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The glomerulus, the filtering part of the nephron, consists of a network of capillaries through which the blood flows under the influence of hydraulic forces generated by contractions of the heart. The nephron consists of a renal, or Malpighian, corpuscle, a proximal tubule, a loop of Henle, and a distal tubule. The interlobular arteries of the kidney branch extensively to form afferent arterioles which supply each glomerulus. The glomerulus nearly fills Bowman’s capsule. The specialized cells that constitute the visceral layer, called podocytes, are stellar in shape and possess cytoplasmic extensions that originate from the nucleated portion of the cell body. The Glomerular Basement Membrane is an extracellular matrix interposed between the epithelial foot processes and endothelium. The location and properties of the glomerular mesangial cells confer a central role for these cells in the regulation of renal function. Glomerular filtration is the initial step in urine formation.

We examined the influence of chronic treatment with ANG-(1–7) on development of hypertension and end-organ damage in spontaneously hypertensive rats (SHR) chronically treated with the nitric oxide synthesis inhibitor l-NAME (SHR-l-NAME). l-NAME administered orally (80 mg/l) for 4 wk significantly elevated mean arterial pressure (MAP) compared with SHR controls drinking regular water (269 ± 10 vs. 196 ± 6 mmHg). ANG-(1–7) (24 μg·kg−1·h−1) or captopril (300 mg/l) significantly attenuated the elevation in MAP due to l-NAME (213 ± 7 and 228 ± 8 mmHg, respectively), and ANG-(1–7) + captopril completely reversed the l-NAME-dependent increase in MAP (193 ± 5 mmHg). l-NAME-induced increases in urinary protein were significantly lower in ANG-(1–7)-treated animals (226 ± 6 vs. 145 ± 12 mg/day). Captopril was more effective (96 ± 12 mg/day), and there was no additional effect of captopril + ANG-(1–7) (87 ± 5 mg/day). The abnormal vascular responsiveness to endothelin-1, carbachol, and sodium nitroprusside in perfused mesenteric vascular bed of SHR-l-NAME was improved by ANG-(1–7) or captopril, with no additive effect of ANG-(1–7) + captopril. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1–7)- or captopril-treated SHR-l-NAME, with additive effects of combined treatment. The beneficial effects of ANG-(1–7) on MAP and cardiac function were inhibited when indomethacin was administered with ANG-(1–7), but indomethacin did not reverse the protective effects on proteinuria or vascular reactivity. The protective effects of the ANG-(1–7) analog AVE-0991 were qualitatively comparable to those of ANG-(1–7) but were not improved over those of captopril alone. Thus, during reduced nitric oxide availability, ANG-(1–7) attenuates development of severe hypertension and end-organ damage; prostaglandins participate in the MAP-lowering and cardioprotective effects of ANG-(1–7); and additive effects of captopril + ANG-(1–7) on MAP, but not proteinuria or endothelial function, suggest common, as well as different, mechanisms of action for the two treatments. Together, the results provide further evidence of a role for ANG-(1–7) in protective effects of angiotensin-converting enzyme inhibition and suggest dissociation of factors influencing MAP and those influencing end-organ damage.

Summary 1 The purpose of this study was to examine the effect of inhibition of the formation of cytochrome P450 metabolites of arachidonic acid with 1-aminobenzotriazole (ABT) on the development of hypertension and end-organ damage in spontaneously hypertensive rats (SHR) chronically treated with nitric oxide synthesis inhibitor l-NAME (SHR-l-NAME). 2 Administration of l-NAME in drinking water (80 mg l−1) to SHR for 3 weeks significantly elevated mean arterial blood pressure (MABP) (223 ± 4 mmHg) as compared to SHR controls drinking regular water (165 ± 3 mmHg). The administration of ABT (50 mg kg−1 i.p. alt diem) for 6 days significantly attenuated elevation of blood pressure in SHR-l-NAME (204 ± 4 mmHg). 3 l-NAME-induced increase in urine volume and protein was significantly lower in ABT-treated animals. 4 The impaired vascular responsiveness to noradrenaline and isoprenaline in the perfused mesenteric vascular bed of SHR-l-NAME-treated animals was significantly improved by ABT treatment. 5 Morphological studies of the kidneys and hearts showed that treatment with ABT minimized the extensive arterial fibrinoid necrosis, arterial thrombosis, significant narrowing of arterial lumen with marked arterial hyperplastic arterial changes that were observed in vehicle treated SHR-l-NAME. 6 In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischaemia was significantly better in ABT-treated SHR-l-NAME. 7 These results suggest that in hypertensive individuals with endothelial dysfunction and chronic NO deficiency, inhibitors of 20-HETE synthesis may be able to attenuate development of high blood pressure and end-organ damage.

