renal cortex (that we are yet to investigate) or others.
(d) Co-oxydextran despite its other toxic effects reduced the gentamicin-induced accumulation of phospholipids in the renal cortex of rats, without decreasing the drug accumulation levels.
(e) Experiments with NH4Cl disclosed that raising the intralysosomal pH results in phospholipidosis and may aggravate the phosphlipidosis induced by gentamicin.
(f) Experiments with leupetin disclosed that gentamicin-induced phospholipidosis can be partly blocked by leupeptin by an unknown mechanism. And in the presence of a proteolytic inhibitor like leupeptin, poly-L-Glu exhibited protective effect against gentamicin-induced phospholipidosis.
1.2 Points not proved or assumed :
(a) We have not demonstrated in vivo that poly-L-Asp or other polyanions bind gentamicin and displace it from the negatively-charged phospholipids and thereby restore the activity of the gentamicin-inhibed lysosomal phospholipases. We assumed all these by extrapolating the in vitro findings made under the conditions existing in vivo.
(b) We have not demonstrated the in vivo rates of degradation of polyanionic peptides in lysosomes of rat kidney and assumed that their in vitro degradation rates by liver lysosomal extracts are comparable to those in vivo rates in kidney cortex.
(c) We have demonstrated that poly-L-Asp and poly-L-Glu are taken up and accumulated in the lysosomes of kidney cortex to the same extent. Based on the preliminary results and the similar biophysical properties of these peptides, we assumed that there may not be large differences in theirs rates of uptake and accumulation by kidney.
(d) We have not demonstrated that poly-L-Glu is not hydrolysed preferentially by plasma proteinases or brush-border peptidases. We simply bypassed these factors and reproduced the observations made in animal models in cell culture models.
1.3 Incidental discoveries
(a) The nephrotoxic potential of poly-D-Glu, a polyanionic peptide. Several aspects of this finding constitute interesting research problems for the future.
(b) Toxicity induced by co-oxydextran is also an interesting aspect that may probably unravel the various modes by which kidney tissue reacts to toxic insults.
(c) Raising the intralysosomal pH by NH4Cl induces phospholipidosis in cultured cells and aggravates the phospholipidosis induced by gentamicin.
(d) Leupeptin, an inhibitor of cathepsin B, stimulated the activity of acid sphingomyelinase in cultured fibroblasts and partly protects against the gentamicin-induced lysosomal phospholipidosis.
1.4 Prospectives :
(a) All the points that were not proved or assumed (section 1.2) constitute the research problems for the immediate future.
(b) Nephrotoxicity of poly-D-Glu can be pursued further to include histochemical or autoradiographic or subcellular fractionation studies.
(c) Work on leupeptin may open new vistas for research.
2 Contributions to the understanding of the aminoglycoside nephrotoxicity :
2.1 Strengthening of the ‘cascade’
The work presented here strengthened the cascade proposed by Laurent and Tulkens (1987). Conversely, the cascade can be a good model to explain the results obtained here on the mechanism of protection afforded by polyaspartic acid. Several points in this regard have already been discussed.
2.2 Disproving Williams’ hypothesis:
As already discussed the work also disproves the Williams’ hypothesis on the mechanism of protection afforded by polyaspartic acid. It also questions her theory that renal membrane binding is the key event in the toxicity of aminoglycosides.
2.3 Other theories of toxicity :
This work also puts the other theories on the neprhotoxicity of aminoglycosides in an inconvenient position, especially in view of the facts (i) that polyaspartic acid did not change the intracellular drug disposition as shown by Lallay and Tulkens (1989), (ii) polyaspartic acid, which blocked the development of lysosomal phospholipidosis, also prevented the cell necrosis, (iii) poly-L-glutamic acid which did not block the development of lysosomal phospholipidosis also could not prevent cell necrosis and (iv) in all these cases, whether protected or not, morphologically the alterations were limited to lysosomes only and no other subcellular oranelles exhibited abnormalities