Enhancement of gentamicin-induced nephrotoxicity by Mg deficiency in non-pregnant rats. Morphological, enzyme histochemical and immunohistochemical studies

Non-pregnant rats fed an Mg-deficient diet showed some degenerated and calcified proximal tubules in the inner region of the medullary rays accompanied by reduced or absent enzyme activities. After gentamicin treatment some damaged convoluted proximal tubules occurred. Enzyme histochemistry revealed decreased activities for brush border and lysosomal hydrolases; using immunohistochemistry lesions were detectable for the cytoskeletal proteins keratin and vimentin. Administration of gentamicin to Mg-deficient rats led to a further decrease of hydrolase activities in obviously intact proximal tubules and drastic structural and enzymatic defects as well as alterations of the cytoskeletal proteins in the convoluted and straight segments of other proximal tubules and to a lesser degree also in glomeruli and further portions of the tubular apparatus including the collecting ducts.     

Effect of phosphate deprivation on enzymes of the cyclic adenosine monophosphate metabolism in rat proximal convoluted and proximal straight tubules.

Phosphate deprivation causes a resistance to the phosphaturic effect of parathyroid hormone (PTH). The present study determined whether acute phosphate deprivation alters basal or stimulated activities of key enzymes of the cyclic adenosine monophosphate (cAMP) metabolism in microdissected proximal convoluted and proximal straight tubules, since blunted cAMP levels in these proximal subsegments might account for refractoriness to the effect of PTH on phosphate reabsorption in the proximal convoluted and proximal straight tubule segments. In the proximal convoluted tubules of rats fed a normal-phosphate diet (NPD), PTH increased the adenylate cyclase activity by tenfold. In the proximal convoluted tubule of rats fed a low-phosphate diet (LPD), PTH also increased the adenylate cyclase activity by tenfold. In addition, forskolin increased the adenylate cyclase activity to levels similar to PTH in the proximal convoluted tubule of rats fed NPD or LPD. In the proximal straight tubule of rats fed NPD, PTH resulted in an approximately fivefold increase in adenylate cyclase activity. In the proximal straight tubule of rats fed LPD, PTH resulted in a fourfold increase in adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was markedly decreased (46%) in the proximal straight tubule of phosphate-deprived rats. The cAMP-phosphodiesterase activity in the proximal convoluted tubule was significantly increased by 26% in phosphate-deprived rats. The cAMP-phosphodiesterase activities in the proximal straight tubules from rats fed NPD or LPD were similar. We conclude that distinct differences in key enzymes of cAMP metabolism exist in the proximal convoluted and proximal straight tubule subsegments. Further, phosphate deprivation affects the cAMP-phosphodiesterase and adenylate cyclase activities differently in these nephron subsegments.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ hybridization and immunohistochemistry of renal angiotensinogen in neonatal and adult rat kidneys

Recent evidence suggests that a local reninangiotensin system is operational in the kidney and that it mediates some of the actions of angiotensin II on renal tubules. In this study the ontogeny and renal distribution of the unique precursor to angiotensin II formation, angiotensinogen, was investigated in rats by use of immunohistochemistry, immuno-electron microscopy and non-isotopic hybridization histochemistry. At the light-microscopic level, intense staining for angiotensinogen was found in the proximal convoluted tubules of the cortex, with lighter staining in the straight proximal tubules of the outer stripe. The strongest immunostaining was found in the kidneys of neonatal rats, where glomerular mesangial cells and medullary vascular bundles were also immunopositive. The angiotensinogen content of the kidneys in late gestation embryos and neonates showed the presence of angiotensinogen by day E18 and a peak content in the neonate. Non-isotopic hybridization histochemistry with biotinylated oligodeoxynucleotide probes confirmed the presence of angiotensinogen mRNA expression in the proximal convoluted tubules of the renal cortex. Electron-microscopic immunohisto-chemistry showed staining of relatively few electron-dense structures close to the apical membrane of proximal convoluted tubule cells in the adult kidney. In the neonatal rat kidney, angiotensinogen immunostaining at the electron-microscopic level was found throughout the proximal tubule cells and was markedly stronger than that seen in adult kidney. The presence of angiotensinogen, from embryonic day 18, in the proximal tubules, mesangial cells and vasculature of the kidney suggests multiple potential sites of intrarenal angiotensin II generation with an ontogeny in late gestation.     