Translation elongation factor-1 (EF-1) forms a primary site of regulation of protein synthesis and has been implicated amongst others in tumorigenesis, diabetes and cell death. To investigate whether diabetes-induced oxidative stress affects EF-1 gene expression, we used a free radical scavenger, vitamin E. The following groups of rats (5/group) were studied: control, vitamin E control, diabetic and diabetic treated with vitamin E. Markers of hyperglycemia, kidney function, oxidative stress, and kidney hypertrophy were elevated in diabetic rats. Increased urinary protein excretion indicated early signs of glomerular and tubular dysfunction. The mRNA and protein levels of the three EF-1 subunits (A, Balpha, and Bgamma) were determined in renal cortex extracts using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis and western blotting. EF-1A mRNA expression in renal cortex extracts was significantly increased by at least 2-fold (p < 0.002) in diabetic rats; however, there was no change in the mRNA levels of EF-1Balpha and EF-1Bgamma subunits. Similar results were observed at the protein level. Treatment of diabetic rats with vitamin E for 10 days suppressed both glycemic and oxidative stresses in renal cortex and kidney hypertrophy. EF-1A mRNA and protein levels were also reduced to control levels. In conclusion, EF-1A but not EF-1Balpha and EF-1Bgamma gene expression is significantly enhanced in the renal cortex of diabetic rats. Normalization of enhanced EF-1A expression by vitamin E treatment suggests a role for EF-1A during diabetes-induced oxidative stress.

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.

The aim of this study was to test the hypothesis that treatment with angiotensin-(1–7) [ANG-(1–7)] or ANG-(1–7) nonpeptide analog AVE-0991 can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(1–7) (576 μg·kg−1·day−1 ip) or AVE-0991 (576 μg·kg−1·day−1 ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(1–7) or AVE-0991. There was a significant increase in urine protein (231 ± 2 mg/24 h) in diabetic animals compared with controls (88 ± 6 mg/24 h). Treatment of diabetic animals with ANG-(1–7) or AVE-0991 resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (183 ± 16 and 149 ± 15 mg/24 h, respectively). Treatment with ANG-(1–7) or AVE-0991 also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-1, angiotensin II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1–7)- or AVE-0991-treated animals. These results suggest that activation of ANG-(1–7)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.

Sodium, potassium adenosine triphosphatase (Na,K-ATPase) is a membrane-bound enzyme that maintains the Na + and K + gradients used in the nervous system for generation and transmission of bioelectricity. Recently, its activity has also been demonstrated during nerve regeneration. The present study was undertaken to investigate the ultrastructural localization and distribution of Na,K-ATPase in peripheral nerve fibers. Small blocks of the sciatic nerves of male Wistar rats weighing 250–300 g were excised, divided into two groups, and incubated with and without substrate, the para -nitrophenyl phosphate ( p NPP). The material was processed for transmission electron microscopy, and the ultra-thin sections were examined in a Philips CM 100™ electron microscope. The deposits of reaction product were localized mainly on the axolemma, on axoplasmic profiles, and irregularly dispersed on the myelin sheath, but not in the unmyelinated axons. In the axonal membrane, the precipitates were regularly distributed on the cytoplasmic side. These results together with published data warrant further studies for the diagnosis and treatment of neuropathies with compromised Na,K-ATPase activity.

