An improved blood substitute. In vivo evaluation of its renal effects.

Nephrotoxicity of free hemoglobin (Hb) based blood substitutes still awaits full elucidation. Previous reports attributed Hb passage through the renal glomeruli to a tendency of the Hb tetramer to dissociate into dimers. Now it has become more evident that the Hb tetramer is able to extravasate. It appears that the electrical charge of proteins plays an important role, with electronegativity and a low isoelectric point favoring intravascular persistence. This effect was utilized in the development of an improved blood substitute, comprising Hb reacted with o-ATP and o-adenosine, to form an intra- and intermolecularly cross linked product, which is reduced with glutathione. The modification reagents possess the desired pharmacologic activities and produce an increase in the electronegative charges on the Hb surface. All Hb polymers and chemically modified tetramers present in this solution have a uniform electronegative charge, with a pl of 6.1-6.2. In this present study, unmodified bovine Hb and an improved blood substitute were used for the replacement of 40% of the total blood volume in rats. The nephrotoxic effect was investigated by the determination of urinary output, glomerular filtration rate (GFR), fractional excretion of sodium (FENa), potassium (FEK), and chloride (FECl), urine/plasma osmolality ratio, and urine N-acetyl-beta-D-glucosaminidase (NAG) level. The free Hb and non heme protein contents in the urine were analyzed by using isoelectric focusing and size exclusion liquid chromatography methods. The results indicate that unmodified Hb is nephrotoxic. An initially elevated urinary output was followed by a significant oliguria associated with decreased GFR, FEK, and FECl and elevated FENa and NAG. Severe hemoglobinuria was associated with proteinuria. Analysis of urine from unmodified Hb treated rats revealed the presence of Hb tetramers. Histopathological examination of the kidneys showed cytoplasmic vacuolization of proximal tubular epithelium. On the contrary, an improved blood substitute did not produce any nephrotoxic reactions. It was found that this Hb solution did not pass through the renal glomerular barrier and was not present in urine samples. In conclusion, such a chemical and pharmacological alteration of Hb molecules reduced their interaction with renal glomeruli and suspended nephrotoxicity.

Corticosterone metabolism and membrane transport.

The mammalian kidney metabolizes virtually all of the steroid hormones. Corticosterone receptors have been found in the cortical collecting tubule, and at least four metabolites of the hormone have been identified in rat renal tissue and urine. The biologic activity of these metabolites is not completely known. In this study, we examined the functional effects of three of the metabolites of corticosterone on membrane transport in toad and turtle bladders; we also analyzed the oxidoreductase pathways for corticosterone metabolism. In the toad bladder, maximal water flow (vasopressin- and cyclic AMP-stimulated) was unaffected by corticosterone, 11-dehydro-20-dihydrocorticosterone (metabolite I) and 11-dehydrocorticosterone (metabolite IV); maximal water flow was significantly inhibited by 20-dihydrocorticosterone (metabolite II). Sodium transport in the toad bladder was stimulated by corticosterone, 11-dehydrocorticosterone and 20-dihydrocorticosterone. Analysis of the oxidoreductase pathways in this tissue revealed that most of the corticosterone was oxidized to 11-dehydrocorticosterone, a biologically active compound; 11-dehydrocorticosterone was further metabolized to 11-dehydro-20-dihydrocorticosterone, a biologically inactive compound. Only 6% of the parent compound was converted to 20-dihydrocorticosterone. In the turtle bladder, none of the metabolites tested altered hydrogen ion secretion over the time period studied; no significant biotransformation of corticosterone occurred in this tissue. As the metabolites of corticosterone found in toad bladder are the same as those identified in mammalian tissues, our studies suggest that some of them may be important modulators of sodium and water transport in the distal nephron. Our data further suggest that these compounds are likely not involved in the regulation of urinary acidification.