Venkatesha U. et al. J Sci Res Pharm, 2018;7(6):64-69

© 2012, JSRP. All Rights Reserved http://www.worldinventiapublishers.com/

World Inventia Publishers

Journal of Scientific Research in Pharmacy http://www.jsrponline.com/

Vol. 7, Issue 6, 2018 ISSN: 2277-9469

USA CODEN: JSRPCJ

Research Article

SEQUENTIAL CHANGES IN ELECTRON MICROSCOPY OF KIDNEYS AND URINARY PARAMETERS IN GENTAMICIN INDUCED NEPHROTOXICITY IN RATS

Venkatesha Udupa 1 * and Veeru Prakash 2

* 1 Department of Toxicology, Glenmark Pharmaceuticals Limited, A607, TTC Industrial Area, MIDC, Mahape, Navi Mumbai 400 709, Maharashtra, INDIA. 2 Department of Biochemistry and Biochemical Engineering, Jacob Institute of Biotechnology and Bioengineering, Sam Higginbottom University of

Agriculture, Technology and Sciences, Allahabad, 211 007, Uttar Pradesh, INDIA.

Received on: 07-06-2018; Revised and Accepted on: 21-06-2018

ABSTRACT

The objective of the study was to evaluate changes in urinary parameters and ultramicroscopic changes in kidneys with Gentamicin induced

nephrotoxicity in male Sprague Dawley rats. Gentamicin was administered subcutaneously once daily at 30 and 100 mg/kg for seven consecutive days.

On day 4 and day 8 post treatment, urine and kidney samples were collected for investigation. Urinary calcium excretion increased significantly at both

doses on both Day 4 and 8 indicating hypercalciuria. With prolonged administration of Gentamicin, significantly increased total protein seen at both

dose levels; increased N-acetyl glucosamine (NAG), urinary protein and microalbumin (mALB), and decreased blood urea nitrogen (BUN), creatinine

and creatinine clearance seen at 100 mg/kg of Gentamicin that correlated with electron microscopic changes. On Day 4, minimal nuclear changes and

indistinct cytoplasmic organelles seen with 30 mg/kg of Gentamicin. Increased myeloid bodies, loose cell junction, pyknotic nucleus, elongated

mitochondria, loose cell junction, moderate margination and dilated capillaries seen with 100 mg/kg of Gentamicin. On day 8, myeloid bodies in

proximal convoluted tubule (PCT), dilated capillaries, dilated inter cellular area, condensed foot processes, indistinct endoplasmic reticulum, dark and

dense mitochondria were seen with 30 mg/kg of Gentamicin. Karyolysis, mild swelling, distribution of blocks of chromatin throughout nucleoplasm,

moderate margination of chromatin, loose cell junction, mild electron lucent and shabby appearance of mitochondria, empty perinuclear cytoplasm,

multiple myeloid bodies, electron dense bodies with different shapes and sizes, podocytes present with 100 mg/kg of Gentamicin.

KEY WORDS: Gentamicin, nephrotoxicity, ultrastructure, myeloid bodies, Calcium.

INTRODUCTION

Drug induced nephrotoxicity is one of the key problem in

human medicine and a reason for attrition of compounds during drug development. Drug or its metabolites are generally excreted through urine, but when they accumulate and reaches high levels, they eventually lead to nephrotoxicity and cause a variety of other symptoms of kidney trouble. The routine method of renal toxicity evaluation of new products is light microscopy of kidney and renal function tests and biochemical biomarkers of renal cell injury in blood and urine. Visual assessment (color, clarity), volume, specific gravity or osmolality, pH, and quantitative or semi-quantitative determination of total protein and glucose are urinary parameters recommended in toxicology studies (Weingand et al., 1996). Commercial available dipstick, which is inexpensive and easy to perform, contains reagents to determine ketones, bilirubin, urobilinogen, hemoglobin, nitrite and leukocyte esterases (Newman and Price, 1999). Urine is an ideal non-invasive source of biomarkers to diagnose and classify kidney diseases. However, conventional urine markers (casts, electrolytes or urine chemistry) and

*Corresponding author: Venkatesha Udupa Department of Toxicology, Glenmark Pharmaceuticals Limited, A607, TTC Industrial Area, MIDC, Mahape, Navi Mumbai 400 709, Maharashtra, INDIA. * E-Mail: venkatesha.udupa@glenmarkpharma.com DOI:

plasma or serum biomarkers (Creatinine and BUN) are nonspecific and insensitive.