The purpose of this study was to examine the effect of inhibition of Ras-GTPase mediated signalling on the development of hypertension and end-organ damage in spontaneously hypertensive rats chronically treated with nitric oxide synthesis inhibitor L-NAME (SHR-L-NAME). Administration of L-NAME in drinking water (80 mg/L) for 3 weeks significantly elevated mean arterial blood pressure (MABP) (223 +/- 4 mmHg) as compared to that of SHR controls (165 +/- 3 mmHg). The administration of Ras-GTPase inhibitor FPTIII (232 ng/min) to SHR-L-NAME during the last 6 days significantly attenuated high blood pressure (192 +/- 4 mmHg). Morphological studies of the kidneys and hearts showed that treatment with FPTIII minimized the extensive arterial fibrinoid necrosis, arterial thrombosis, narrowing of arterial lumen with marked arterial hyperplastic arterial changes that were observed in vehicle treated SHR-L-NAME. L-NAME-induced increase in urine volume and protein was also significantly lower in FPTIII-treated animals. The impaired vascular responsiveness to isoprenaline in the perfused mesenteric vascular bed of SHR-L-NAME-treated animals was significantly attenuated by FPTIII treatment. In isolated per-fused hearts, recovery of left ventricular function from a 40 min of global ischemia was significantly better in FPTIII-treated SHR-L-NAME. Treatment with FPTIII also significantly reduced expression of cardiac sodium-hydrogen exchanger-1 (NHE-1) which was elevated in SHR-L-NAME. These data indicate that inhibition of Ras-GTPase-mediated signalling can attenuate end-organ damage during severe hypertension and endothelial dysfunction. (c) 2005 Elsevier Ltd. All rights reserved., The purpose of this study was to examine the effect of inhibition of Ras-GTPase mediated signalling on the development of hypertension and end-organ damage in spontaneously hypertensive rats chronically treated with nitric oxide synthesis inhibitor L-NAME (SHR-L-NAME). Administration of L-NAME in drinking water (80 mg/L) for 3 weeks significantly elevated mean arterial blood pressure (MABP) (223+/-4 mmHg) as compared to that of SHR controls (165+/-3 mmHg). The administration of Ras-GTPase inhibitor FPTIII (232 ng/min) to SHR-L-NAME during the last 6 days significantly attenuated high blood pressure (192+/-4 mmHg). Morphological studies of the kidneys and hearts showed that treatment with FPTIII minimized the extensive arterial fibrinoid necrosis, arterial thrombosis, narrowing of arterial lumen with marked arterial hyperplastic arterial changes that were observed in vehicle treated SHR-L-NAME. L-NAME-induced increase in urine volume and protein was also significantly lower in FPTIII-treated animals. The impaired vascular responsiveness to isoprenaline in the perfused mesenteric vascular bed of SHR-L-NAME-treated animals was significantly attenuated by FPTIII treatment. In isolated perfused hearts, recovery of left ventricular function from a 40 min of global ischemia was significantly better in FPTIII-treated SHR-L-NAME. Treatment with FPTIII also significantly reduced expression of cardiac sodium-hydrogen exchanger-1 (NHE-1) which was elevated in SHR-L-NAME. These data indicate that inhibition of Ras-GTPase-mediated signalling can attenuate end-organ damage during severe hypertension and endothelial dysfunction.

Publisher Summary This chapter reveals that kidney plays a key role in the body by functioning as an integrating and regulatory organ that maintains the homeostasis of the extracellular fluid and the pH of blood in the normal physiological range. The high excretory capacity of the kidney requires a large blood flow. A total of 20-25% of the cardiac output flows through the kidneys, 90% of which passes through the renal cortex. The kidneys continuously eliminate substances such as creatinine, urea, and absorbed xenobiotics (drugs and chemicals) and their metabolites in the urine. At the same time, normal renal function is crucial for retaining valuable blood components, such as glucose and amino acids. As an endocrine organ, the kidney is the site of synthesis for mediating factors, such as renin, erythropoietin, prostaglandins, cytokines, and kinins.

First-, second-, and third-generation cephalosporins were investigated for their peroxidative and nephrotoxic potential. Renal cortical slices from male Wistar rats were incubated at 37° C for 1 h in a phosphate-buffered medium containing the cephalosporin (1.25, 2.5, 5 or 10 mg/ml). In another series of experiments 5 mg/ml cephalosporin was incubated under the same conditions for 30, 60, 90 and 120 min. Subsequently, slices were incubated for 60 or 90 min in a bicarbonate- or phosphate-buffered medium containing pyruvate or tetraethylammonium (TEA) to determine gluconeogenesis and TEA accumulation, respectively. The peroxidative potential was determined at the end of the first incubation by measuring the increase in the malondialdehyde (MDA) content in renal cortical slices. The nephrotoxic potential was determined at the end of the second incubation by measuring the decrease in accumulation of the organic ion (TEA) and decrease of pyruvate-stimulated gluconeogenesis in renal cortical slices. First-generation cephalosporins, cephaloridine and cephalothin showed a time- and concentration-dependent increase in MDA content and a decrease in TEA accumulation and gluconeogenesis by renal cortical slices. Cefazolin, another first generation cephalosporin, showed a weak peroxidative and practically no nephrotoxic potential. In the group of second-generation cephalosporins, cefotiam showed a weak peroxidative potential comparable to that of cefoxitin but had a much greater nephrotoxic potential which was similar to that of cephaloridine. The third-generation cephalosporins, cefotaxime and cefoperazone showed a low peroxidative and no nephrotoxic potential. Based on their nephrotoxic potential, the cephalosporins investigated in the present study could be ranked as follows: cephaloridine > cefotiam > cephalothin > cefoxitin > cefazolin > cefoperazone ≧ cefotaxime.

Rat kidney microsomes reduced cephaloridine when incubated anaerobically with NADPH. Superoxide anion was generated in a concentration- and time-dependent manner when cephaloridine was incubated with rat kidney microsomes. Cephaloridine increased the in vitro peroxidation of rat kidney microsomal lipids in a concentration- and time-dependent manner. Cephaloridine-induced lipid peroxidation was inhibited by a combination of superoxide dismutase and catalase, by the hydroxyl radical scavengers, mannitol, (+)-cyanidanol-3 and by the singlet oxygen scavenger histidine in a concentration-dependent manner. It is proposed that cephaloridine nephrotoxicity may occur through the transfer of an electron from reduced cephaloridine to oxygen and subsequent formation of the superoxide anion, hydrogen peroxide, the hydroxyl radical and singlet oxygen. These activated oxygen species then are very likely to react with membrane lipids to induce lipid peroxidation and nephrotoxicity.

Protein degradation in the kidney occurs mainly in lysosomes, organelles which may also accumulate nephrotoxic chemicals. The goal of this study was to evaluate the effects of intracellular accumulation of gentamicin, cephaloridine and cisplatin on lysosomal digestion of the protein lysozyme. Gentamicin (15 or 30 mg/kg/day for 3 or 5 days), cisplatin (2.5 or 5 mg/kg) or cephaloridine (500, 1000, 2000 or 2500 mg/kg) was administered ip to male Wistar rats. The main site of the nephrotoxic effects of these compounds was the proximal tubule where these agents differentially affected S1, S2 and/or S3 segments. A 2- and 4-fold increase of the excretion of N-acetyl-β-D-glucosaminidase (NAG) was observed in the urine from cisplatin- and gentamicin-treated rats, respectively; no change in enzyme excretion occurred after cephaloradine. One hour prior to sacrifice, rats were given 0.3 mg of unlabelled lysozyme in combination with 125I-lysozyme in 0.3 mL saline. Renal cortical slices were prepared and incubated for 15, 30, 60 and 90 min. Release of trichloroacetic acid (TCA) soluble radioactivity into the medium was assumed to quantify lysosomal degradation of lysozyme. Accumulation of p-aminohippurate (PAH) in renal cortical slices and changes in blood urea nitrogen (BUN) concentration were used as indices of renal damage. TCA-soluble radioactivity increased in the medium from kidney slices from control rats to 50% of the total radioactivity after 90 min incubation. In gentamicin-treated rats, lysozyme degradation was significantly decreased by doses of 15 and 30 mg/kg/day after 3 and 5 days of exposure in the absence of any changes in BUN or PAH accumulation. Increased BUN and decreased PAH accumulation was accompanied by reduced release of lysozyme degradation products from kidney slices of rats treated with both doses of cisplatin. Cephaloridine appeared to have no effect on renal catabolism of lysozyme at 500 and 1000 mg/kg; however at higher doses lysozyme degradation decreased and changes in BUN and PAH occurred. Thus, all three compounds used were effective in decreasing renal catabolism of lysozyme. Protein degradation was an early and sensitive indicator of aminoglycoside-induced nephrotoxicity since a decrease in protein degradation in kidney slices from gentamicin-treated rats was not associated with alterations in BUN or PAH accumulation but was associated with ultrastructural changes.

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7-ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.

Gentamicin and other aminoglycoside antibiotics in high doses may produce proteinuria and other signs of nephrotoxicity. Proteinuria may result from general renal damage or may reflect alterations in specific steps in the renal handling of proteins. To distinguish between these two possibilities, experiments were designed to quantify the effects of nephrotoxic doses of several aminoglycosides on the renal handling of proteins in the isolated perfused rat kidney with the cationic low-molecular-weight protein lysozyme as a representative protein. Each aminoglycoside was administered ip to male Wistar rats (30 mg/kg/day) for 7 days. Lysozyme and 125I-lysozyme were added to the perfusate to achieve a lysozyme perfusate concentration of about 100 mg/liter. Clearances of inulin and lysozyme, release of tyrosine and trichloroacetic acid-soluble radioactive metabolites into the perfusate, and the glomerular sieving coefficient of lysozyme were determined. Scanning and transmission electron microscopy indicated that gentamicin and tobramycin decreased the number and diameter of the endothelial fenestrae of the glomerular capillaries. Concurrently, gentamicin and tobramycin decreased the glomerular sieving coefficient of lysozyme from 0.8 to 0.6 and 0.5, respectively. Netilmicin did not affect the percentage reabsorption of lysozyme whereas gentamicin and tobramycin decreased lysozyme reabsorption from 71.7 to 35.4 and 34.4% of the filtered load, respectively. Lysozyme degradation, estimated by the release of tyrosine into the perfusate during a 150-min perfusion period, was decreased from a control value of 12 mumol/liter to 4.43 and 4.65 mumol/liter in kidneys from rats treated with gentamicin and tobramycin, respectively. This study demonstrates that polycationic aminoglycosides may affect several processes involved in renal handling of lysozyme including glomerular permeability, tubular reabsorption, and intracellular proteolytic degradation.

The aminoglycosides gentamicin, netilmicin and tobramycin were each administered (30 mg/kg/day) to male Wistar rats for 21 days. At 7, 14 and 21 days rats were anesthetized and renal clearances of inulin and egg-white lysozyme were quantified. Plasma concentration of lysozyme varied between 20 and 120 mg/liter in control and aminoglycoside treated rats. Inulin clearance (GFR) average 0.96 ml/min/kidney in control rats. Whereas GFR decreased to an average value of 0.47 ml/min/kidney in gentamicin treated rats, GFR did not fall in tobramycin treated rats, averaging 0.97 ml/min/kidney over the entire treatment period. In netilmicin rats GFR declined to 0.53 ml/min/kidney after 7 days of treatment and then increased to 0.78 ml/min/kidney after 14 days and 21 days of treatment. Reabsorption of lysozyme averaged about 94% of the filtered load in control rats. Reabsorption of lysozyme decreased in gentamicin treated rats to about 58% after 7 days and 14 days of treatment and increased to 79% after 21 days of treatment. After tobramycin reabsorption of lysozyme decreased to about 67% after 7 days of treatment and increased to 85% after 14 days and 21 days of treatment. Reabsorption of lysozyme remained unchanged when compared to controls over the entire treatment period in netilmicin treated rats. Thus, gentamicin reduced both GFR and renal reabsorption of lysozyme. Netilmicin reduced GFR somewhat without altering lysozyme reabsorption whereas tobramycin compromised lysosyme reabsorption without altering GFR. These differences might suggest differences in site(s) of action of various aminoglycosides.

