AMELIORATES GLOMERULAR BASEMENT MEMBRANE ULTRASTRUCTURAL CHANGES CAUSED BY GENTAMICIN ADMINISTRATION IN RATS

Gentamicin is commonly used for the treatment of severe gram negative bacterial infections but inevitably cause renal failure during prolonged use. Th e aim of our study was to emphasize protective eff ects of pentoxifylline on glomerular basement membrane (GBM) alterations induced by gentamicin in rats. Experiments were done on  male Wistar rats divided in three experimental groups. GM-group was treated daily with gentamicin in dose of  mg/kg during  days. PTX-group was treated daily with pentoxifylline in dose of  mg/kg and the same dose of gentamicin as in GM-group during  days. Th e control group received  ml/day saline intraperitoneally. Morphometric parameter measured during the analysis was glomerular basement membrane thickness. In GM-group of animals glomeruli were enlarged and GMB was diff usely and unequally thickened with neutrophil cells infi ltration. In proximal tubules epithelial cells, vacuolization of cytoplasm with coagulation-type necrosis were observed. In PTXgroup of animals glomeruli were somewhat enlarged and GBM was thickened only in some segments. Coagulation-type necrosis was not found. Blood urea and serum creatinine concentration in GM-group were signifi cantly elevated in comparison with PTX-group while potassium level was decreased. Our results suggest that PTX has protective eff ects on GBM and proximal tubules in GM-treated rats.


Introduction
Gentamicin (GM) is an aminoglycoside antibiotic commonly used as an agent for the treatment of severe Gram-negative infections (). Although nephrotoxicity as a major side eff ect of aminoglycosides accounting for - of all acute renal failure cases (), the exact mechanisms of this adverse action still remain unclear. GM has been shown to enhance the generation of reactive oxygen species (ROS) (, ) which are putative causative agents of cell death in diff erent pathological conditions including various models of renal diseases (, , , ). Accordingly, the administration of several compounds with antioxidant activity has been successfully used to prevent or ameliorate GM-induced nephrotoxicity (, , , ). Th e usage of gentamicin in clinical practice would be undoubtedly increased if potent protective agent for its undesirable kidney side eff ects could be found. Th us, a potential therapeutic approach to protect or reverse renal gentamicin damage would have very important clinical consequences (). Pentoxifylline (PTX) is a hemorheological agent used in the treatment of peripheral vascular diseases. Recently, PTX has gained considerable interest as a ROS scavenger. Several in vitro studies have confi rmed the potential antioxidant eff ects of this drug (, , ). Although its mechanism of action is not well understood, pentoxifylline decreases blood viscosity by altering erythrocyte deformability (), reducing platelet aggregability and fibrinogen levels (), and increasing neutrophil mobility (). Also, pentoxifylline is a potent stimulator of prostacyclin in vascular beds () and like other xanthines such as theophylline, is an adenosine receptor antagonist (). Th erefore, PTX is a potent anti-infl ammatory agent capable of ameliorating kidney infl ammation by acting on various targets including the synthesis of pro-infl ammatory cytokines and chemokines, as well as the growth and activation of inflammatory mononuclear cells. The aim of our experimental investigation was to determine the potential protective effect of pentoxifylline on glomerular basement membrane ultrastructural changes caused by gentamicin in rats.

Material and Methods
All studies were performed on adult male Wistar rats, weighing  - g. Animals were housed in a central facility under controlled conditions ( h light/ dark cycle and room temperature of ºC ± ºC) and with free access to food and water. All experimental procedures were conducted in accordance with the principles for the care and use of laboratory animals in research. The investigation conforms to the regulations of the European Union and USA Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (National Academy of Science Press, Washington, DC, ).

Experimental protocol
Th e total number of  animals was divided in  groups, one of which was used as a sham control. The experimental group of animals or GM-group ( rats) received gentamicin (Galenika AD, Belgrade, Serbia) intraperitoneally in a daily dose of  mg/kg. PTX-group animals ( rats) were treated daily with pentoxifylline (Jugoremedija A.D. Niš, Serbia) intraperitoneally in dose of  mg/kg and the same dose of gentamicin as in GM-group. Th e control group of animals or C-group ( rats) received  ml/day saline intraperitoneally. Both experimental and control group were treated over the period of  consecutive days. Following the last application, nine days after the beginning of the experiment, all animals were anaesthetized using mg/kg ketamine (Ketamidor , Richter Pharma AG, Wels, Austria) and then sacrifi ced. Immediately after vivisection ml blood from aorta was taken for biochemical analysis.

