Novel mechanisms of cytoprotection in renal proximal tubules

The amino acid glycine prevents cell death in numerous models of hypoxia and toxicant-induced injury. Strychnine, a neuronal glycine receptor antagonist, has recently been shown to mimic the cytoprotective actions of glycine in renal proximal tubules (RPT). The purpose of this dissertation was to characterize glycine and strychnine cytoprotection in rabbit RPT.

Glycine and strychnine cytoprotection was reversible and occurred in the late stages of cell injury. Glycine and strychnine (1 mM) prevented cell death induced by the diverse nephrotoxicants tetrafluoroethyl-L-cysteine, bromohydroquinone, mercuric chloride, sodium cyanide, and t-butylhydroperoxide.

Binding studies with $\sp3$H-strychnine revealed that $\sp3$H-strychnine bound in a saturable and reversible manner to the plasma membrane of RPT (IC$\sb{50}$ = 0.8mM). $\sp3$H-strychnine was displaced by unlabelled strychnine, but not by cytoprotective amino acids. In the central nervous system, strychnine and glycine modulate chloride flux via the glycine receptor, therefore, the effects of the toxicant exposure on chloride flux were determined. Chloride influx occurred in the late stages of cell injury induced by a variety of nephrotoxicants. Chloride influx preceded swelling and cell lysis, and was specifically inhibited by glycine and strychnine.

Immunoblot and immunofluorescence analyses identified the $\beta$-subunit of the neuronal glycine receptor and the associated protein gephyrin along the basolateral portion of RPT. The $\alpha$-subunit was not identified. The absence of the $\alpha$-subunit prompted a search for other potential subunits. GABA$\sb{\rm A}$ receptor agonists were found to be cytoprotective to RPT. GABA, THIP HCl, and muscimol prevented toxicant-induced cell death, with muscimol being the most potent (IC$\sb{50}$ of 50 $\mu$M). Immunoblot and immunofluorescence analyses revealed that the $\beta\sb3$-subunit, but not the $\beta\sb2$- or $\alpha\sb1$-subunit of the GABA$\sb{\rm A}$ receptor was present in RPT. Further the GABA$\sb{\rm A}$, $\beta\sb3$ subunit shared the same distribution as the $\beta$-subunit of the glycine receptor and the associated protein gephyrin.

Therefore, it is proposed that RPT contain a novel cytoprotective receptor that is composed of neuronal glycine and GABA$\sb{\rm A}$ receptor subunits. Further, this receptor modulates chloride influx in the late stages of cell injury and is responsible for strychnine and glycine cytoprotection.