Effects of the aminoglycosides, gentamicin, netilmicin and tobramycin (30 mg/kg/day X 7, i.p.), on glomerular ultrastructure were determined in male Wistar rats. Glomerular ultrastructure was studied by scanning and transmission electron microscopy. The average number of capillary endothelial fenestrae per 5 microns of capillary wall decreased from a control value of 10.8 to 8.2 and 8.1 in gentamicin- and tobramycin-treated rats, respectively, as shown by transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that the density of endothelial fenestrae decreased to about 80% of control in gentamicin- and tobramycin-treated rats. In addition, gentamicin and tobramycin produced a marked reduction in the number of granulated myoepithelioid++ cells of the juxtaglomerular apparatus. Netilmicin appeared to have no effect on glomerular ultrastructure. Both TEM and SEM, indicate that the nephrotoxic aminoglycosides gentamicin and tobramycin alter the ultrastructure of the fenestrated endothelia of the glomerular capillaries. These ultrastructural changes appear to be correlated to the impairment of the glomerular permeability for different compounds.

Previous renal clearance studies provided quantitative data concerning renal reabsorption of proteins while the simultaneous processes of renal accumulation and degradation remain, to a great extent, insufficiently investigated. Thus, it was the aim of this study to measure renal reabsorption of egg-white lysozyme at various lysozyme concentrations and to relate the corresponding accumulation and degradation of lysozyme to the lysozyme transport rates in intact rats and isolated perfused rat kidneys. Lysozyme (with125I-lysozyme in certain experiments), was continuously infused i.v. or added to the perfusate to achieve plasma (or perfusate) concentrations of lysozyme (PLY) of approximately 50, 500 or 1000 mg·l−1 for periods of time varying between 3 and 120 or 150 min. Clearances of inulin and lysozyme or the total content of radioactivity and the trichloroacetic acid (TCA)-soluble radioactivity in the kidney tissue were determined at the end of clearance or accumulation periods. Additionally the perfusate concentration of the metabolite tyrosine was measured by high performance liquid chromatography (HPLC). The reabsorption rates of lysozyme (TLY) were concentration-dependent in both intact rats and isolated perfused rat kidney. After 25 min of lysozyme infusion, the lysozyme reabsorption rates amounted to 37, 245 and 331 μg·min−1·g−1 kidney at the above lysozyme concentrations. After the same infusion time, the accumulation rates of lysozyme were 8, 59 and 118 μg·min−1·g−1 kidney. The difference between the transport rate and accumulation rate should represent the renal degradation rate of lysozyme. The renal accumulation and degradation of lysozyme appeared to increase in a time- and concentration-dependent manner. The renal lysozyme degradation is of limited capacity as shown by measuring directly the release of the amino acid tyrosine by using HPLC. Renal degradation of lysozyme was almost totally inhibited by gentamicin in the presence of significant transport of lysozyme. The results of this study also demonstrate the ability of the rat kidney to reabsorb and accumulate large amounts of the cationic low molecular weight protein lysozyme without ultrastructural changes at plasma concentrations of lysozyme as high as 500 mg·l−1. Transmission electron microscopy indicated an increase in the number of endocytic vesicles and lysosomes at 1000 mg·l−1 plasma concentration of lysozyme after a 30 min infusion.

In order to elucidate the molecular mechanisms of cephaloridine (CPH) nephrotoxicity, the effect of cephaloridine treatment on the protein composition of different subcellular fractions from rat kidney cortex was investigated. After intravenous treatment of male Wistar rats with 250–1200 mg/kg/d CPH for 1–3 days, kidneys were removed and the homogenate from renal cortex was separated into lysosomal, cytosolic and microsomal fractions. The polypeptide composition of the different subfractions was analyzed by one-dimensional SDS-gel electrophoresis and quantified by densitometry. Significant differences in the polypeptide composition between treated and non-treated animals were seen in the microsomal fraction. CPH-treatment induced a polypeptide with an apparent molecular weight of 44,000 and decreased the content of cytochrome P-450 isoenzymes in the microsomal fraction. Solubilization experiments showed that the CPH-induced microsomal polypeptide of molecular weight 44,000 is a peripheral membrane protein rather than an integral membrane protein. The induction of this protein by CPH was dose- and time-dependent. Preliminary experiments using the kidney slice technique indicate that the induction of this polypeptide correlates with the nephrotoxicity measured as decrease in renal cortical accumulation of organic ions. Thus, the results of the present study indicate that treatment of rats with CPH resulted in the induction of a microsomal polypeptide of molecular weight 44,000 which could be a sensitive parameter of cephaloridin nephrotoxicity.

To quantify the effects of gentamicin, kanamycin and netilmicin on renal protein reabsorption and accumulation, these drugs were administered to rats intraperitoneally (30 mg/kg/day) for 7, 14 or 21 days. Scanning electron microscopy of the glomerular endothelia, urinary measurements of sodium, potassium, endogenous lysozyme, N-acetyl-beta-D-glucosaminidase (NAG) as well as clearance and accumulation experiments after i.v. administration of egg-white lysozyme and measurements of inulin clearance (GFR) were done in each treatment group. Gentamicin administration decreased diameter, density and shape of endothelial fenestrae. Kanamycin and netilmicin appeared to have no effect at the dose used. All three aminoglycosides decreased GFR and increased urinary excretion of sodium and potassium. While gentamicin and kanamycin decreased the percentage reabsorption and accumulation of lysozyme after i.v. administration of egg-white lysozyme netilmicin had no effect. Daily excretion of total protein, endogenous lysozyme and NAG increased only after treatment with kanamycin and gentamicin. Thus, aminoglycosides may act as nephrotoxicants at glomerular and/or tubular level inducing impairment of renal reabsorption and accumulation of proteins.

The polycationic aminoglycoside gentamicin has been reported to compete with other aminoglycosides and cationic compounds for uptake into renal tubular cells at brush border membranes and during subsequent endocytosis. Gentamicin has also been reported to decrease the activity of lysosomal proteolytic enzymes in the rat kidney. In vivo and in vitro experiments were carried out to determine the effect of acute exposure to gentamicin on renal handling of the cationic low molecular weight protein, lysozyme. Thirty or 60 mg/kg of gentamicin was given to male Wistar rats (250-300 g) intravenously or isolated rat kidneys were perfused with gentamicin concentrations of 0.25, 0.50, 1.0 and 2.5 mg/ml. Subsequently, clearances of lysozyme (CLY) and inulin (GFR, glomerular filtration rate) were measured in the intact rat and in the isolated perfused rat kidney. The glomerular sieving coefficient of lysozyme was determined in control and gentamicin perfused kidneys after complete inhibition of tubular lysozyme reabsorption by sodium iodoacetate. Renal degradation of 125I-labelled lysozyme was quantified in control and gentamicin perfused kidneys by measuring the release of [125I] monoiodotyrosine to the perfusate. The glomerular sieving coefficient of lysozyme increased in kidneys perfused with gentamicin from a control value of 0.8-1.0. A dose-dependent increase of the ratio CLY/GFR in intact rats treated with gentamicin and dose-dependent decrease of percentage reabsorption of lysozyme in the isolated perfused kidneys demonstrate inhibition of renal reabsorption of lysozyme by gentamicin. Perfusion of kidneys with 0.25 mg/ml gentamicin reduced renal degradation of lysozyme to about 50% after 2 h perfusion; at gentamicin concentrations of 0.5 mg/ml and higher almost all degradation of lysozyme was inhibited. Thus, acute exposure to gentamicin causes impairment of filtration, tubular reabsorption and catabolism of the protein lysozyme, effects which are due to pharmacological interactions between the molecules of the 2 cations gentamicin and lysozyme.