Histological analysis
The kidneys were sectioned and fixed in  paraformaldehyde (in , mol/dm phosphate buffer saline), dehydrated in graded alcohols and processed for paraffi n wax embedding. Th en, kidney slices were cut on  μm thick sections using HistoRange microtome (model: LKB , LKB-Produkter AB, Bromma, Sweden) and stained with hematoxylineosin (HE), PAS (Periodic Acid Schiff ) and Jones methenamine silver according to conventional staining protocols as described by Bancroft and Stevens ().

Morphometric analysis
Histological slides were analyzed using light microscope (Olympus BX, Tokyo, Japan) and Micro Image . (Olympus, Tokyo, Japan) image analysis and processing software were used for the morphometric analysis. Spatial calibration, by object micrometer (:), as well as optical density calibration was performed before each analysis. Morphometric parameter measured during the analysis was glomerular basement membrane thickness (μm). Th e glomerular basement membrane thickness was estimated as a mean distance after manual tracing of two lines along both sides of NENAD STOJILJKOVIĆ ET AL.: PENTOXIFYLLINE AMELIORATES GLOMERULAR BASEMENT MEMBRANE ULTRASTRUCTURAL CHANGES CAUSED BY GENTAMICIN ADMINISTRATION IN RATS the basement membrane. In each animal at least  glomeruli were measured, excluding columns of Bertin.

Biochemical analysis
As previously mentioned, after finishing the experiment, blood samples were taken from aorta and analyzed for markers of renal impairment. Plasma creatinine, blood urea, sodium and potassium concentrations were measured using an automatic biochemical analyzer (A Biosystems, Barcelona, Spain).

Statistical analysis
Statistical analysis included estimation of mean values and standard deviation (SD) for parameters obtained during the morphometric and laboratory analysis. Th e statistical significance for the differences between the experimental and control group of animals' parameters was tested by Student's t -test using NCSS statistical software (NCSS Kaysville, Utah). In all cases, statistical significance was inferred for p<,.

. Histological analysis
Th e kidney sections taken from the experimental GMgroup of animals showed areas of proximal tubule epithelial cells necrosis and apoptosis, vacuolization of cytoplasm and epithelial desquamation ( Figure ). Glomeruli were enlarged, glomerular basement membrane was irregularly thickened, with neutrophil cell infiltration. In the cytoplasm of viable proximal tubule epithelial cells numerous black dots ("myeloid bodies") were found in the sections stained with silver methenamine ( Figure ). In PTX-group of animals glomeruli were somewhat enlarged, and the glomerular basement membrane was thickened only in some segments of the glomer-uli. Coagulation-type necrosis was not found. In some epithelial cells of proximal tubules dark inclusions and cytoplasm vacuolization were observed ( Figure ).

. Morphometric analysis
The morphometric analysis of glomerular basement membrane thickness between animals from the experimental GM, PTX and control groups are presented in the (Table ).
Morphometric analysis of glomerular basement membrane thickness showed statistically significant differences between the control and the experimental GM and PTX groups ( Table ). The mean glomerular membrane thickness was significantly greater in gentamicin treated group of animals compared to PTX and control group of rats (p< ,) ( Table ). Glomerular basement membrane thickness of PTXgroup of animals was greater than in control group of rats, but without statistical significance (Table ).

. Biochemical analysis
Using multivariate t-test, statistically signifi cant diff erences were found for values of BUN and creatinine (p<,).
The mean values of BUN and creatinine serum concentrations found in GM and PTX groups of rats were statistically significantly elevated in comparison to the control group of rats (p<,

Discussion
Because of its strong bactericidal eff ect, gentamicin is widely used antibiotic in the treatment of infections caused by gram-negative microorganisms. However, the ample data also speak of its nephrotoxic effect demonstrated in a numerous experimental studies in which gentamicin acute renal insufficiency was induced (, ). Oxidative stress is probably the most common pathogenic factor of cell damage in the complex pathogenesis of gentamicin nephrotoxicity. The data obtained from experiments suggest that both vascular (glomerular) and tubular targets are involved in drug-induced nephrotoxicity (). In our study, gentamicin given in a supratherapeutic dose (mg/ kg) induced acute renal insufficiency (ARI) in rats. Pentoxifylline penetrates the kidney tissue, where it reduces the production of free oxygen radicals due to its anti-oxidant eff ect. When it is applied alone or together with the vitamin E, pentoxifylline shows satisfactory eff ects on renal damage (, ). Th e increased production of TNF-alpha which induces apoptosis () and other cytotoxins which are important factors of cellular proliferation and ECM synthesis of mesangial cells of glomeruli and interstitial fi broblasts can be prevented with pentoxifylline (). Th ese data suggest that pentoxifylline has an ameliorative eff ect on the course of experimental ARI, which is in accordance with the results of other authors (, ). The mechanism of amelioration probably involves the stimulation of renal vasodilator prostaglandins as well as the prevention of vascular congestion (). On the other hand, it has been shown that PTX aff ects calcium homeostasis and inhibits calcium entry in the human erythrocyte (). It has been reported that calcium channel blockers are effective in the prevention of GM-induced nephrotoxicity (by inhibiting calcium accumulation in the kidney) (). Th e most important eff ect of PTX in gentamicin nephrotoxicity is the increased blood flow through blood vessels which leads to signifi cant improvement of the renal microcirculation. It also reduces the tubular dysfunction and glomerular fi ltration, which leads to the reduction of BUN and creatinine in plasma. Th e histopathological changes observed in GM-group of animals consisted of enlargement of glomeruli and glomerular basement membrane alterations with unequal thickness in some of its segments (). Th e neutrophilic leucocytes were present in some glomeruli capillaries. Th e changes in the proximal tubules were dominant and manifested in the form of segmented necrosis of the coagulation type, cytoplasm vacuolization of tubular epithelial cells with preserved nuclei and multitude of dark inclusions ("myeloid body"). Th e structural changes in the distal tubules were not found. Th ese changes mostly coincide with the changes already described by other authors (, ). The presence of neutrophils in the glomerular capillaries confi rms that the renal microcirculation and glomerular hemodynamics were impaired by the administration of gentamicin. If the changes in the kidneys and glomeruli are primarily due to changed microcirculation and hemodynamics in the capillaries, the removal of those would abolish gentamicin eff ects in renal nephrotoxicity. Th e PTX-group of rats showed lesser morphological and functional kidney damage in comparison to the rats treated with gentamicin only. Glomeruli were insignificantly enlarged and the basement membrane of glomerular capillaries was thickened only in some segments of glomeruli. Th ere were no signs of necrosis in the proximal tubules, but only vacuolization and the presence of dark inclusions were noted in the cytoplasm of some cells, which points out the protective eff ect of pentoxifylline. Th e glomerular basement membrane (GBM) is progressively formed by two membranes: the lamina rara interna (LRI; formed from the endothelial cells) and the lamina rara externa (LRE; formed from the pedicels of the epithelial cells), which fuse and generate a common lamina densa (LD) in the central part. In the rat neonate, all these stages can be observed, the fi rst diff erentiated (older) nephrons being in the juxta -medullary area. Th e urine is formed in mature glomeruli by fi ltration through the GBM, which stops the large molecules, such as proteins. Th is permselectivity of the GBM is considered to be mainly due to an electrostatic shield made of highly negatively charged heparan sulfate proteoglycans con-  () found that the developing GBM was abnormal in the treated neonates, and that morphological and functional alterations of the GBM were still present in the juxtamedullary glomeruli of the adult animals, i.e., month-old rats, prenatally exposed to gentamicin. In the early stages of GBM maturation, the fi brillar material appeared more important in the LRE and LRI before they joined to form the LD; in later stages, the LD itself looked larger and denser. Morphometric analysis showed that the GBM layers were diff erent from those of controls in treated neonates: the LRI and LRE were thinner while the LD was enlarged. The counting of anionic sites showed an increase of their number per square micrometer of the LRE or LRI and thus an increased density; glycosaminoglycans were more sulfated and quantita-tively more abundant than in controls (). Our results revealed that glomerular basement membrane thickness was signifi cantly larger in gentamicin treated rats than in the control group, as previously described (). Morphometric analysis revealed that GMB was highly signifi cantly thickened in GM-group of rats, compared to control and PTX groups. Th e thickness of GBM was higher in PTX-group compared to control group of rats, but this diff erence was not statistically signifi cant.
In the GM-group of animals, the biochemical analysis showed the most significant increase in serum urea and creatinine as a sign of the functional alterations of kidney. This is rather customary having in mind that the above laboratory parameters and electrolytes are secreted predominantly by glomerular filtration.
Th e values of serum creatinine and BUN in the PTXgroup of rats were enhanced in relation to the control group but reduced in relation to the GM-group.

Conclusion
Our results suggest that PTX has ameliorative eff ects on GBM and proximal tubules in GM-treated rats.

Acknowledgment
Th is work was supported by the Ministry of Science Republic of Serbia grant .