Comparative investigation of kidney mesangial cellsfrom increased oxidative stress-induced diabetic rats by usingdifferent microscopy techniques

Ayse Kose Sargin • Belgin Can • Belma Turan

Received: 8 October 2013 / Accepted: 19 December 2013 / Published online: 29 December 2013

� Springer Science+Business Media New York 2013

Abstract High glucose and increased oxidative stress

levels are the known important mediators of diabetic

nephropathy. However, the effects of these mediators on

tissue damage basically due to extracellular matrix

expansion in mesangial cells have yet to be fully examined

within the context of early stage diabetic nephropathy. In

this study, we attempted to characterize changes in mes-

angial cells of streptozotocin-induced diabetic rats with a

comparative investigation of kidney tissue by using dif-

ferent microscopy techniques. The serum levels of urea and

creatinine of diabetic rats, as biomarkers of kidney

degeneration, decreased significantly compared to those of

age-matched controls. In diabetic rats, there are increased

malondialdehyde and oxidized-glutathione levels as well as

reduced-glutathione and glutathione-peroxidase activity

levels in renal tissue compared to those of the controls. By

using light and electron microscopies, we showed that there

were marked thickening in Bowman’s membrane and

glomerular capillary wall, increased amount of extracellu-

lar matrix often occupying Bowman’s space, degenerations

in tubules, an increased number of mesangial cells in the

network of glomerular capillary walls, and increased

amount of lipid accumulation in proximal tubules in the

renal tissue of diabetic rats. Our confocal microscopy data

confirmed also the presence of irregularity and widened in

glomerular capillaries, their attachment to the Bowman’s

capsule, degenerated heterochromatin, thickening in foci of

glomerular basement membrane, and marked increase in

mesangial cells. These results suggest that a detailed

structural investigation of kidney tissue provides further

information on the important role of mesangial cells in

pathogenesis of diabetic nephropathy.

Keywords Mesangial cells � Diabetes � Oxidative stress �Antioxidant defense � Kidney

Introduction

Diabetes mellitus is a worldwide health issue that is highly

related to vascular dysfunction and chronic vascular

remodeling [1, 2]. It has been suggested that, vascular

dysfunction is the main sign involved in the pathogenesis

of diabetic cardiovascular complications [3, 4], and dia-

betic nephropathy is the major cause of end-stage renal

disease in the industrialized world, resulting, in part, from

both altered matrix metalloproteinase (MMP) activity and

higher concentrations of reactive oxygen species (ROS)

[5]. Therefore, the concert of all pathogenetic changes in

the system under hyperglycemia results in a particular

sequence of events, including structural changes, changes

in the contractile apparatus and the receptors, extracellular

matrix protein deposition, and fibrosis in the smooth

muscle cells [6–8].

Damages in vascular smooth muscle cells, in part, due to

increased ROS level play important role in the pathogen-

esis of diabetes-induced cardiovascular diseases [9, 10].

Basically, marked thickening of total aortic wall, presented

with the thickness of collagen fibers in the region of tunica

adventisia, was observed in the aortic rings from diabetic

A. K. Sargin � B. Can

Department of Histology-Embriology, Faculty of Medicine,

Ankara University, Ankara, Turkey

B. Turan (&)

Department of Biophysics, Faculty of Medicine, Ankara

University, Ankara, Turkey

e-mail: belma.turan@medicine.ankara.edu.tr

123

Mol Cell Biochem (2014) 390:41–49

DOI 10.1007/s11010-013-1953-7

rats, while these changes were prevented by treating dia-

betic animals with antioxidants, accompanied by a clear

reduction in oxidative stress markers of diabetes [9, 10].

Furthermore, it has been also demonstrated that diabetes

induced significant increases in both MMP-2 and MMP-9

plasma gelatinolytic activities [11]. A number of studies

demonstrate important contribution of MMPs into patho-

logical processes, including diabetes [12, 13]. In this pro-

cedure, ROS can drive both activation and expression of

MMPs in cardiovascular system [14, 15].

