THE EFFECT OF CALCIUM CHANNEL BLOCKER VERAPAMIL ON GENTAMICIN NEPHROTOXICITY IN RATS

Aminoglycoside antibiotics are obligated nephrotoxins and inevitably cause renal failure during prolonged use. Experimental models of gentamicin-induced nephrotoxicity have shown histopathological, ultrastructural and functional alteration with blood urea nitrogen and serum creatinine increase leading to acute renal insuffi ciency (ARI). Th e aim of our study was to emphasize eff ects of verapamil, a calcium channel blocker, on gentamicin-induced ARI in rats. Experiments were done on  male Wistar rats (- g) divided in three experimental groups. G-group animals ( rats) were treated daily with gentamicin in dose of  mg/kg during  days. GV-group animals ( rats) were treated daily with verapamil in dose of  mg/kg and the same dose of gentamicin as in G-group during  days. Th e control group ( rats) received  ml/day saline intraperitoneally. Histological examinations were done using hematoxylin and eosin, periodic acid Schiff and methenamine silver staining methods. Morphometric parameters included measurement of glomerular area, major and minor axis, perimeter, diameter, roundness, and mean optical density. Biochemical analyses were used to determine concentrations of blood urea, serum creatinine, sodium and potassium. In G-group rats’ glomerular basement membrane was diff usely and unequally thickened with polymorphonuclear neutrophils infi ltration, while coagulation-type necrosis and vacuolization of cytoplasm of proximal tubules epithelial cells were observed. In GV-group rats' glomeruli were slightly enlarged with thickened basement membrane in some segments but without coagulation-type necrosis. Morphometric analyses showed statistically signifi cant diff erences between the G-group and control group of animals in glomerular size, mean optical density and average roundness (p<,). On the other hand, morphometric analyses between GV-group and control group animals did not show statistically signifi cant diff erences in any of parameters measured. Blood urea and serum creatinine concentration in G-group were signifi cantly elevated in comparison with GV-group (p<,) but sodium and potassium levels in G-group were decreased compared to GV-group without statistical signifi cance. Our results show that verapamil modify some of morphological and functional kidney alterations induced by gentamicin.


Introduction
A rather frequent administration of the aminoglycoside antibiotic gentamicin in the clinical practice has shown its unquestionable nephrotoxic effect (). Even in low concentrations, gentamicin is bactericidal, but apart from vestibulotoxicity, it also shows a particularly high nephrotoxicity which can lead to acute renal insufficiency (ARI). Because of the potential nephrotoxicity of gentamicin, the administration of substances which would prevent or reduce the alteration of kidneys, and thus development of renal insufficiency, has been investigated in recent years. More recent study () has showed that the administration of gentamicin in a single daily dose significantly reduce a risk of gentamicin nephrotoxicity, although there is still a high incidence of gentamicin-induced ARI. Nephrotoxicity induced in experimental models (,,) showed histopathological, ultrastructural and functional renal impairments in the form of tubular desquamation and necrosis as well as the enhanced blood urea nitrogen and creatinine. Th e predilection sites of damage are the renal cortex, i.e. glomeruli and proximal tubules. Th e exact mechanism of the cell impairment and the way in which gentamicin enters the cell are unclear. Th e stimulation of nephrotoxic eff ect of gentamicin is availed by the morphological and functional characteristics of the kidneys such as a large renal blood fl ow and therefore by a great proportional exposure of the renal parenchyma to toxic eff ects, high oxygen consumption, susceptibility of the renal tissue to hypoxia and the degree of the permeability of proximal tubules. Th ere are numerous publications pointing out to the protective effect of verapamil possibly associated with blocking the calcium infl ux to the impaired cells or to the vasodilatory effects of verapamil which improve the renal blood fl ow and thereby moderate the development of acute renal insufficiency (, , , ). The aim of our experimental investigation was to determine the eventual preventive effect of the calcium channel blocker verapamil on gentamicin induced acute renal insufficiency 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, ). The total number of  animals was divided in  groups, one of which was used as a sham control. Th e experimental group of animals or G-group ( rats) received gentamicin (Galenika AD, Belgrade, Serbia) intraperitoneally in a daily dose of  mg/kg. GV-group animals ( rats) were treated daily with verapamil (Galenika AD, Belgrade, Serbia) in dose of  mg/kg and the same dose of gentamicin as in G-group during  days. 