Gentamicin, a broad-spectrum aminoglycoside antibiotic is nephrotoxic because a small amount of the administered dose is retained and accumulate in the endosomal and lysosomal vacuoles and the Golgi apparatus of the proximal convoluted tubules (Sandoval et al., 1998). Approximately 30% of gentamicin treated patients develop signs of nephrotoxicity in seven days (Cuzzocrea et al., 2002). The nephrotoxicity of Gentamicin has been shown to involve an inflammatory process in humans and experimental animals (Kourilsky et al., 1982). It has been demonstrated that gentamicin causes increase in serum creatinine and urea levels and severe proximal tubular necrosis, followed by deterioration and renal failure (Silan et al., 2010). Membranous structures that can be damaged by gentamicin include lysosomes, mitochondria and microsomes. The cells of the proximal renal tubules have the ability to concentrate gentamicin several folds more than plasma levels. Gentamicin binds to the phospholipids of the cell membrane of the renal tubules and enter inside the cells then it binds to subcellular organelles altering the mitochondrial respiration and small amount may be taken up by lysosomes (Erdem et al., 2000). The characterization of nephrotoxicity in terms of serum markers level and histological changes have been extensively reported already, however, the sequential changes in urinary parameters and ultrastructural changes in kidneys are lacking. Hence, in this study, we aimed to explore the temporal and dose relation in urinary parameters and ultrastructural changes in kidneys with Gentamicin.

Venkatesha U. et al. J Sci Res Pharm, 2018;7(6):64-69

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MATERIALS AND METHOD

Materials: Gentamicin (Genticyn Abbott, 80 mg/2 ml) was administered

at 30 and 100 mg/kg/day subcutaneously for 7 consecutive days. Doses were selected based on the results obtained in an exploratory dose-finding toxicity study (results not discussed in this manuscript), where dose dependent increase in serum blood urea nitrogen, creatinine and histological changes were seen in kidneys. Two dose levels of Gentamicin, 30 and 100 mg/kg/day (single daily dose, subcutaneous) were selected in this study in order to establish clear kidney toxicity without lethality.

Animals and study design:

In brief, 30 male Sprague Dawley rats supplied by Glenmark Pharmaceuticals Limited, Navi Mumbai, were acclimatized for at least 10 days prior to dosing. The rats were aged between 7–8 weeks, weighing between 175 - 190 g at the start of dosing. These 30 rats were randomized into three groups of 10 in each group. The groups were designated control (distilled water, subcutaneous), 30 and 100 mg/kg of Gentamicin (subcutaneous) as 1, 2 and 3 groups, respectively. Five animals in each group were euthanized on Day 4 and Day 8 respectively, for evaluating urine and ultrastructural pathology of kidneys. These animals were euthanized using isoflurane and oxygen on Day 4 and 8. The experimental animals were observed daily for clinical signs and mortality if any. Body weight and feed consumption were recorded once in 3 days and until the day of sacrifice.

All animals were maintained in an individually ventilated cage (IVC) with Corn cob (BioCobb, AT&T) as the bedding material. Commercial pellet diet (Altromin, Germany) and community tap water passed through a RO system (Millipore) were given. Feed and water was provided ad libitum throughout the study period unless restricted by experimental requirements. All animals were identified by tail marking for individual identification throughout the study period. CPCSEA guidelines were followed regarding animal welfare practices and the experimental protocol was approved by Glenmark’s Institutional Animal Ethics Committee (IAEC).

Urine collection and analysis: Rats were housed individually in Nalgene rat metabolic cages

on Day 3 and Day 7, overnight and urine was collected in chilled 25 ml standard conical polypropylene tubes kept in ice container (thermocol box) throughout the collection period of approximately 16 hours. During urine collections, animals were fasted but had free access to water. At the end of the collection period, urines were examined for color, appearance, measurement of volume, specific gravity and Semiquantitative parameters. After low speed centrifugation (400 g) at 4 degree C for 5 min, multiple aliquots of the supernatant urine were prepared and analyzed for various traditional parameters (Albumin, blood urea nitrogen, glucose, total protein, microalbumin, N-acetyl glucosamine and Creatinine) assessment using Randox clinical chemistry analyser. Urine microscopy assessment was done using sediment. Semi-quantitative parameters (bilirubin, ketone, blood, pH, urobilinogen, nitrite and leukocytes) using Multistix 10 SG reagent strips (Clinitek), specific gravity using refractometer and sediments microscopy. Electrolytes (sodium, calcium, potassium and chloride) were estimated using AC 9801 Automatic Electrolyte analyser (Allied Biotechnology India Pvt Ltd). Creatinine clearance was calculated from the creatinine concentration in the collected urine sample, the urine flow rate (volume/min), and the creatinine serum concentration.

Statistical analysis: Statistical analysis was performed on body weights, body

weight gain and urinalysis parameters. Results for all these parameters were expressed as mean ± standard deviation of the mean (SD). All the comparisons were made between treatment groups and vehicle control group. The data was evaluated using one-way ANOVA with Dunnett's post test (multiple comparison test). A p < 0.05 was considered significant in all evaluations. The complete analysis was performed using GraphPad Prism statistical software version 5.02 for Windows, GraphPad Software, San Diego California USA.

Transmission electron microscopy: On the day of terminal sacrifice i.e. on day 4 and day 8 all the

experimental animals were subjected to inhalation of isoflurane: oxygen

for euthanasia followed by exsanguination. The rats were observed for gross necropsy examinations of kidney and other organs. Kidney samples were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.2) for 24 h at 40C, and washed with PBS for 4 times each 45 minutes, then post fixed in 1% aqueous Osmium Tetroxide for 2h later washed with deionized distilled water for 6 times each 45 minutes, dehydrated in series of graded alcohols one time each for 45 minutes, infiltrated and embedded in araldite 6005 resin or spur resin. Incubated at 800C for 72h for complete polymerization. Ultra thin (60 nm) sections were made with a glass knife on ultra microtome (Leica Ultra cut UCT-GA-D/E-1/00), mounted on copper grids and stained with saturated aqueous Urenyl acetate (UA) and counter stained with Reynolds lead citrate (LC). Transmission electron microscopy was carried under TEM (Model: Hitachi, H-7500 from JAPAN) at required magnifications as per the standard procedures at RUSKA Laboratory. Representative photographs of the various changes noticed were taken, wherever essential.

RESULT AND DISCUSSION

The purpose of this study was to evaluate the time course of

changes in urinary parameters and ultrastructural pathology noticed in kidneys after seven consecutive days of Gentamicin (subcutaneous, single dose daily) administration in male Sprague Dawley rats. Animals treated with Gentamicin (30 and 100 mg/kg/day) showed no clinical signs and tolerated the treatment. On day 8, significantly reduced body weight and food consumption was seen in the high dose rats treated with Gentamicin. There is paucity of reported information on change in body weight or food consumption with similar type of experiments/doses of Gentamicin administration.

Urinalysis: No significant treatment related findings seen on Day 4 and 8

with regard to presence of bilirubin, ketone, blood, changes in pH, urobilinogen, nitrite, leukocytes and semi-quantitative parameters. Microscopic changes in urine sediments was characterized by presence of granular cast in both Day 4 and 8 with Gentamicin treated animals. On Day 4, significantly increased calcium with Gentamicin at 30 and 100 mg/kg, and significantly increased sodium with Gentamicin at 30 mg/kg. On Day 8, significantly increased total protein and calcium seen at both dose levels of Gentamicin; increased NAG, urinary protein and mALB, and decreased BUN and creatinine at 100 mg/kg of Gentamicin. Trend in increased urinary glucose was seen with 100 mg/kg of Gentamicin on Day 8. Significantly, increased potassium with Gentamicin at 30 mg/kg and decreased sodium with Gentamicin at 100 mg/kg was observed. These changes were considered as incidental due to lack of dose and temporal relationship. All other parameters estimated on Day 4 and 8 were comparable to the respective control groups (Table 1). The proteinuria and damage to podocytes were seen with gentamicin administration on day 8 in the current study that demonstrates the correlation of the findings with pathophysiology of renal injury. NAG is a lysosomal enzyme that is well characterized, and is found predominantly in lysosomes of the PCT. The results showed lack of change in urinary NAG excretion on day 4 in Gentamicin treated animals that correlated with minimal degree of tubular degeneration or necrosis. However, on day 8, significantly increased urinary NAG seen with 100 mg/kg of Gentamicin correlated with moderate or severe degeneration in tubules. Creatinine is synthesized in the liver and PCT at a constant rate, transported to muscles via blood, freely filtered and do not get reabsorbed. In this study, excretion of creatinine on day 4 was comparable among, Gentamicin and control animals. However, decreased creatinine seen in 100 mg/kg of Gentamicin on day 8. This has correlated with loss of weight (cachexia), reduced food consumption and electron microscopic changes in kidneys indicating decline in kidney function.

BUN and creatinine are nitrogenous waste products of metabolism removed from the blood by the kidneys. During glomerular filtration, urea passes from blood to the glomerular filtrate, the fluid that is the precursor of urine. The concentration of urea in the filtrate as it is formed is similar to that in plasma so the amount of urea entering the proximal tube of the nephron from the glomerulus is determined by the glomerular filtration rate (GFR). During the passage of the filtrate, urea is reabsorbed and secreted back to filtrate which results in 30-50 % of the filtered urea appearing in urine. In case of kidney damage, the urea

Venkatesha U. et al. J Sci Res Pharm, 2018;7(6):64-69

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will not properly get filtered into urine. This was exactly reflected in the current study where there were no significant changes in BUN excretion on day 4 with Gentamicin that correlated with minimal electron microscopic changes seen in kidneys. However, decrease in urinary BUN excretion seen in 100 mg/kg of Gentamicin that correlated with severe electron microscopic changes.

Urinary calcium increased by several folds (2.5 and 3.1 folds on day 4 over control; 6.1 and 7.0 folds on day 8 over control) in dose dependent and temporal manner on day 4 and day 8 with Gentamicin at 30 and 100 mg/kg respectively. There are abundant evidence of experiments in animals that single and repeated infusion of Gentamicin decrease tubular reabsorption of calcium leading to increased excretion of this divalent cation in to urine (Parsons et al, 2000). Increased loss might be because of interference in parathormone (PTH- parathyroid hormone) activity. It has been observed that Gentamicin caused increased urine calcium in PTH stimulated control rats as it interferes with renal PTH-mediated calcium transport (Elliott et al, 1987). Parsons et al, 2000 explained that the gentamicin acts to reduce tubular calcium reabsorption is early distal tubule in case of hypercalciuria.

Transmission electron microscopy: Control kidney samples showed epithelial cells with normal

uniform nucleus with healthy nucleolus, tight junctions with normal nucleus, brush border area with normal nucleus and epithelial cells (Figure 1). On Day 4, minimal nuclear changes and indistinct cytoplasmic organelles seen with 30 mg/kg dose group of Gentamicin. Similarly, kidneys of animals administered Gentamicin at 100 mg/kg showed increased myeloid bodies, loose cell junction, pyknotic nucleus, elongated mitochondria, loose cell junction, moderate margination and dilated capillaries (Figure 1).

Kidneys of animals administered Gentamicin at 30 mg/kg on Day 8 showed many lysosomal /myeloid bodies in PCT, dilated capillaries, dilated inter cellular area, condensed foot processes, indistinct endoplasmic reticulum, dark and dense mitochondria (Figure 2). Similarly, kidneys of animals administered Gentamicin at 100 mg/kg on Day 8 showed karyolysis, mild swelling, distribution of blocks of chromatin throughout nucleoplasm, moderate margination of chromatin, loose cell junction, mild electron lucent and shabby appearance of mitochondria, empty perinuclear cytoplasm (or increased

perinuclear space), multiple myeloid bodies, electron dense bodies with different shapes and sizes, podocytes present (Figure 2).

Alarifi et al., 2012, Ashraf et al., 2017, Constantin Craciun and Cristina Pasca, 2014, reported similar to these study findings. The earliest changes seen in Gentamicin at 100 mg/kg on Day 4 with increased myeloid bodies, loose cell junction, pyknotic nucleus and elongated mitochondria is correlated with Girard Hottendorf and Patricia Williams, 1986, who reported presence of myeloid bodies that consists of aggregates of concentrically whorled membranes giving a lamellar structure with Gentamicin administered to rats at 160 mg/kg, single intraperitoneal dose. Donald et al., 1976 reported few of these changes with Gentamicin at 30 mg/kg, S/C on Day 8. These changes include mitochondrial swelling and destruction, vacuolization of cytoplasm, simplification and loss of basal membrane infolding, disarray of microvillus borders and cytoplasmic debris including numerous myeloid bodies. Epithelia of the pars recta, distal convoluted tubules, and cortical collecting tubules containing scattered dense but intact lysosomes with myeloid bodies. The earliest ultrastructural changes in proximal tubule involves an increase in size and number of secondary lysosomes (cytosegresomes). Cytosegresomes contain electron-dense, membranous material called myeloid bodies. Myeloid bodies consist of aggregates of concentrically whorled membranes, giving them a lamellar structure. Mitochondria and cisternae of rough endoplasmic reticulum are swollen. Large vacuoles are present near the base of the cell. Multicentric and unicentric myeloid bodies lie free in the cytoplasm.

The transmission electron microscopic findings with Gentamicin treated animals in the current study is in parallel to report from Donald Houghton et al., 1976 who studied effect Gentamicin in rats at 40 mg/kg on Day 7, 10, 12 and 14 days. On Day 7, they recorded epithelial cells containing increased number of large, irregular and dense lysosomes, swollen mitochondria, vacuolation and dilated cisternae of the rough endoplasmic reticulum. Gentamicin induced nephrotoxicity is due to direct perturbation of cellular or subcellular organelle function occurs most commonly in the highly metabolically active proximal tubules and less so in the distal tubules and collecting ducts. The injury can be related to accumulation of gentamicin in the lysosomes. Lysosomal overload of gentamicin results in compromised lysosomal membrane integrity.

Table No. 1: Urine markers levels on Day 4 and Day 8

Treatment POT SOD CHLO CAL GLU CREA BUN TP NAG mALB CrCL

(mmol/L) (mg/dL) (g/dl) (U/L) (mg/l)

Day 4

Control Mean 93.77 65.44 405.64 2.62 10.30 32.03 784.12 0.16 11.74 7.32 2138.05

SD 45.59 12.18 200.86 0.99 9.09 20.32 373.23 0.11 7.82 3.34 535.08

Gentamicin 30 mg/kg

Mean 81.23 86.85** 288.25 6.63* 7.90 26.27 733.65 0.16 12.74 13.41 2389.17

SD 18.01 12.52 248.55 3.26 3.54 6.81 255.98 0.05 9.91 11.23 1066.26

Gentamicin 100 mg/kg

Mean 82.12 76.34 355.59 8.19*** 7.80 21.96 605.60 0.14 14.01 8.85 2000.53

SD 18.57 12.45 161.36 3.92 2.66 5.86 139.32 0.06 2.45 2.14 672.12

Day 8

Control Mean 81.81 67.53 325.24 2.95 7.00 29.78 781.31 0.16 19.04 7.12 2154.04

SD 15.43 12.80 187.17 1.22 1.56 6.80 193.08 0.04 8.69 4.02 386.24

Gentamicin 30 mg/kg

Mean 97.66* 71.70 298.19 18.18** 7.20 29.98 630.37 0.24* 20.37 17.73 2153.36

SD 8.81 19.44 151.47 10.10 1.69 5.20 253.93 0.05 9.00 16.76 497.18

Gentamicin 100 mg/kg

Mean 74.02 50.93* 364.68 20.57*** 35.1 14.85*** 392.06*** 0.31*** 46.30*** 77.74*** 567.94***

SD 11.83 9.41 140.15 7.87 49.6 2.87 108.23 0.09 21.09 11.12 346.45 N=5, POT – Potassium, SOD-Sodium, CHLO-Chloride, CAL-Calcium, GLU-Glucose, CREA-Creatinine, BUN-blood urea nitrogen, TP-Total protein, NAG-N acetyl glucosamine, mALB-microalbumin, CrCL-creatinine clearance

* One way ANOVA followed by Dunnett’s multiple comparison test (P<0.05);** One way ANOVA followed by Dunnett’s multiple comparison test (P<0.01); *** One way ANOVA followed by Dunnett’s multiple comparison test (P<0.001)

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Control. Magnification 7K

Epithelial cells showing normal uniform nucleus with healthy nucleolus (arrow).

Gentamicin at 30 mg/kg, Magnification 4K

Minimal nuclear changes (arrow) and indistinct cytoplasmic organelles

Gentamicin at 100 mg/kg, Magnification 4K

Elongated mitochondria (circle), increased myeloid bodies, loose cell junction, pyknotic nucleus and dilated capillary (arrow).

Fig. 1: Transmission electron microscopy of control and Gentamicin administered at 30 and 100 mg/kg, Day 4

Venkatesha U. et al. J Sci Res Pharm, 2018;7(6):64-69

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Control, Magnification 5K Tight junctions with normal nucleus, brush border area with normal nucleus (arrow) and epithelial cells (dotted arrow).

Gentamicin at 30 mg/kg, Magnification 1K Presence of many lysosomal bodies in PCT (arrow), dilated capillaries, dilated inter cellular area, condensed foot processes (circle) and indistinct endoplasmic reticulum.

Gentamicin at 100 mg/kg, Magnification 5K Karyolysis (dotted arrow), multiple myeloid bodies (circle), blocks of chromatin distributed in entire nucleoplasm, margination of chromatin, loose cell junction, mild electron lucent and shabby appearance of mitochondria, perinuclear cytoplasm is empty and presence of podocytes.

Fig. 2: Transmission electron microscopy of control and Gentamicin administered at 30 and 100 mg/kg, Day 8

CONCLUSION

Current experimental data characterizes the dose and

duration dependent urinary parameters and ultrastructural changes in kidneys with Gentamicin administration in male Sprague Dawley rats. The presence of myeloid bodies in the tubular epithelial cells following gentamicin treatment as early as Day 4 of the treatment suggests that they represent lysosomal isolation of gentamicin bound cytoplasmic structure and serve as an ultrastructural marker for gentamicin induced nephrotoxicity.

ACKNOWLEDGEMENTS

Authors thank Glenmark Pharmaceuticals Limited, Mumbai

for providing facility for this work. No external grant has been used for this work.

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How to cite this article:

Venkatesha Udupa and Veeru Prakash. SEQUENTIAL CHANGES IN ELECTRON MICROSCOPY OF KIDNEYS AND URINARY PARAMETERS IN GENTAMICIN INDUCED NEPHROTOXICITY IN RATS. J Sci Res Pharm 2018;7(6):64-69.

Conflict of interest: The authors have declared that no conflict of interest exists.

Source of support: Nil