A number of early observations have shown that the

renal morphologic lesions of diabetic nephropathy in type 1

diabetic patients occur in the glomeruli, arterioles, inter-

stitium, and tubules [16, 17], while glomerulopathy is

considered as the most important structural change char-

acterized by thickening of glomerular basement membrane

and mesangial expansion. Furthermore, increasing evi-

dence suggests that tubular epithelial cells participate in

epithelial–mesenchymal trans-differentiation, and ulti-

mately interstitial fibrosis [18], which suggests that tubular

cells also participate in the progression of diabetic

nephropathy. He et al. [19] studied the role of ROS in early

diabetic nephropathy induced by streptozotocin (STZ) in

rats. Their data demonstrated that an altered redox system

shown by an increased malondialdehyde and decreased

activity of glutathione peroxidase, and superoxide dismu-

tase in the renal cortex, an enhanced inducible nitric oxide

synthetase, total nitric oxide synthase, and constitutive

nitric oxide synthase and a declined nitric oxide were

accompanied by increased extracellular matrix markers

[20].

Clinically, the renal functional parameters are strongly

related with the structural changes, especially with the

degree of mesangial expansion in both type 1 and type 2

diabetes, and glomerular lesions are present in type 1

diabetic patients before the onset of the clinical mani-

festations of diabetic nephropathy [21] as well as mes-

angial expansion is the lesion leading to the loss of

glomerular filtration rate in diabetic nephropathy [16].

Mesangial cells are critical determinants in the accumu-

lation of extracellular matrix in the glomeruli. In mes-

angial cell cultures, high glucose concentrations increase

the synthesis [22] and decrease the degradation of extra-

cellular matrix [23]. Although these changes can result

from direct actions of a high glucose concentration, it is

not entirely clear how the high glucose concentration

exerts its effects on mesangial cells. Therefore, in order to

understand the cellular events causing pathological

extracellular matrix accumulation in diabetic nephropathy,

we aimed to clarify the histopathological alterations in

kidney mesangial cells to have more detailed information

by using light, confocal, and electron microscopy exam-

inations in STZ-diabetic rat kidneys.

Materials and methods

Induction of diabetes

Diabetes was induced in 3-month-old male Wistar rats as

described previously [9]. A week after injection of STZ

(50 mg/kg, single injection as intraperitoneal), blood glu-

cose level was measured by using a glucose analyzer

(Glucotrend, Roche). Rats with blood glucose level at least

over threefold of the control group rats (CON group) are

evaluated as diabetic rats (DM group). All rats were kept

for 5–6 weeks following induction of diabetes. All rats had

free access to standard rat chow and water. A syringe with

a 17-gage needle containing heparin was used to draw

blood from the chest cavity. Then, kidneys were immedi-

ately removed and cleaned. One kidney from each animal

was fixed in 10 % formaldehyde, while another one was

cut longitudially into 2 parts: one part was fixed in 4 %

paraformaldehyde and second part was in 2.5 % glutaral-

dehyde. The plasma fraction was obtained following cen-

trifugation and stored at -80 �C for later determination of

parameters including oxidant stress markers in the rats.

All animal care and experimental procedure were per-

formed by following Ankara University ethics guidelines

(No: 2008-19-76).

Biochemical analysis in blood samples

Blood sample was collected from the tail vein and the glucose

level was measured by using a glucose analyzer (Glucotrend,

Roche). To measure serum enzymes, blood samples were

centrifuged for 20 min at 4,0009g, and then the sera were

stored at -20 �C for subsequent measurement of levels of

urea, uric acid, and creatinine by using conventional colori-

metric assay kits according to the manufacturer’s instructions

(renal function assessment kits, RANDOX).

Biochemical estimation of redox status in renal

homogenate

To perform redox status in kidney tissue, first renal

homogenate was prepared as described previously [24]. The

homogenate was centrifuged at 14,0009g for 1 h at 4 �C.

Protein content in the tissue was determined by an earlier

reported [24] by using bovine serum albumin (BSA) as the

standard. The supernatant was used to measure lipid perox-

idation, as well as both reduced and oxidized glutathione

levels (Amersham Biosciences and Cayman Chemical, Ann

Arbor, MI, USA). The lipid peroxidation in the tissue

homogenate was determined by using the thiobarbituric acid

reactive substances (TBARS) assay kit (ZeptoMetrix Cor-

poration, Buffalo, NY, USA) for the estimation of mal-

ondialdehyde (MDA) content. Glutathione peroxidase

42 Mol Cell Biochem (2014) 390:41–49

123

(GPX) activity in the same supernatant was performed by

using a colorimetric assay [24]. In this study, we determined

GPX activity by measuring the decrease in glutathione, GSH

content after incubating the sample in the presence of H2O2

and NaN3 [25].