Relative contribution of V-H+ATPase and NA+/H+ exchanger to bicarbonate reabsorption in proximal convoluted tubules of old rats

With aging, the kidney develops a progressive deterioration of several structures and functions. Proximal tubular acidification is impaired in old rats with a decrease in the activity of brush border Na+/H+ exchange and a fall of H-ion flux measured with micropuncture experiments. In the present work we evaluate the contribution of 5-N-ethyl-n-isopropyl amiloride- (EIPA) and bafilomycin-sensitive bicarbonate flux (JHCO3-) in proximal convoluted tubules of young and aged rats. We performed micropuncture experiments inhibiting the Na+/H+ exchanger with EIPA (10(-4) M) and the V-H+ATPase with bafilomycin (10(-6) M). We used antibodies against the NHE3 isoform of the Na+/H+ exchanger and the subunit E of the V-H+ATPase for detecting by Western blot the abundance of these proteins in brush border membrane vesicles from proximal convoluted tubules of young and old rats. The abundance of NHE3 and the V-H+ATPase was similar in 18-month-old and 3-month-old rats. The bicarbonate flux in old rats was 30% lower than in young rats. EIPA reduced by 60% and bafilomycin by 30% in young rats; in contrast, EIPA reduced by approximately 40% and bafilomycin by approximately 50% in old rats. The inhibited by bafilomycin was the same in young and old rats: 0.62 nmol.cm-2.s-1 and 0.71 nmol.cm-2.s-1, respectively. However, the EIPA-sensitive fraction was larger in young than in old rats: 1.26 nmol.cm-2.s-1 vs. 0.85 nmol.cm-2.s-1, respectively. These results suggest that the component more affected in bicarbonate reabsorption of proximal convoluted tubules from aged rats is the Na+-H+ exchanger, probably a NHE isoform different from NHE3.     

OCCURRENCE OF PHAGOSOMES AND PHAGO-LYSOSOMES IN DIFFERENT SEGMENTS OF THE NEPHRON IN RELATION TO THE REABSORPTION, TRANSPORT, DIGESTION, AND EXTRUSION OF INTRAVENOUSLY INJECTED HORSERADISH PEROXIDASE

The size, number, and location of lysosomes, phagosomes, and phago-lysosomes in different segments of the proximal and distal tubules, in the collecting tubules, and in invading macrophages of the kidneys of rats were compared by staining lysosomes (acid phosphatase) red, and phagosomes (injected horseradish peroxidase) blue in separate sections, and by staining phago-lysosomes purple by successive application of the reactions for the two enzymes in the same sections. It was concluded from these observations that the absorption of the foreign protein from the lumen and its gradual digestion in large phago-lysosomes took place mainly in the cells of the proximal convoluted tubules of the outer cortex. Several segments of the proximal convoluted tubules were distinguished on the basis of differences in the size and location of the phago-lysosomes and the amounts of peroxidase ingested. The distal tubules showed, in addition to moderate numbers of phago-lysosomes, many small phagosomes in the apical and basal zones of the cells. Moderate numbers of phagosomes and phago-lysosomes were observed in the cells of the collecting tubules. Macrophages showing very large phago-lysosomes were seen in the peritubular capillaries of the medulla, after injection of peroxidase. When high doses of peroxidase were administered, enlarged phago-lysosomes, parts of which seemed to be extruded into the lumen, were formed in the terminal segments of the proximal convoluted tubules.     

Histochemical and electron microscopical changes in the kidney of pregnant rats at the time of partus (author's transl)]

In the convoluted proximal tubules of the kidney of pregnant rats alkaline phosphatase activity decreases from 21 days gestation till the 2nd day after birth. During the same time the reaction product of the histochemical succinate dehydrogenase assay exhibits an atypical granular character in some of the proximal tubules, which sometimes additionally contain sudanophilic structures. Electron microscopy reveals dilated mitochondria, an increased number of autophagosomes and big lipid vacuoles of low density.     

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat.

The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60 mg kg-1 day-1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-beta-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of 1H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by 1H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.     

Tubular stress proteins and nitric oxide synthase expression in rat kidney exposed to mercuric chloride and melatonin.

Stress proteins such as HSP70 members (HSP72 and GRP75) and metallothionein (MT) protect the kidney against oxidative damage and harmful metals, whereas inducible nitric oxide synthase (iNOS) regulates tubular functions. A single dose of mercuric chloride (HgCl(2)) can cause acute renal failure in rats, its main target being the proximal tubule. Oxidative damage has been proposed as one of its pathogenic mechanisms. In this study we tested whether melatonin (MEL), a powerful antioxidant compound, is effective against HgCl(2) nephrotoxicity. Rats were treated with saline, HgCl(2) (3.5 mg/kg), MEL (5 mg/kg), and MEL + HgCl(2) and examined after 24 hr for HSP72, GRP75, MT, and iNOS by immunohistochemistry and immunoblotting. Tubular effects of the treatment were then characterized by ultrastructure. In the HgCl(2) group, all markers were overexpressed in convoluted proximal tubules and sometimes in distal tubules. In the MEL + HgCl(2) group, GRP75 and iNOS decreased in convoluted and straight proximal tubules, whereas HSP72 and MT persisted more than the saline and MEL-only groups. Tubular damage and mitochondrial morphometry were improved by MEL pretreatment. In conclusion, the beneficial effect of MEL against HgCl(2) nephrotoxicity was outlined morphologically and by the reduction of the tubular expression of stress proteins and iNOS. These markers could represent sensitive recovery index against mercury damage.     

Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.

Four monoclonal antibodies (MAb) specific for the L-type isoenzyme of rat pyruvate kinase (L-PK) were produced and characterized. They detect at least two different epitopes of the isoenzyme, as shown in interference binding assay and Western blot analysis after peptide mapping. The MAb were used in immunohistology to demonstrate the L-PK isoenzyme in paraffin-embedded normal rat tissues. L-PK was found only in hepatocytes, kidney proximal tubules, islet cells of pancreas, and epithelial cells of the villi of small intestine. The content of L-PK in hepatocytes was often lower in the periportal areas as compared with the periveneous zone. In kidneys a clearcut difference in L-PK content and distribution existed between male and female rats. Male animals possessed more L-PK in the kidney cortex than females. The L-PK content in the inner cortical zone (straight proximal tubules) was higher than in the outer cortical zone (convoluted proximal tubules) in kidneys of males. In contrast, female rats displayed less L-PK in the inner than in the outer cortical zone of the kidneys. Only some of them exhibited the same amount of the isoenzyme in both parts of the kidney proximal tubules.     

Na-K]ATPase activity in proximal and distal tubules of the rat kidney: modification and application of a quantitative cytochemical technique

A modified cytochemical assay for [Na-K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na-K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na-K]ATPase activity in several distal sites. The concentration of ouabain necessary for maximal inhibition of activity was 3.0 mM and half-maximal inhibition was obtained in all regions with 30-100 microM ouabain. In distal sites, [Na-K]ATPase formed a higher proportion of total ATPase activity (60-80 per cent) than in proximal sites (20-40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na-K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na-K]ATPase per length of tubule. The highest activities over the basal basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin greater than MALout greater than PCT greater than PD greater than PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na-K]ATPase activity in the major functional regions of the rat nephron.     

Effects of subacute 3-monochloropropane-1,2-diol treatment on the kidney of male albino rats

3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food contaminant. Although the kidney is thought to be a target organ for 3-MCPD toxicity, nephrotoxic structural changes are relatively unstudied. We investigated the renal alterations caused by 3-MCPD in male albino rats. 3-MCPD was administered orally, at a dose of 60 mg/kg for 7 days. 3-MCPD caused significant elevation of serum creatinine and urea levels together with hydropic degeneration, necrosis and shedding of the cells of the proximal convoluted tubules, urinary casts in the distal convoluted tubules and interstitial inflammatory cell infiltration. Administration of 3-MCPD for a period as short as 7 days causes acute renal failure in male albino rats.     

Enzyme histochemical and histological changes in the adult rat kidney after prenatal gentamicin exposure

Treatment of rats between day 15 and 20 of gestation with gentamicin caused histological as well as enzyme histochemical lesions in the kidney of the one year old offspring (F1 generation). Other organs were not significantly affected. Primarily in the female kidney dilated convoluted proximal tubulus with reduced or absent staining for brush border and lysosomal proteases, phosphatases and glycosidases and mitochondrial dehydrogenases were observed. In comparison, glomeruli were less frequently damaged and contained fewer capillary loops or irregularly arranged tissue elements with lowered or no activities for plasma membrane-associated proteases as well as specific and non-specific phosphatases. In addition, the activities of proteases and phosphatases in cortical and medullary endothelial cells of capillaries were reduced or even abolished in the kidney of the female and male F1 generation after treatment of their mothers with gentamicin.     

Expression and function of arginine-producing and consuming-enzymes in the kidney

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.     

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat

The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60 mg kg^-1 day^-1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-β-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of ¹H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by ¹H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.     

Proximal tubular atrophy: Qualitative and quantitative structural changes in chronic obstructive nephropathy in the pig

Summary Kidneys of pigs with various degrees of induced chronic obstructive nephropathy were studied by light- and electron microscopy to assess the structural changes of proximal convoluted tubules with increasing degrees of atrophy. A particular aim was to evaluate the quantitative relationship between proximal tubular and interstitial changes in early tubular atrophy. The kidneys were subjected to varying degrees of ureteral obstruction and were fixed by in vivo vascular perfusion. Quantitative (morphometric) analyses were carried out on montages of electron micrographs representing randomly selected cortical areas and cross sections of individual proximal convoluted tubules. The results demonstrated that ureteral obstruction was followed by significant reductions in proximal tubular epithelium, in volume of proximal tubular mitochondria and in surface area of proximal tubular basolateral membranes. These changes were present even in the absence of any demonstrable increase in cortical interstitium or alterations in the relationships between proximal tubules and peritubular capillaries. With increase in the volume of cortical interstitium the proximal tubules were further simplified in ultra-structure with a reduced number of interdigitating lateral cell processes. Concomitantly there were significant quantitative changes in the spatial associations between tubules and capillaries due to increase in tubulo-capillary distances. The present study shows that ultrastructural changes in proximal tubules during early atrophy precede the volume increase in cortical interstitium associated with chronic obstructive nephropathy. It is suggested that the early tubular changes are due to decreased functional loads, whereas the further progression of tubular atrophy may be a result of impaired nourishment of the tubular cells due to increased interstitial tissue and altered relationships between tubules and capillaries.This work was supported by grant no 12-0727 from the Danish Medical Research Council     

Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.     

Lysosomal enzymes are decreased in the kidney of diabetic rats

The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of β-glucuronidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.     

鸡肾发生的组织学观察

采用组织学方法和电镜技术,对9个不同发育时期的鸡(Callus domestiaus)胚胎进行了观察.通过对鸡胚胎肾组织发生过程的观察,探讨鸡胚中肾的发生与退化,后肾的发生、分化规律和特点.结果表明,孵育到第16期在中肾前端附近出现一些中肾小泡.孵育到第18期形成中肾小管.孵育到第26期,中肾小管的盲端内陷,原始的肾小囊和肾血管球形成,中肾小管显著伸长并迂回曲折.孵育到第33～37期,体前后部中肾组织均已形成完整的肾单位.第37～46期体前部至后部的中肾组织依次退化.孵育到第26期从泄殖腔附近发出的输尿管芽向生后肾组织侵入生长,生后.肾组织产生许多生后肾小泡.第33期出现肾小囊和肾小管,肾小管伸长并发生折叠,出现集合小管、近端小管和远端小管的形态分化.第37～46期肾小体逐渐发育成熟,肾小管继续分化出现细段.鸡的中肾具有排泄功能.鸡后肾的发生与分化存在明显的时间差异.肾单位的分化中,同一胚龄肾组织内可存在不同发育阶段的肾小体,集合小管分化较早,诱导近端小管和远端小管分化,细段分化较迟.     

Histochemical studies on the uptake of horseradish peroxidase by rat kidney slices

When rat kidney slices were incubated in the presence of horseradish peroxidase, there was an energy-dependent uptake of the protein by the cells of the kidney tubules. The uptake was greatest in the proximal convoluted tubules and in the thick ascending limbs of the loops of Henle; it was abolished by cold, anoxia, 2,4-dinitrophenol, and fluoroacetate, and was more readily depressed by unfavorable metabolic conditions in the proximal convoluted tubules than in the thick ascending limbs. Protein uptake was inhibited when the kidney slices were incubated in electrolyte-free media. In sodium chloride solutions, uptake was reduced as sodium was progressively replaced by choline, and ouabain inhibited uptake in the proximal convoluted tubules, but not in the thick ascending limbs. To a limited extent, lithium could replace sodium in the incubation medium with no depression of peroxidase uptake. These results suggest that a sodium-stimulated, ouabain-sensitive ATPase may be involved in the uptake of protein by cells of the kidney tubule. The intracellular transport of peroxidase in cells of the proximal convoluted tubules was abolished by cold, anoxia, and 2,4-dinitrophenol, but it was not affected by concentrations of ouabain which inhibited the uptake of the protein.     

Metabonomics with 1H-NMR spectroscopy and liquid chromatography-mass spectrometry applied to the investigation of metabolic changes caused by gentamicin-induced nephrotoxicity in the rat

Abstract

The model nephrotoxin gentamicin was administered to male Wistar-derived rats daily, for 7 days, at 60?mg?kg^?1?day^?1, subcutaneously, twice daily. Conventional clinical chemistry urinalysis showed a significant increase in N-acetyl-β-D-glucosaminidase (NAG) activity from day 3. At necropsy on day 9, clear histological damage to the kidney was noted with all animals showing a generally severe nephropathy primarily focused on the proximal convoluted tubules. The urinary excretion pattern of endogenous metabolites over the time course of the study was studied using a combination of ¹H-NMR spectroscopy and HPLC-TOF-MS/MS using electrospray ionization (ESI). Changes in the pattern of endogenous metabolites as a result of daily administration of gentamicin were readily detected by both techniques with significant perturbations of the urinary profile observed from day 7 onwards. The findings by ¹H-NMR included raised glucose and reduced trimethylamine N-oxide (TMAO). Changes in metabonomic profiles were observed by HPLC-MS in both positive and negative ESI. The MS data showed reduced xanthurenic acid and kynurenic acid, whilst neutral loss experiments also revealed a changed pattern of sulphate conjugation on gentamicin administration.