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. The kidneys were sectioned and fixed in  paraformaldehyde (in , mol/dm  phosphate buffer saline), dehydrated in graded alcohols and processed for paraffin wax embedding. Then, 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 (). Histological slides were analyzed using light microscope (Olympus BX, Tokyo, Japan) and Micro Image . (Olympus, Tokyo, Japan) image analysis and processing software was used for the morphometric analysis. Spatial calibration, by object micrometer (:), as well as optical density calibration was performed before each analysis. Morphometric parameters measured during the analysis of glomeruli were area, major and minor axis, perimeter, diameter, roundness and mean optical density. 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). Th e data obtained from morphometric and functional measurements of each experimental group were expressed as mean value and standard deviation and were analyzed by multivariate analysis of variance (MANOVA) using NCSS statistical software (NCSS Kaysville, Utah, USA). Paired comparisons between animal groups were performed using Student's t-test. In all cases, statistical signifi cance was inferred for p<,.

Results
The kidney sections taken from the experimental Ggroup of animals showed that the glomerular basement membrane was thickened diffusely, and it was of unequal thickness ( Figure ). Th e presence of polymorphonuclear neutrophils was noticed in certain glomerular capillaries as well ( Figure ). In this group of experimental animals, the fields of necrosis of coagulation type were found in a large number of proximal tubules as well as the vacuolization of the cytoplasm of cells still possessing nucleus. Th e distal tubules were of normal ap-pearance. Th e presence of dark inclusions in the epithelial cell cytoplasm of proximal tubules was observed in the sections stained with silver methenamine ( Figure ).
In GV-group of animals glomeruli were somewhat enlarged, and the glomerular basement membrane was thickened only in some segments of the glomeruli ( Figure ). In this group of animals, the fi elds of coagulation-type necrosis were not found. In some epithelial cells of proximal tubules dark inclusions and cytoplasm vacuolization were observed ( Figure ). The application of the MANOVA test for all glomerular morphometric parameters showed statistically significant differences between the control and the experimental G and GV groups: (Wilks Lambda = ,; Rao's R = ,; p< ,). Th e application of the t-test showed the following statistically signifi cant diff erences: area, optical density, minor axis, perimeter and roundness (p<, for each parameter). Statistically significant differences were found between the G-group of rats and the control group as follows: in the size of glomeruli (area, major axis, minor axis, average diameter and perimeter) (p<,); optical density of glomeruli (p<,) and roundness of glomeruli (p<,). Statistical analysis of glomerular morphometric parameters in the GV-group of rats did not show statistically signifi cant diff erences in relation to the control group. Statistically signifi cant diff erences found between the experimental G and GV groups were as follows: in the size of glomeruli area, minor axis, perimeter, optical density and roundness of glomeruli (p<,) ( Table ). The application the MANOVA test for all laboratory parameters showed high statistically significant differences between the experimental G and GV groups and the control groups of rats: (Wilks Lambda = ,; Rao's R = ,; p< ,) Th e application of the t-test showed a statistically signifi cant diff erence in the values of urea and creatinine (p<,). Th e mean values of urea and creatinine serum concentrations found in the G-group of rats were statistically sig-nifi cantly enhanced, while the mean values of potassium serum concentrations in the G group were statistically signifi cantly reduced, in relation to the same values in the control group (p<,). Th ere were no statistically signifi cant diff erences between the values of sodium serum concentrations in the G group and the control group. Th e values of urea and creatinine serum concentrations in the GV-group of rats were statistically signifi cantly enhanced, while the values of potassium serum concentrations were statistically signifi cantly reduced, in relation to the same values in the control group of rats (p<,). Th e values of urea and creatinine serum concentrations found in the G-group of rats were statistically signifi cantly enhanced in relation to the same values found in the GV-group (p<,). Th ere were no statistically signifi cant diff erences between the values of sodium and potassium serum concentrations found in the G group in relation to the same values found in the GV group (Table ).

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 data found in the literature also speak of its nephrotoxic eff ect demonstrated in a number of experimental studies in which gentamicin acute renal insuffi ciency was induced (, , ). In our study, gentamicin given in a supratherapeutic dose (mg/kg) induced acute renal insuffi ciency (ARI) in rats. Th e histopathological changes observed in these animals consisted of enlargement of glomeruli and glomerular basement membrane alterations with unequal thickness in some of its segments. The neutrophilic leucocytes were present in some glomeruli capillaries. Th e changes in the proximal Data are presented as the mean ± SD. * p<0,001 vs. control #p<0,05 vs. GV-group TABLE 2. Biochemical analysis of serum levels of electrolytes, blood urea, and creatinine in G-group, GV-group and control group of rats.
Data are presented as mean ± SD. * p<0,01 vs. control **p<0,05 vs. control #p<0,05 vs. G-group 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 are not found. Th ese changes mostly coincide with the changes already described by other authors (, , ). In relation to the control group, morphometric analysis of the glomeruli of G-group of rats showed signs of glomerular impairment such as the enlargement of glomeruli with reduction in optical density of glomeruli and roundness (p<,). Th e above mentioned results point out that there are morphological and functional changes in the glomeruli caused by the eff ect of gentamicin. In the mentioned group of animals, the biochemical analysis showed the most signifi cant increase in serum urea and creatinine as a sign of the functional alterations of kidney, whereas the values of serum sodium and potassium were reduced in relation to the GV-group. However, the obtained values were not statistically signifi cant. Th is is rather customary having in mind that the above laboratory parameters and electrolytes are secreted predominantly by glomerular fi ltration. Th e morphological changes in the proximal tubules reduced sodium and potassium reabsorption, and consequently increased the urinary excretion of these electrolytes. In his study Matsuda (), showed that the electrolyte composition of the renal tubular cells in the gentamicin nephrotoxicity was diff erent in relation to the necrotic and non-necrotic tubular cells of the proximal tubules. It was showed that the histological impairment was present only in the proximal tubules with sodium and potassium concentration in the necrotic tubular cells which was lower than in control, whereas the concentrations of sodium in the non-necrotic cells were somewhat higher than those observed in proximal tubules of control group. This showed that the sodium and potassium serum levels correlate with the histopathological fi ndings in the cells of proximal tubules where gentamicin expressed its main nephrotoxic eff ect, which coincides with our fi ndings. Th e 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. As a consequence of gentamicin toxic eff ect, there comes to the impairment and decay of different cell organelles (lysosomes, mitochondria). Th e impairment of organelles leads to necrosis and desquamation of the cells. Our results showed that the most severe necrosis and desquamation was registered in the proximal tubules of animals treated with gentamicin only. Gentamicin increases the entry of Ca ++ in the mesangial cells and the intracellular concentration of cytosol Ca ++ . Th e increased concentrations of Ca ++ cause the contraction of the mesangial cells along with consequent glomerular contraction. Th e contraction of glomeruli and the mesangial cells leads to the reduction in ultrafi ltration coeffi cient, and the reduction in glomerular fi ltration. Th is would be a logical explanation of the given hemodynamic impairments as a main characteristic of toxic renal failure. Th e main components of disturbed renal hemodynamics are the following: reduction in renal blood fl ow in accordance with the increase in renal vascular resistance, reduction in ultrafi ltration surface and ultrafi ltration coeffi cient, tubular necrosis with consequent tubular obstruction, and impairment of water and electrolytes reabsorption (). Further, our experiments showed a minor impairment of glomeruli and tubules in animals treated with both gentamicin and verapamil (mg/kg b.w./h). Glomeruli were insignifi cantly 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. Th e results of morphometric investigations of glomeruli of GV-group did not show statistically signifi cant diff erence in relation to the control group of rats. In relation to the G-group the diff erences were