Histopathologic examination

For light microscopic evaluation, the removed kidney

samples were fixed in phosphate buffer 10 % formaldehyde

for 2 days and washed after the fixation to remove the

excess fixative and then dehydrated by passing it through

graded alcohol solutions (50, 75, 96, and 100 %). As a

clearing agent, an organic solvent xylol was used to

remove the alcohol. After clearing process, the tissue was

infiltrated with the embedding agent melted paraffin. Fol-

lowing infiltration, the paraffin was allowed to solidify so

that a firm homogeneous mass containing the embedded

tissue was obtained. Embedded samples were sectioned to

3-lm thickness by Leitz-1512 microtome. For this study,

sections were stained with hematoxylin and eosin, Mas-

son’s trichrome, Mallory Azan, PAS, Alcian blue-PAS,

silver metamine, or oil red O. All samples were photo-

graphed by Nikon-Eclipse E600 photomicroscope.

Confocal microscopy investigation

Kidneys were fixed in 4 % paraformaldehyde for 24 at room

temperature. Then, they were first followed for 24 h in 20 %

sucrose and second, waited in 30 % sucrose until the tissue

sinks to the bottom. Then, all the samples were immersed

into cryomatrix. By using a cryomicrotome adjusted to

-20 �C working temperature, the samples were sectioned to

10-lm thickness and placed into poli-L-lysine plated lamels.

These were stained for laminin B2/c1 Ab-2 (Clone D18)

mouse monoclonal anti-rat antibody (Thermo Scientific,

catalog number: MS-1356-R7, UK) and a-smooth muscle

actin (ASMA) mouse monoclonal anti-rat antibody (Sigma,

catalog number: A5228, USA). Briefly, sections washed

with PBS for 30 min, stained for laminin and ASMA, and

then incubated with appropriate fluorescein conjugated

secondary antibody as FITC-conjugated goat anti-mouse

antibody (Jackson, catalog number: 115-095-100, USA) at

37 �C in the dark. For counter staining, 1 lg/mL Hoechst-

33258 was used. After mounting medium, all slides were

stored at -20 �C until microscopic investigations. Immu-

nofluorescence images were obtained by using a scanning

laser confocal microscope (Zeiss-LSM510).

Electron microscopy investigation

Small kidney samples were fixed in a solution of 2.5 %

glutaraldehyde in a phosphate buffer at pH 7.2 for 2 or 4 h

and postfixed in 1 % osmium tetroxide. Later, the materials

were dehydrated in graded ethanol solutions and embedded

in araldite 6005 (Ciba Geigy, Summit, NJ, USA). Sections

were cut on a Leica Ultracut R (Leica, Solms, Germany)

ultramicrotome with a glass knife, semithin sections

(700–1,000 nm) were stained with toluidin blue/azur II,

while ultrathin sections were stained with uranyl acetate

and lead citrate, and viewed on LEO 906-E transmission

electron microscope (LEO Elektronenmikroskopie,

Oberkochen, Germany).

Results

Baseline characteristics of the rats

STZ-treated rats (diabetics) displayed progressive hair loss,

decreased activity, and impaired body weight gain compared

with CON group rats. The diabetic rats have a mean body

weight as 182 ± 11 g, while the controls have 266 ± 10 g,

although their initial body weights were in the similar range

(220–230 g). The final blood glucose level of diabetic group

was 48.1 ± 3.6 mmol/L, whereas this value was

10.1 ± 2.1 mmol/L in CON group. These observed changes

in the general features of STZ-treated rats, such as hair loss

and impaired body weight curve, are general consequences

of hyperglycemia shown previously by our team [26, 27].

In order to confirm a mimic of type 1 diabetes by STZ

injection in rats and its consequence in the renal system, we

monitored the serum levels of urea, uric acid, and creati-

nine of STZ-injected rats (diabetic rats, DM group). As can

be seen from Table 1, the serum levels of urea and creat-

inine decreased in this group significantly compared to

those of the controls, while their serum uric acid levels

were not significantly different among these two groups.

Oxidative stress status of the experimental animals

We also monitored basic biomarker levels of oxidative

stress and antioxidant-defense system in the renal tissue of

DM group rats compared to those of the CON group rats.

As can be seen in Table 2, renal MDA level, as an oxi-

dative stress marker, from diabetic rats is significantly

higher than that of the age-matched controls. Renal redox

state in the rats is presented as tissue reduced glutathione

(GSH) and oxidized glutathione (GSSG) levels. Renal GSH

in diabetic group was found to be significantly lower, while

the GSSG level was significantly higher compared to those

of the age-matched controls. As a third biomarker param-

eter of antioxidant-defense system in the tissue, we mea-

sured GPX activity in the same tissue supernatant, which

was significantly less in diabetic group compared to that of

the age-matched control.

Mol Cell Biochem (2014) 390:41–49 43

123

Table 1 Body weight (BW), blood glucose (BG), urea, uric acid, and creatine levels in serum of experimental animals

Groups BW (g) BG (mmol/L) Urea (mmol/L) Uric acid (lmol/L) Serum creatinine (lmol/L)

CON (n = 8) 266 ± 10 10.1 ± 2.1 6.8 ± 0.4 119.7 ± 7.9 61.8 ± 3.9

DM (n = 10) 182 ± 11* 48.1 ± 3.6* 11.2 ± 0.5* 109.6 ± 5.8 88.7 ± 4.1*

Values are presented as mean ± SEM, and number of animals is given in parentheses

CON control group, DM diabetic group

* P \ 0.05 vs. CON

Table 2 Effects of diabetes on redox state of kidney tissue

Groups MDA (lmol/g protein) GSH (lmol/g protein) GSSG (lmol/g protein) GPX (U/g protein)

CON (n = 6) 112.8 ± 4.3 10.1 ± 2.1 2.1 ± 0.1 590.1 ± 10.7

DM (n = 6) 192.2 ± 5.5* 5.2 ± 1.1* 8.1 ± 1.6* 433.2 ± 10.9*

Values are presented as mean ± SEM, and number of animals are given in parentheses

MDA malondialdehyde, GSH reduced glutathione, GSSG oxidized glutathione, CON control group, DM diabetic group

* P \ 0.05 vs. CON

Fig. 1 Investigation of diabetes-induced alterations on kidney tissue

by light microscopy. Control group (a) results are presented as data on

normal appearance of basement membrane of parietal epithelium,

proximal and distal tubule (arrow), proximal tubule (p), distal tubule

(d) with Alcian Blue-PAS. Bar represents 40 lm (a1). In b1, normal

appearance of proximal tubule (p), distal tubule (d), lumen of blood

vessel (Lu) with silver-metamin, and bar represents 20 lm. In c1 and

d1, there are normal urinary space (Us), proximal tubule (p), distal

tubule (d) with oil red O (bar 40 lm) and mesangial cell (M),

podocyte (po), parietal epithelium cell (pe), and endothelial cell

(E) with Toluidine Blue-Azur II (bar 20 lm). The results for diabetic

group are represented in (b). Basically urinary space (Us), proximal

tubule (p), distal tubule (d), thickening in Bowman’s membrane

(arrow) apparent with an increased amount of extracellular matrix

often occupying Bowman’s space, degenerations in tubules (arrow

head), thickening in glomerular capillary wall (asterisk) obtained with

Alcian Blue-PAS (Bar20 lm) are seen in (a’1). b’1 Proximal tubule

(p), distal tubule (d), lumen of blood vessel (Lu), inflammation and

edema in interstitial tissue (asterisk), thickening in the tubules

membranes (arrow), and tubules with transparent cytoplasm (arrow

head) are seen in silver-metamin stained tissue samples (bar 40 lm).

c’1 Proximal tubule (p), distal tubule (d), lipid accumulation in

proximal tubules (arrow) are apparent in oil red O stained samples

(bar 20 lm), while ruined mesangial cell (M), podocyte (po), parietal

epithelium cell (pe) and endothelial cell (E), urinary space (Us),

inflammatory cells in interstitial region, thickening in basement

membranes, and hyaline degeneration in vessel wall are seen in

Toluidine Blue-Azur II stained samples with bar representing 20 lm

(d’1)

44 Mol Cell Biochem (2014) 390:41–49

123

The present data clearly showed that STZ-induced dia-

betes caused significant disbalance of antioxidant/oxidant

ratio not only in rats but also in their renal system com-

pared to that of the age-matched controls.

Histologic analysis of kidney tissue by using light

microscopy

Each kidney specimen was examined under a light

microscope. There were irregular urinary spaces, degen-

erations in proximal and distal tubules, thickening in

Bowman’s membrane and glomerular capillary walls, and

the urinary space was irregular in renal corpuscle from

diabetic group, while these were in normal appearance in

the CON group (Fig. 1a, b; diabetics vs. controls). In the

same samples from diabetic group, we detected marked

inflammation and edema in the interstitial tissue, thicken-

ing in the tubules membranes by means of connective tis-

sue, and tubules with transparent cytoplasm. In some cases,

the lumen of the tubules was unnaturally widened, the

epithelial cells ruined, tubular basement membrane was

broken and resembled bristles.

Although we did not aim to quantify these parameters in

this study, we measured some parameters of glomerular

capillaries and Bowman capsule. The diameter of glo-

merular capillaries in the diabetic group was increased by

20 % in comparison to that of the CON group, while the

Bowman diameter was not different among the groups. On

the other hand, we observed about 30 % thickening in the

Bowman’s membrane of the samples from the diabetic

group.

Confocal microscopy analysis of kidney tissue

Examination of renal tissue of diabetic group for immu-

nofluorescence images stained with laminin and ASMA by

using confocal microscopy showed that there were marked

enlargement in the glomerular capillary and basement

Fig. 2 Confocal findings in diabetic rat kidney tissue. Control group

(a) results are presented as data on normal appearance of glomerular

capillary (asterisk), basement membrane of parietal layer of Bow-

man’s capsule (arrow) with a slight laminin signal (a2). Arrow head

shows that ASMA positive vessel (b2). Bars in (a2) and (b2) are

20 lm. Diabetic group findings (b) presenting increase in laminin

signals (arrow) (a’2) and marked increase in ASMA signals (arrow)

(b’2). Bars in (a’2) and (b’2) are 20 lm

Mol Cell Biochem (2014) 390:41–49 45

123

membrane of parietal layer of Bowman’s capsule (Fig. 2b)

compared to that of the CON group. Particularly, in dia-

betic group, there was a strong positive laminin signal in

the mesangium. Similarly, the sections from diabetic group

showed a positive ASMA signal increase in the glomerular

mesangium compared to that of the control (Fig. 2a).

Electron microscopy analysis of kidney tissue

Electromicroscopically, an increased number of mesangial

cells in the network of glomerular capillary walls and an

increased amount of lipid accumulation in proximal tubules

were detected in diabetic group. Degenerative glomerulus,

glomerular basement membrane, and tubules appeared in

diabetic rat kidneys. The cells in mesangial matrix had

nucleus with differentially degenerated heterochromatin,

and foci of glomerular basement membrane thickening

were seen in the same sections. In addition, accumulation

of electron dense material in tubular mitochondria and

mitochondrial degeneration were observed in the diabetic

group (Fig. 3b). The tubular basement membranes in dia-

betic rats were thickened and wavy.

The nucleus membrane of mesangial cells in diabetic rat

samples had infiltrated into cell–matrix, which implies the

existence of some contractile filaments such as myosin in

the nucleus. In addition, in the same sections, thin filaments

inside the cytoplasm were concentrated near the nucleus

membrane. Compared with control rats, some modest

glomerular lesions were noted in diabetic rats: glomerular

capillaries were irregular, widened, and attached to the

Bowman’s capsule while mesangial cell number was

significantly higher in diabetic rats. The degree of

Fig. 3 Representative mesangial cell findings in renal tissue of

diabetic rats, sections stained with uranyl acetate and lead citrate, and

viewed on a transmission electron microscope. a Control group

findings in (a3) presenting basal lamina (bl), endothelial cell (E),

nucleus of mesangial cell (N), podocyte (po), and parietal epithelium

cell (pe) with a scale of bar 4.34 lm, and in (b3) presenting lysosome

(L), mitochondrion (m), cytoskeleton elements of cytoplasm (arrow),

nucleus of mesangial cell (N), and rough endoplasmic reticulum (rER)

with a scale of bar 0.56 lm. b Diabetic group results in (a’3)

podocyte (po), thickening in basal lamina (arrow), nucleus of

mesangial cell (N), podocyte cell process (arrow head), mesangial

matrix (asterisk), and lumen of a glomerular capillary (Lu) with bar

scale of 2.01 lm, and in (b’3) representing podocyte (po), nucleus of

mesangial cell (N), erythrocyte (e), basal lamina (arrow head),

mitochondrion (m), podocyte cell process (arrow), nuclear envelope

(curly arrow) with a bar scale of 1.56 lm. In addition, there are

appearances of nucleus of mesangial cell (N), mitochondrion (m),

rough endoplasmic reticulum (rER), polyribosome (r), and clathrin

coated vesicle (arrow head) in (c’3) with a bar scale of 0.72 lm, and

nucleus of mesangial cell (N), basal lamina (bl), branching cytoplas-

mic processes of mesangial cell into the adjacent basement membrane

(arrow) in (d’3) with a bar scale of 0.56 lm

46 Mol Cell Biochem (2014) 390:41–49

123

tubulointerstitial damage was modest. There were some

widened tubuli with incipient atrophy of the epithelial

cells. In addition, slight focal interstitial fibrosis was

observed. The intrarenal arterial vessel showed modest

thickening of the walls.

Mesangial expansion, diffuse thickening of the capillary

basement membrane, and adhesions between adjacent

loops as well as between loops and the parietal layer of

Bowman’s capsule were noted. Trapping of lipid droplets

was observed within the capillaries and collagen deposition

with hyalinization was also found.

The sections from control rats were in normal appear-

ance for all investigated items (Fig. 3a).

Discussion

The present study demonstrated that STZ injection of rats

could induce marked renal damage, as evidenced by the

alterations in the glomerular mesangial matrix clustering as

well as irregularity and widened in glomerular capillaries,

their attachment to the Bowman’s capsule, degenerated

heterochromatin, thickening in foci of glomerular basement

membrane, and marked increase in mesangial cell number.

By using confocal microscopy, the above data were con-

firmed with immunofluorescence images stained with

laminin and ASMA.

The data from the present and previous studies suggest

that oxidative stress is involved in the etiology of diabetes-

induced damage in renal tissue via depressed endogenous

antioxidant-defense mechanism. In this study, the circula-

tory and the renal tissue oxidative stress levels in STZ-

diabetic rats were markedly increased, while their antiox-

idant-defense mechanism was significantly depressed.

Indeed, oxidative stress plays several critical roles in the

development of diabetic renal complications. Diabetes-

induced renal dysfunction is associated with a very low

plasma insulin level, and an unbalanced level of oxidative

stress to antioxidant defense ratio, while oxidative stress in

diabetic animals is reversed with antioxidants [28–32]. The

end products of an advanced glycosylation accumulate in

the plasma and the tissue proteins of diabetic patients

displaying a correlation with the severity of the disease [33,

34]. In chronic renal failure resulted from induction of

diabetes mellitus in animal and human models, high

molecular weight growth factors as hepatocyte growth

factor (HGF) and TGF-b, expression of which increase by

oxidative stress and advanced glycosylation, were shown to

have effects [35]. The strong fibrosis effect of TGF-bresults from both elevation of matrix synthesis and defec-

tive removal of abundant amount of matrix by TGF-b [35].

Diabetic nephropathy is a common complication seen in

diabetic subjects. Poor glycemic control plays a significant

role in this pathology, as shown by both clinical and

pathological studies [36]. Early studies showed that dia-

betic nephropathy is morphologically characterized by the

accumulation of mesangial matrix and thickening of the

glomerular basement membrane, mostly due to the accu-

mulation of matrix proteins [37]. Overproduction of these

matrix proteins considered to be due to the phenotypic

change of mesangial cell. The most prominent lesion in

DM is the accumulation of glycogen in the epithelial cells

of proximal straight tubules, distal tubule of ascending loop

of Henle [38], and macula densa [39] of juxtaglomerular

complex. Former work demonstrates that secondary to

hyperglycemia, renal tubular cells reabsorb glucose

excessively leading to an accumulation in the cell [40].

Kang et al. [41] have indicated that the accumulation has

occured after 1 month in distal tubular and thin piece

epithelial cells, while proximal tubule cells have shown

accumulation after 6 months when they evaluated glyco-

gen accumulation at 1, 3, and 6 months after a single dose

of alloxan treatment. Light microscopic observations of our

study revealed a translucent appearance in some proximal

and distal tubule epithelial cells by diverse stains. On the

other hand, in control group there was no translucent

staining and no findings of degeneration owing to glycogen

accumulation were noted.

In order to search whether the degeneration in renal

tubules were originated from fat accumulation, we per-

formed oil red O, a dye that demonstrates intracellular

neutral fats as discrete round red bodies. Interestingly

enough, there was no staining in the control group sections,

while frozen sections of diabetes group displayed promi-

nent lipid accumulation only in the proximal tubule epi-

thelial cells close to basal location. This basal engagement

of staining was thought to be related to degeneration of

numerous mitochondrial membranes regarding ATP syn-

thesis fulfilled for active transport in the area. In the study

by Wang et al. [42] about lipid metabolism and accumu-

lation in mouse kidneys by oil red O staining, it has been

exhibited marked neutral lipids accumulation in glomerules

and tubule of transgenic mouse.

Myofibroblasts are specialized cells with the properties of

both fibroblasts and smooth muscle cells. They can be

observed in fibrotic tissues and show de novo synthesis of

smooth muscle related proteins including ASMA. ASMA is

produced by smooth muscle cells of vessels and with myosin

it regulates vessel tonus. In addition, this molecule functions

as the source of inflammatory cytokines and matrix [43].

ASMA expression is a well-known feature for myofibro-

blasts and for this reason, ASMA is commonly used as a

marker. ASMA molecules located on actin filaments con-

tract collagen fibrils and decrease motility of cells by

increasing the adhesion of cells to intercellular matrix. This

contractile function allows wound healing in the skin and the

Mol Cell Biochem (2014) 390:41–49 47

123

formation of a closure force in the connective tissue. On the

other hand, myogenic properties in pathological conditions

of visceral organs (as lung, liver, kidney) were not clearly

understood [43]. Chebotevera et al. [44] have declined that

together with CD34, ASMA can be used as a marker to

evaluate the activation and prognosis of disease in glomer-

ulonephritis patients. In control group of our study, ASMA

was expressed only in the smooth muscle layer of vessels, as

expected in a normal tissue. In the experimental group,

although ASMA was present in a granular form specifically

at mesangium of glomerules, renal tubular and interstitial

locations showed no expression for ASMA. Essawy et al.

[45] contrarily, have detected seldom positivity of ASMA in

some glomerules and tubules of patients with diabetic

nephropathy.

In conclusion, our present data indicate that the ultra-

structural investigation of mesangium, strategic unit of

renal corpuscle, by diverse microscopic techniques can

help identifying the effect of damaging factors at cellular

level and can be a beneficial source for related clinical

studies. Since mesangium is accepted as the functional and

the structural unit of a glomerule, it is widely affected and

damaged by glomerular diseases. A better understanding of

mesangial cell behavior and matrix biology may aid new

therapeutic approaches in treatment of renal diseases.

References

1. Bader R, Bader H, Grund KE, Markensen S, Christ H, Bohle A

(1980) Structure and function of the kidney in diabetic glomer-

ulosclerosis: correlations between morphologic and functional

parameters. Pathol Res Pract 167:204–216

2. Ziyadeh FN, Simmons DA, Snopes ER, Goldfarb S (1991) Effect

of Myo-inositol on cell proliferation and collagen transcription in

proximal tubule cells cultured in elevated glucose. J Am Soc

Nephrol 1:1220–1229

3. Kakizawa H, Itoh M, Itoh Y, Imamura S, Ishiwata Y, Matsumoto

T, Yamamoto K, Kato T, Ono Y, Nagata M, Hayakawa N, Suzuki

A, Goto Y, Oda N (2004) The relationship between glycemic

control and plasma vascular endothelial growth factor and

endothelin-1 concentration in diabetic patients. Metabolism

53(5):550–555

4. Feener EP, King GL (2001) Endothelial dysfunction in diabetes

mellitus: role in cardiovascular disease. Heart Fail Monit 1(3):

74–82

5. Spitaler MM, Graier WF (2002) Vascular targets of redox sig-

nalling in diabetes mellitus. Diabetologia 45(4):476–494

6. Khan ZA, Chakrabati S (2003) Growth factors in proliferative

diabetic retinopathy. Exp Diabesity Res 4:287–301

7. Dogra G, Rich L, Stanton K, Watts GF (2001) Endothelium-

dependent and independent vasodilation studies at normoglyca-

emia in type I diabetes mellitus with and without microalbu-

minuria. Diabetologia 44(5):593–601

8. Kamata K, Miyata N, Kasuya Y (1989) Involvement of endo-

thelial cells in relaxation and contraction responses of the aorta to

isoproterenol in naive and streptozotocin-induced diabetic rats.

J Pharmacol Exp Ther 249(3):890–894

9. Zeydanli EN, Turan B (2009) Omega-3E treatment regulates matrix

metalloproteinases and prevents vascular reactivity alterations in

diabetic rat aorta. Can J Physiol Pharmacol 87(12):1063–1073

10. Zeydanli EN, Bilginoglu A, Tanriverdi E, Gurdal H, Turan B

(2010) Selenium restores defective beta-adrenergic receptor

response of thoracic aorta in diabetic rats. Mol Cell Biochem

338(1–2):191–201

11. Zeydanli EN, Kandilci HB, Turan B (2011) Doxycycline ame-

liorates diabetic vascular endothelial and contractile dysfunction

in thoracic aorta. Cardiovasc Toxicol 11(2):134–147

12. Dollery CM, McEwan JR, Henney AM (1995) Matrix metallo-

proteinases and cardiovascular disease. Circ Res 77:863–868

13. Creemers EE, Cleutjens JP, Smits JF, Daemen MJ (2001) Matrix

metalloproteinase inhibition after myocardial infarction: a new

approach to prevent heart failure? Circ Res 89(3):201–210

14. Moshal KS, Metreveli N, Tyagi SC (2008) Mitochondrial matrix

metalloproteinase activation and dysfunction in hyperhomocy-

steinemia. Curr Vasc Pharmacol 6:84–92

15. Nelson KK, Melendez JA (2004) Mitochondrial redox control of

matrix metalloproteinases. Free Radic Biol Med 37(6):768–784

16. Osterby R (1974) Early phases in the development of diabetic

glomerulopathy. Acta Med Scand Suppl 574:3–82

17. Brito PL, Fioretto P, Drummond K, Kim Y, Steffes MW, Basgen

JM, Sisson-Ross S, Mauer M (1998) Proximal tubular basement

membrane width in insulin-dependent diabetes mellitus. Kidney

Int 53:754–761

18. Okada H, Inoue T, Suzuki H, Strutz F, Neilson EG (2000) Epi-

thelialmesenchymal transformation of renal tubular epithelial

cells in vitro and in vivo. Nephrol Dial Transplant 15:44–46

19. He H, Yang X, Zeng X, Shi M, Yang J, Wu L, Li L (2007) Pro-

tective effect of Liuwei Dihuang decoction on early diabetic

nephropathy induced by streptozotocin via modulating ET-ROS

axis and matrix metalloproteinase activity in rats. J Pharm Phar-

macol 59(9):1297–1305

20. Rysz J, Banach M, Stolarek RA, Pasnik J, Cialkowska-Rysz A,

Koktysz R, Piechota M, Baj Z (2007) Serum matrix metallo-

proteinases MMP-2 and MMP-9 and metalloproteinase tissue

inhibitors TIMP-1 and TIMP-2 in diabetic nephropathy. J Neph-

rol 20(4):444–452

21. Chavers BM, Bilous RW, Ellis EN, