Indian Journal of Experimental Biology
Vol. 52, April 2014, pp. 332-343
Impairment of renal structure and function following heterogeneous chemical
mixture exposure in rats
Kiran Morya & Kauresh D Vachhrajani*
Division of Environment and Toxicology, Department of Zoology, Faculty of Science,
The Maharaja Sayajirao University of Baroda, Vadodara 390 002, India
Received 14 January 2013; revised 27 December 2013
Renal structural and functional alterations following an exposure to a heterogeneous chemical mixture (HCM) of
phthalic acid di butyl ester, 1, 2–dichlorobenzene, cadmium chloride and chromium trioxide, administered through oral
gavage in low doses (1/100 and 1/1000 of LD50 value of individual chemical) for 60 days, followed by withdrawal till
120 days resulted in significant rise in kidney lipid peroxidation and fall in the activities of enzymatic antioxidants.
However, withdrawal of HCM treatment restored most of these altered parameters. Degenerative changes in the kidney
included proximal convoluted tubules devoid of brush boarder with cytoplasmic blebbing, dissolution and sloughing of
nuclei. Cortical glomeruli were also affected with epithelial disintegration, pyknosis of podocyte nuclei and mesengial cell
hyperplasia. The morphological alterations recovered fully in the low dose compared to the high dose treatment group.
Keywords: Heterogeneous chemical mixture, Kidney structure and function, Lipid peroxidation, Low doses, Oxidative stress
Although the ecosystem or even the humans are
exposed to complex chemical mixtures the majority of
existing toxicological data are for single compound or a
mixture of few chemicals, mostly of the same class1-5.
The studies on exposure of individual chemical fail to
deal efficiently with the toxic potentials of chemical
mixtures. A variety of environmentally persistent
chemicals are being released into the environment and
are consistently found as contaminant in vegetables,
fruits, fishes and even in human blood, milk, urine
and hair samples6,7. However, in several cases the
detected concentrations are below the levels shown to
cause adverse effects in laboratory animals when
administered individually8,9. Much of the literature on
the toxicity of individual substances has detailed the
toxic responses at relatively higher dosage of
exposure than those actually detected as
environmental levels. Several substances, such as
cadmium, arsenic, chromium, lead, mercury, nickel,
dioxins, polycyclic aromatic hydrocarbon, phenolic
compounds, phthalates, and pesticides are considered
to be carcinogenic, however, evidences that these
substances cause cancer at detected environmental
levels are very less10-12. Epidemiological studies over
_________
*Correspondent author
Mobile: 91-9427839382
E-mail: [email protected]; [email protected]
a last few decades have described the health hazards
by chemical pollution. Although combined chemical
exposures are common events, epidemiology of
multiple chemical exposures is a relatively
unexplored field in occupational and environmental
health. A survey of chemical body burden in US
population by Department of Health and Human
Services, Centre for Disease Control and Prevention,
USA noted presence of 212 chemicals in the blood
and/or urine samples wherein 75 of these compounds
were recorded for the first time13. It is suspected that
total number of environmental pollutants and other
chemicals as body burden could be much higher than
detected. Although, the presence of chemicals at very
low levels does not necessarily indicate that it is
biologically available at toxic levels or it is the
causative agent of a particular toxic response, mere
presence of such several chemicals does magnify the
potential risk to an individual. Unfortunately, it is not
known how many such chemicals are present at very
low or undetectable levels in our body. The use of
different combination drugs/herbal extracts in
therapeutics/cosmetics has also increased in past two
decades14,15. The therapeutic doses of drugs, if not
cleared out from body completely, may result into low
level body burden with great potentials to interact
with already existing toxicant burden16. This is bound
to lead to a very challenging situation where the
MORYA & VACHHRAJANI: LOW DOSE CHEMICAL MIXTURE TOXICITY IN KIDNEY
toxicants and drugs form much different mixture of
chemicals in the body than one could expect.
The toxicity outcomes of chemical mixture are
based on the interactions of component chemicals.
The mixtures are classified based on either number or
the variety of component chemicals17. The
toxicological evaluation of the entire mixture
describes dose-response relationship and potential
hazards, although the interactions of component
chemicals and the mode of action of the mixture
cannot be determined18-20. However, these aspects are
studied in detail earlier and therefore do not require
retesting of the toxicity of individual component of
the mixture21,22. Further, the aim of the present
experiment is to show that very low level of
heterogeneous chemical mixture exposure can be
potentially toxic. In the present investigations, the
heterogeneous mixture has been assessed for its
potentials of structural and functional alterations in
kidney of the rat. The test mixture included phthalic
acid di butyl ester, 1, 2 –dichlorobenzene, cadmium
chloride and chromium trioxide, administered through
oral gavage at low doses for 60 days following
withdrawal for next 60 days. These chemicals are
environmentally persistent constituents of the
discharges from various industries of central and
south Gujarat which are released into the Gulf of
Khambhat, Gujarat, India and thus pollute various
resources and pose health hazards23-25.
Materials and Methods
AnimalsAdult male Wistar rats weighing
300-350 g were obtained from Sun Pharma Advance
Research Company LTD., Vadodara. The animals were
allowed to acclimatize in departmental animal house for
15 days. These animals were housed in standard
conditions (22±3 °C, L:D 12:12), fed with commercial rat
chow and water ad libitum. The experiments on animal
333
were approved by Institutional Animal Ethics Committee;
animal handling and all procedures on animals were
carried out in accordance with the guidelines.
All the chemicals were purchased from SISCO
Research Laboratories, Gujarat. The chemicals used
in the study were of analytical grade or of the highest
grade commercially available. For the preparation of
chemical mixture phthalic acid di butyl ester and 1,
2–dichlorobenzene were mixed in corn oil while
cadmium chloride and chromium trioxide were
dissolved in distilled water at a concentration equal to
1/100 and 1/1000 of LD50 value of individual
chemical26-29 (Table 1). The administered doses are
even lower than the Non Observable Adverse Effect
Level (NOAEL) of individual experimental
chemical30-33. The doses were also compared with the
Lowest Observable Adverse Effect Levels (LOAEL)
of individual chemical. At the time of dosing all the
toxicants were mixed to a total volume of 0.8 mL and
administered daily through oral gavage.
Protocol for toxicity and recovery study Each
group comprised of 20 rats except for group I
designated as zero or initial day control group that
comprised of 10 rats (Table 2). Rats of group I
(day zero control group) were sacrificed on day 1.
Rats of group II (control group- 60 day) were
maintained on normal diet and that of group III
(vehicle control group- 60 day) were administered
0.8 mLof corn oil mixed with water. Group IV (high
dose HCM- 60 day) and V (low dose HCM- 60 day)
rats were exposed to chemical mixture (phthalic acid
di butyl ester, 1, 2 –dichlorobenzene, cadmium
chloride and chromium trioxide) at a dose level equal
to 1/100 and 1/1000 of LD50 value of each chemical
compound, respectively (Table 2). On 61st day 10 rats
from groups II-V were sacrificed after urine and
blood collection and remaining 10 animals from each
group were kept for withdrawal study for further
Table 1 Toxicological profile of selected compounds
LD 50 (oral)
NOAEL/LOAEL* Doses
Administered Doses
(mg/kg body weight)
(mg/ kg body weight)
LD50/100
LD50/1000
(mg/kg body weight) (mg/kg body weight)
Cadmium chloride
88
9-4
0.88
0.088
(Onwuka et al., 2010)
(Guilhermino et al., 1998)
Chromium trioxide
80
2.4
0.80
0.080
(Szelag et al., 2003)
(EPA, 1991)
Phthalic acid dibutyl ester
8000
50-138
80
8
(ATSDR, 2001)
(NTP, 1995)
1,2-dichloro benzene
500
25
5
0.5
(NTP, 1985)
(Robinson et al., 1991)
NOAEL: No Observable Adverse Effect Level
LOAEL: Lowest Observable Adverse Effect Level
Selected compound
INDIAN J EXP BIOL, APRIL 2014
334
Table 2Distribution of experimental groups and treatment schedule
Groups
I
II
III
IV
V
VI
VII
VIII
IX
Treatment
Zero (initial) day control
Control (no treatment)
Control (vehicle administration for 60 days)
Heterogeneous chemical mixture high dose treated group (1% of LD50 dose/animal/day) (administered for 60 days)
Heterogeneous chemical mixture low dose treated group (0.1% of LD50 dose/animal/day) (administered for 60 days)
Control (no treatment)
Control (vehicle administration for 60 days followed by no treatment for withdrawal phase further till 120 days)
Heterogeneous chemical mixture high dose treated group (1% of LD50 dose/animal/day) for 60 days followed by no
treatment for withdrawal phase until 120 days.
Heterogeneous chemical mixture low dose treated group (0.1% of LD50 dose/animal/day) for 60 days followed by no
treatment for withdrawal phase until 120 days.
60 days (total 120 days experimentation). In the
withdrawal set the normal and vehicle control groups
were designated as Groups VI (control group- 120 day)
and VII (vehicle control group- 120 day), they were
not administered even vehicle and supplied normal
diet and water ad libitum. The high and low dose
toxicant withdrawal groups were designated as
Groups VIII (high dose HCM- 120 day) and IX
(low dose HCM- 120 day), were not administered any
toxicant during withdrawal phase and were supplied
normal diet and water ad libitum (Table 2).
Analytical proceduresUrine samples were
collected in flasks (maintained in ice pack) over a 24h
period by placing individual animal in metabolic
cages. Blood samples were collected from retro
orbital sinus plexus to obtain serum. The serum and
urine samples were stored at -4°C until assayed. The
urine volume and urinary concentration of creatinine
were estimated and from this data Glomerular
Filtration Rate (GFR) was calculated34 as below:
GFR (mL/min) =
Urinary creatinine ( mg/dL ) × urine volume ( mL ) × 1000 ( g )
Serum creatinine ( mg/dL ) × body weight ( g ) × 1440 ( min )
Serum urea and creatinine concentration were
determined according to Urease method based on
Berthelot’s reaction (Bayer Diagnostic India Ltd.,
India) and kit method of Jaffe reaction (Transasia
Biomedicals Ltd., India), respectively. Potassium and
sodium concentrations were determined by flame
photometry. After blood collection the animals were
sacrificed for tissue collection. The kidneys were
excised, blotted free of blood, weighed and utilized for
histological examination and biochemical estimations.
Biochemical estimations in renal tissues
Lipid peroxidation assayThe level of lipid
peroxidation (LPO) was measured as the
Autopsy
day
01
61
61
61
61
121
121
121
121
malondialdehyde (MDA) content35. Tissue homogenate
was shaken with thiobarbituric acid reagent and
centrifuged. The supernatant was taken and
malondialdehyde content was determined at 532 nm.
Non enzymatic antioxidant assayThe reduced
glutathione (GSH) was estimated using the procedure
described by Beutler et al36. The assay mixture
containing homogenate and precipitating reagent were
centrifuged to obtain supernatant. The supernatant
was removed and disodium hydrogen phosphate and
Ellman’s reagent were added. The colour developed
was read against blank containing phosphate solution
and Ellman’s reagent at 412 nm.
Antioxidant enzyme assayThe dismutation of
superoxide anion was assayed according to the
method of Marklund and Marklund37. The assay
mixture for the enzyme superoxide dismutase (SOD)
contained Tris–HCl buffer, distilled water,
homogenate and pyrogallol. The samples were
immediately read at 470 nm against blank containing
all the content except homogenate and pyrogallol at
every 1 min interval, for 3 min. The protein content
was measured as per Lowry et al38. The activity of
catalase (CAT) of decomposing H2O2 into water and
oxygen was assayed by the method of Sinha39. The
assay mixture contained H2O2, sodium phosphate
buffer and distilled water. Subsequently homogenate
was added to initiate the reaction. To stop the reaction
dichromate-acetic acid reagent was added after
30 and 60 seconds. The tubes were then heated for
10 min and read at 570 nm. The reduction of free
H2O2 to water by glutathione peroxidise (GPx) was
assayed by the method of Rotruck et al40. The assay
mixture contained sodium phosphate buffer, sodium
azide, reduced glutathione, H2O2, homogenate and
distilled water. The reaction was terminated by
addition of trichloro-acetic acid and centrifuged. The
supernatant was removed and disodium hydrogen
MORYA & VACHHRAJANI: LOW DOSE CHEMICAL MIXTURE TOXICITY IN KIDNEY
phosphate and Ellman’s reagent were added. The
colour developed was read at 412 nm against blank
containing phosphate solution and Ellman’s reagent.
The activity of glutathione-S-transferase (GST) was
assayed by method of Habig et al41. The assay
mixture contained potassium phosphate buffer,
homogenate, distilled water and 1-chloro-2,
4-dinitrobenzene and incubated at 37 °C for 10 min.
Reduced glutathione was added and the optical
density was measured against reagent blank at 340 nm
immediately for 3 min at 30 seconds interval.
Histological examinationThe right kidney was
dissected out and fixed in 10% neutral formalin. The
tissues were processed for paraffin embedding and
5 µm thick sections were stained with hematoxylin
and eosin for microscopic examination.
Statistical analysisAll the data were expressed as
mean±SE. The statistical analysis of the data was done
using one way ANOVA followed by Bonferroni
comparison test using Graph Pad Prism. All statistical
tests were run at 95% confidence interval and P<0.05 was
taken as the level of statistical significance. Statistical
comparisons were made between HCM treated and
vehicle control/control group on respective days.
Results
Renal functionsFollowing the exposure to
heterogeneous chemical mixture the rate of water
uptake was reduced in the treatment groups till day 60
and exhibited a progressive increase comparable to
the control values by 120 days. A corresponding
change was noted in the volume of the urine
formation which significantly reduced in the
treatment goups on day 60 (Table 3). The parameters
of kidney function test like serum and urinary
creatinine levels increased significantly after 60 days
HCM exposure in groups IV and V as compared to
335
control group III (Table 3). However, withdrawal of
HCM treatment showed recovery to the levels as in
control rats (group VII). A mild increase (statistically
insignificant) in serum urea level was observed in
both HCM exposed and withdrawal groups (Table 3).
In toxicity study of HCM, a statistically significant
(P<0.01) decrease in 24 h urine volume was found in
high dose group IV, whereas a mild decrease in urine
volume observed in low dose group V was found to
be statistically insignificant. A slight decrease in
glomerular filtration rate (GFR) was found in sixty
days HCM treated groups.
Table 4 compares the results of serum electrolytes
level of HCM exposed and recovery groups with
control. A significant (P<0.001) rise in the serum
sodium concentration was observed in the 60 days
HCM treated rats. At the end of the withdrawal period
the serum sodium concentration was comparable to
that of control group VII. In HCM toxicity study, the
level of serum potassium increased insignificantly in
high dose treated group, whereas significant (P<0.01)
increase was observed in low dose treated group.
Table 4Serum analysis for electrolyte concentrations
[Values are mean ± SE]
Groups
Serum sodium
(mmol/L)
Serum
potassium
(mmol/L)
Sodium/potassium
ratio
I
II
III
IV
V
VI
VII
VIII
IX
142.90 ±1.30
141.90 ±0.84
140.80 ±0.81
152.00 ±1.01c
154.20 ±1.35c
141.80 ±1.79
144.30 ±1.99
145.00 ±1.36
145.10 ±2.03
4.57 ±0.11
4.29 ±0.26
4.55 ±0.14
5.03 ±0.19
5.69 ±0.29b
5.27 ±0.11
5.33 ±0.15
5.12 ±0.27
5.43 ±0.27
31.45 ±0.93
35.04 ±2.26
30.96 ±1.18
30.59 ±1.46
27.91 ±1.48
26.94 ±0.48
27.26 ±1.40
28.83 ±1.91
26.16 ±1.37
P values a<0.05, b<0.01 and c<0.001
Table 3Serum and urine analysis for indicators of kidney function
[Values are mean ± SE]
Groups
Serum urea
(mg/dL)
Serum creatinine
(mg/dL)
Urine creatinine
(mg/dL)
I (Initial Day Control)
II (General Control- 60d)
III (Vehicle Control- 60d)
IV (HCM high dose- 60d)
V (HCM low dose- 60d)
VI (General Control- 120d)
VII (Vehicle Control- 120d)
VIII (HCM high dose- 120d)
IX (HCM low dose- 120d)
20.82 ±0.45
20.61±0.96
21.52±0.80
23.97±0.93
24.82 ±1.74
21.04 ±0.84
21.48 ±0.59
24.80 ±1.01
25.00±0.58
0.40 ±0.021
0.53 ±0.057
0.49 ±0.038
0.88 ±0.044c
0.69 ±0.025a
0.49 ±0.021
0.51 ±0.021
0.58 ±0.051
0.49 ±0.041
11.23 ±0.22
11.01±0.58
12.58 ±0.81
20.67 ±0.81c
17.35 ±1.03b
12.6 ±0.82
12.9 ±1.03
12.28 ±0.63
11.57±0.39
P values a<0.05, b<0.01 and c<0.001
Urine volume Glomerular filtration
(mL)
rate (mL/min)
10.93 ±0.12
12.01±0.71
11.40 ±0.29
8.03±0.17b
9.60 ±0.53
13.62 ±0.21
14.00±0.71
12.80 ±0.54
13.00 ±0.68
0.64 ±0.07
0.57±0.09
0.61 ±0.05
0.49 ±0.03
0.56±0.04
0.58 ±0.03
0.57 ±0.07
0.68 ±0.08
0.59±0.03
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INDIAN J EXP BIOL, APRIL 2014
Biochemical estimations in renal tissues
Lipid peroxidation assayIn toxicity study groups,
high dose HCM treated rats exhibited a significant
higher LPO compared to normal rats (Fig. 1a). But the
increase was non-significant in low dose HCM treated
and recovery groups (VIII and IX) when compared
with their respective controls. Thus withdrawal from
HCM treatment showed recovery in membrane lipid
peroxidation when compared with HCM treated
toxicity study groups.
Effect on enzymatic and non enzymatic
antioxidantsIn toxicity study, a marginal change in
the enzyme activity and metabolite levels were
observed in the 60 days HCM groups in comparison
to control group III (Fig. 1b-f). A statistically
significant (P<0.05) decrease in the SOD activity was
found in high dose treated HCM group IV, whereas
non-significant decrease in the activity was observed
in low dose HCM treated group V. A statistically
highly significant decrease in the activities of GPx
and CAT were observed in HCM treated groups.
Kidney GST activity was decreased (P<0.05)
significantly in high dose HCM treated group whereas
the decrease was non-significant in low dose HCM
treated group. In recovery group, withdrawal from
HCM treatment for sixty days showed recovery in all
antioxidant parameters, except CAT in group VIII
(high dose HCM treated + recovery) which showed a
significant decrease (P<0.05) when compared to
120 days control group VII (Fig. 1b-f).
Effect on histoarchitecture of kidney The
functional and structural unit of the kidney, the
nephron, is subdivided into glomerulus and tubular
segments or regions. These are proximal convoluted
and straight tubule (PCT), descending and ascending
thin limbs and thick ascending limb of Henle (LH),
macula densa; located within the distal portion of the
thick ascending limb, distal convoluted tubule (DCT),
connecting and collecting tubule (CT) (Fig. 2 a-d).
The space between the capillary loops is filled by the
Fig. 1—Parameters of oxidative stress as indicators of kidney function [(a) lipid peroxidation, (b) catalase, (c) superoxide dismutase,
(d) glutathione, (e) glutathione peroxidase, (f) glutathione S-transferase].
MORYA & VACHHRAJANI: LOW DOSE CHEMICAL MIXTURE TOXICITY IN KIDNEY
Fig 2—Transverse section of kidney of control rats [(a)
photomicrograph depicting the distribution of glomeruli (G) in the
cortical (Cort G) and juxtamedullary (Jx Med G) regions. 100X.
(b) showing the details of histoarchitecture of the renal glomeruli.
The glomeruli are lined by parietal epithelilum (PE) while the tuft
of blood capillaries (Cap) is lined by visceral epithelium (VE). The
glomerular capillary tuft distinctly shows the lobule formation; at
least 4 lobes are clearly seen (L1-L4) the darkly stained nuclei of
podocytes (POD) in the capillary lobes are seen.400X. (c and d)
Section of proximal convoluted tubule (PCT). 400X]
mesangial cells, which are modified smooth muscle
cells and have macrophage like functions. The other
cell type is podocyte (parietal layer) which wrap the
capillaries by their branched processes, the
pedicels. Under light microscope, the prominently
stained oblong nucleus of the podocyte can be seen
(Fig. 2 b).
The exposure to heterogeneous chemical mixture at
high doses for 60 days led to severe changes both in
the cortical and medullary regions of the kidney, the
glomeruli had distorted capillary lobes where both the
parietal and visceral epithelia were significantly
disintegrated leading to prominent shrinkage of the
capillary tuft (Fig. 3 a, b). Of all the animals studied,
one of the animals exhibited the mesengial cell
hyperplasia and degeneration of the epithelia lining
the corpuscle (Fig. 3 b). Most severe degenerative
changes were observed in the proximal convoluted
337
Fig. 3—Transverse section of kidney from rats exposed to high
doses of heterogonous chemical mixture for 60 days. [(a) Showing
the external cortical region (cortical glomaruli). The glomaruli are
shrunken therefore exhibiting a wider gap in the bowmen’s
capsule. The stromal connective tissue exhibited loosening and
some extent of degenerative changes. Some of the tubules are
severely damaged. 200X. (b) The structural integrity of the
glomerular tuft is disorganized. The darkly stained nuclei are
plenty which indicate mesengial cell (MC) hyperplasia. The
macula densa (MD) and the adjacent extra glomerular mesengial
cells (EGMC) are also seen. 200X. (c to e); Showing the severe
degenerative changes like loss of brush border, cytoplasmic
blabbing, cytoplasmic dissolution and sloughing of nuclei in
proximal convoluted tubule. The degenerated mass of
cytoplasm/nuclei accumulated in the tubular lumen. 400X].
tubules where the loss of brush border, cytoplasmic
blebbing, cytoplasmic dissolution and sloughing of
nuclei were typically observed (Fig. 3 c-e). The
degenerated mass of cytoplasm/nuclei accumulated in
the tubular lumen and blocked the passage (Fig. 3 d-e).
The cells of the Loop of Henle, distal convoluted
tubules and the collecting ducts also exhibited
necrotic changes where the tubules were significantly
depopulated and the height of the epithelium quite
diminished. The changes in the low dose exposure
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INDIAN J EXP BIOL, APRIL 2014
group on day 60 were much comparable with those
noted in the high dose exposure group; however, these
were less severe (Fig. 4 a-c).
Following withdrawal for next 60 days, the
recovery was seen in the low dose group but not
significantly in the high dose group (on day 120). The
cortical glomeruli were still damaged while the juxta
medullary glomeruli were comparatively normal;
although several of these exhibited podocyte and
mesengial cell damage (Fig. 5 a, b). The epithelium of
the tubular cells also exhibited significant recovery
and the degenerative changes noted on day 60 were
conspicuously diminished on day 120 in the low dose
exposure group (Fig. 5 e-g). It was interesting to note
that the lumen of most of the tubular cross sections
were free from cellular debris in the low dose
exposure group (Fig. 5 c, d and g).
toxic effect of HCM. This is justifiable since different
regions of the tubules have complicated transport
mechanism supported with enzyme system that may
be used for toxicant or chemicals elimination and may
be damaged by such agents. These findings support
the observations of Herak-Kramberger and Sabolic42
and Cristofori et al43 who found that many chemicals
had a direct toxic action and exerted their effects
Discussion
In the present investigation, renal tubules showed
region specific variable degenerative lesions under
Fig.4—Transverse section of kidney from rats exposed to low
doses of heterogonous chemical mixture for 60 days. (a) Showing
the response of renal cortical region to the toxicant exposure
where cortical glomeruli exhibit mesengial cell hypertrophy and
damage to the tubular epithelium. 200X. (b) The accumulation of
cell debris in lumen of tubule. 400X. (c) The parietal
epithelium of Bowman’s capsule exhibit epithelial hypertrophy
(arrow). 400X].
Fig. 5—Transverse section of kidney from the rat exposed to
heterogeneous chemical mixture for 60 days followed by recovery
phase for further 60 days [(a) and b) Disorganized and
disintegrated epithelium resulted into loss of structural features of
cortical glomeruli and the juxta medullary glomeruli. a 200X,
b 400X. (c and d) Cytoplasmic blabbing and cell sloughing in
proximal convoluted tubules (arrow). 400X. (e-g) The glomeruli
and tubular cells exhibited significant recovery and the
degenerative changes noted on day 60 were conspicuously
diminished on day 120 in the low dose exposure
group. e, f 200X, g 400X]
MORYA & VACHHRAJANI: LOW DOSE CHEMICAL MIXTURE TOXICITY IN KIDNEY
principally on the proximal convoluted tubules. It was
clear from present studies that the glomurular
capillary epithelia as well as the podocytes were
damaged and hence can lead to structural disruption
of the filtration unit (the slit formed by pedicels of
podocytes and the capillary fenestrae)44. Although this
can be confirmed by electron microscopic evaluation,
the light microscopic observations and the functional
analysis in present studies are also appropriate
indicators of such disruptive changes. It is significant
to note that the epithelial damage resulted into
sloughing of cells and cytoplasmic blebs, which
blocked the tubular lumen, more importantly in the
PCT. Such blockage would lead to back leak of the
filtrate and hence the glomerular functions are further
altered (Fig. 6a). This feature was prominently seen
on day 60, particularly in the higher dose HCM
treatment group, with significant recovery noted on
day 120 indicating the restoration of the structural and
functional integrity of the glomerular and tubular
epithelium.
The renal tubule consists of discrete segments
which distinctly differ in their sub cellular
organization and hence structural and functional
peculiarities. The transportation across the membrane
is extensively compromised in the PCT since the
microvilli are conspicuously damaged. The
experimental metals, when administered individually,
significantly destroyed the microvilli and membrane
transport associated enzymes45-47. The experimental
organicals at various doses, when administered
individually, altered the membrane bound enzymes
and inhibited the activity of various enzymes involved
in energy metabolism48. The metals potentially
accumulate in the lysosomes, mitochondria and other
organelles altering the functions of tubular segments
and later may lead to autolysis of such over burdened
cells49-52. This was observed as profuse cytoplasmic
blebbing and cell sloughing into the lumen. The
reabsorption is largely affected due to tubular
epithelial damage, however, the rate of urine
formation is also reduced which is further indicative
of the structural alterations of the filtration apparatus
and overall kidney functions.
The administration of HCM brings about
alterations in renal risk factors and impairment of
renal function which is suggested by elevations in
serum creatinine53,54, decrease in urine volume and
glomerular filtration rate55,56, variations in electrolyte
levels57,58 etc. A non-significant decrease in the
339
glomerular filtration rate supports the fact that HCM
causes renal damage and that a decreased GFR may
be due to decrease in fluid intake59 and/or renal
inadequacy60. The decline in GFR may result from
tubular cell injury and its loss in lumen resulting in
tubular obstruction and back leak of glomerular
filtrate (Fig. 6b).
Metals and organicals have been demonstrated to
generate free radicals and induce oxidative stress61,62.
This can lead to lipid peroxidation, inactivation of
cellular enzymatic and non enzymatic antioxidants
and induction of DNA breakages63,64. Therefore, lipid
peroxidation status can directly suggest the extent of
oxidative damage caused by any toxic agent65.
Intoxication with HCM causes a significant increase
Fig. 6—[(a) Diagrammatic representation of the toxic effects on
the renal tubular structure and function. (b) An outline of the
mechanisms of the toxicant action leading to decrease in
glomerular filtration rate].
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INDIAN J EXP BIOL, APRIL 2014
in lipid peroxidation and lead to condition of
oxidative stress. Glutathione is one of the important
antioxidant; therefore, depletion of GSH is correlated
with increased lipid peroxidation. Superoxide
dismutase catalyzes the breakdown of the superoxide
ions where glutathione and catalase help in removing
hydrogen peroxide66 while GST catalyses the
conjugation of reduced glutathione67 and thus the
activity of these enzymes are significant in reducing
the oxidative stress. Activities of these enzymes were
reduced after 60 day HCM exposure. This indicates
the compromised condition of cellular defence
mechanism against such stress. The withdrawal of
HCM treatment appears to moderate the oxidative
stress condition caused by 60 day HCM exposure, the
activity/levels of all assessed antioxidants were
recovered comparatively.
To understand the action of HCM, an overall
insight into the effect on kidney structure and
functions is required. As explained in Fig. 6b, toxicant
exposure may influence the pre/post renal factors.
However, none of such parameter was assessed in
present experiment therefore; the hypothetical
explanations are not discussed; although they may
play significant role in induction of kidney toxicity. It
is believed that the intra renal factors may be
influenced following the HCM exposure. The
glomerular factors, the vascular factors and the cell
damage are significant contributors leading to
structural alterations affecting the glomerular
filtration rate. The cellular exfoliation may be an
important contributor to lumen blockage leading to
cast formation and induce functional changes by
altering physiology and responses of various cell
types of kidney, like induction of oxidative stress.
Earlier studies reported that the selected
experimental chemicals exhibited several targets of
toxicity when administered individually at various
doses68-72. Tarasub et al.73 investigated the toxic
potentials of cadmium chloride at 200 mg/kg body
weight for 5 days and reported renal toxicity by
inducing lipid peroxidation and varying degree of
morphological alterations including swelling and
hypertrophy of proximal tubular cells. In another
study, 3 mg Cd/kg body weight produced only minor
histopathological alterations in the renal tissues74.
Chromium hexavalent was regarded as nephrotoxic at
dose level of 15 mg/kg body weight via
intraperitoneal injection for 1, 2, 4 and 8 days where
serum creatinine and urea nitrogen levels were
altered75. It induced oxidative and nitrosative stress
and adversely affected the activities of brush border
enzymes of renal tubules76.
Adult male rats exposed to 1.0% of phthalate ester
showed signs of kidney toxicity32. Oxidative DNA
damage was reported at 0.5 g/kg dose of phthalate
ester for ten days in an oral gavage study77.
1, 2-dichlorobenzene at 300 mg/kg dose for 10 days
in rats resulted in significant decrease in organ
weight33. Exposure to industrial effluent, reported to
consist of the experimental chemicals and other
toxicants23, at 1 ppm doses for 60 days altered the
structure and functions of adult and pre pubertal male
rat kidneys where the proximal tubular segment was
conspicuously affected22. Studies on components of
effluent and industrial chemicals demonstrated
toxicity on structure and function of male
reproductive organs78-80.
Present studies suggested that low dose exposure to
heterogeneous chemical mixture potentially alter the
structure and function of kidney and these are more
severe than the changes induced by the component
chemicals individually at such low doses. Further, the
types of the changes induced by chemical mixture are
more or less similar to the individual component
chemical toxicity and thus the mode of action of the
component chemical is probably not modified to any
significant level. However, the combination of the
mixture seems to have potentiated the action of the
component chemicals and hence very low doses of
chemical mixture prove to be potentially hazardous.
Acknowledgement
KM is thankful to University Grants Commission,
New Delhi for the award of JRF under RFSMS
Scheme.
Conflict of interest
There is no conflict of interest or of financial
disclosures between the authors.
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