Autoradiographic Localization of [ ³ H]Gentamicin in the Proximal Renal Tubules of Mice

Abstract

The site of localization of [3H]gentamicin within mouse kidney is shown to be the proximal renal tubule by coincidence of the radioactivity, as visualized by autoradiography, and the mucopolysaccharide-rich microvilli characteristic of proximal convoluted tubules, as visualized by histochemical staining.The extensive accumulation and avid retention of aminoglycoside antibiotics by the kidneys of both experimental animals (4, 9, 13) and humans (1, 3) may be related to the nephrotoxicity associated with these drugs. If these processes are related, then an understanding of the precise anatomic localization of aminoglycoside accumulation and retention may be fundamental to the prevention and treatment ofaminoglycosideinduced nephrotoxicity. We have investigated the intrarenal localization of the aminoglycoside gentamicin in the mouse kidney.Three ,uCi/mg with gentamicin sulfate from Sigma) in 0.2% ethanol. Three control male mice were injected with 5 ml of 0.2% ethanol per kg, and both kidneys from each of these mice were prepared and examined in the same manner as for the treated mice. Twenty-four hours later, both kidneys from each mouse were removed under pentobarbital anesthesia, and sections of the medial parts of the kidneys (both axes were sampled in different kidneys) were mounted on a copper microtome chuck and frozen in liquid nitrogen slush. Four-micrometer sections of these tissues were either thaw-mounted or drymounted onto emulsion-coated (Kodak NTB-3) slides and stored for approximately 1 month. This autoradiographic procedure has been developed to prevent or minimize the diffusion of small molecules from tissue sites (11). After development of the emulsion, the tissues were stained either with pyronine-y or by the periodic acid-Schiff technique, which visualizes mucopolysaccharide-rich microvilli characteristic of cells in the proximal convoluted tubules.A representative photograph of the resultant autoradiograph and histochemical staining is shown in Fig. 1. All kidney preparations from treated animals showed similar findings. Dense clusters of autoradiographic grains (ARG) were restricted to cortical regions of the kidney, where they were associated with most, but not all, tubule profiles. The ARG were strikingly absent over glomeruli and over structures in the medulla including the thin limbs, collecting ducts, and blood vessels. A very slight enrichment of ARG was observed over the straight parts of the tubules below the cortical layers containing the glomeruli. In periodic acid-stained sections, the ARG were found exclusively over tubules that stained positively for mucopolysaccharides near the lumen. Furthermore, the convoluted tubules with the dense cluster of ARG had larger cells when compared with the tubules with no ARG. Control slides prepared for positive and negative chemography showed no spurious generation of or fading of latent images. The same localization has also been visualized in rat kidney preparations (data not shown).From these data it is concluded that [3H]gentamicin is very ...

Maturation of Renal Tubular Transport of Gentamicin

“…Although renal gentamicin elimination is primarily by filtration and its cellular uptake predominantly a luminal one [3], a possible transtubular influx of gentamicin contributing to its renal clearance should also be considered. The site of localization of gentamicin in the kidney has been shown to be primarily in the proximal tubular cell whereas uptake by the distal convo luted tubule or medullary tissue were low [8]. In our study we observed accumulation of gentamicin in cortical tissue.…”

Autoradiographic study of tobramycin uptake by proximal and distal tubules of normal and pyelonephritic rats

“…DISCUSSION In recent years the capacity of aminoglycosides to accumulate in the renal cortex and their persistence have been intensely studied. To understand the mechanism of aminoglycoside accumulation, several investigators (11,18,20) have used autoradiography in an attempt to localize the intrarenal site of aminoglycoside accumulation. These studies have shown that the antibiotic enters the proximal tubule cells by pinocytosis at the luminal cell membrane into endocytotic vacuoles and fuses with lysosomes (18,20).…”

“…The present study shows that the transport of tobramycin within the infected nephron is disturbed. These data might shed some light on the influence of infection on the intrarenal pharmacology of aminoglycosides.To understand the mechanism of cellular accumulation as well as the cellular transport of aminoglycosides, several investigators have studied the intrarenal disposition of gentamicin by autoradiography (11,18,20). Coupled with electron microscopy, this technique has the unique advantage of allowing recognition of the presence of a labeled compound within tissue and individual cell organelles.…”

Disposition of quinine in rats with induced renal failure

“…Glycerolinduced renal failure is haemodynamically mediated by renal ischaemia, while mercuric chloride and gentamicin induce renal failure as a result of a direct nephrotoxic effect on cellular integrity [14][15][16]. Although renal impairment induced by gentamicin and mercuric chloride are aetiologically the same, gentamicin produces a nephrotoxic effect only on the proximal convoluted tubule [16][17][18], while mercuric chloride produces renal failure due to widespread necrosis in the pars convoluta and cortical and medullary pars recta [19 20].…”