found in the size of glomeruli, glomerular optical density and roundness as well (p<,). Th e values of the area were lower in relation to the G-group pointing out to certain protective properties of verapamil in gentamicin induced ARI. The values of serum creatinine and urea in the GVgroup of rats were enhanced in relation to the control group, but reduced in relation to G-group. Th e sodium and potassium serum concentrations were enhanced in relation to the group treated with gentamicin alone, but signifi cantly reduced (p<,) in relation to the control group. With its vasodilatory eff ects, verapamil increased the kidney blood fl ow. Th e increase in blood fl ow (microcirculation correction) also corrected the morphological changes and reduced the urea and creatinine retention. Th e experiments showed that verapamil did not fully abolish the nephrotoxic eff ect of gentamicin. Further, this suggests that the change in the microcirculation was not the only reason of the impairment of the glomeruli and capillaries. On the other hand, a better hemodynamics at the level of glomerular capillaries (re-NENAD STOJILJKOVIĆ ET AL.: THE EFFECT OF CALCIUM CHANNEL BLOCKER VERAPAMIL ON GENTAMICIN NEPHROTOXICITY IN RATS duced contractions of the mesangial cells) also means a higher exposure of the glomeruli and their structures to gentamicin. Partly, it could lead to a certain vicious circle at the level of the kidneys. Th e protective role of verapamil was proved in some experimental models of ARI. Th is eff ect can be associated with the blockade of Ca ++ infl ux in the impaired cells or with vasodilatory eff ects (). Th e protective function of verapamil in gentamicin nephrotoxicity can also be emphasized by proving the existence of competitive relationship between verapamil and gentamicin to the common transport system of the cell membrane, monitoring their eff ects on the transport system in brush border membrane vesicles. Th e results of this investigation demonstrated that both gentamicin and verapamil are substrates for the transport system of renal organic cations. Th e authors conclude that a high similarity of substrates should have a protective function in the gentamicin-induced nephrotoxicity (). In the conditions of gentamicin-induced impairment i.e. the cell apoptosis and necrosis, the increased concentration of intracellular Ca ++ activates phospholipases, nucleases and proteases which lead to the impairment of the plasma membrane and further entry of Ca ++ ions leading to irreversible impairment of the cells. In these conditions, verapamil as a Ca ++ channel blocker ceases further entry of Ca ++ from the extracellular space into the cell by blocking the entry of Ca ++ through the slow channels which are opened by activating the corresponding receptors "receptor calcium channels". It is considered that verapamil deforms the slow channels and interferes with Ca ++ liberation from the sarcoplasmatic reticulum. In this way, verapamil interrupts the mechanisms of further impairment of the cell in which the main part plays the redistribution of intracellular Ca ++ . Th e intrarenal eff ects of Ca ++ blockers verapamil on Ca ++ infl ux are expressed by the reduction in cellular and functional changes and impairments. Structural alterations in the proximal tubules are signifi cantly reduced after the treatment with verapamil and range within normalization, absence of necrosis, but also visibly damaged cells in the form of vacuolization and reduction in the brush border. Th e vascular eff ects of verapamil on the epithelium are of protective character considering that verapamil slows down the increase of intracellular Ca ++ in the renal microcirculation epithelium thus improving the endothelial dysfunction (). Th e recovery of renal perfusion is evident in the reduction of the overall renal vascular resistance resulting from the vasodilatory eff ects of verapamil on glomerular structures that are aff erent arterioles and contractile elements of the mesangial cells ().

Conclusion
Th e results of our investigation showed the unquestionable nephrotoxic eff ect of gentamicin with pronounced changes in glomeruli and proximal tubules. Th e concomitant administration of verapamil with gentamicin led to the reduction in the morphological and functional changes. Our experimental study showed that administration of verapamil ameliorates gentamicin-induced acute renal insuffi ciency.

List of Abbreviations
ARI -acute renal insuffi ciency G-group -experimental group of animals treated with gentamicin GV-group -experimental group of animals treated with gentamicin and verapamil C-group -control group of animals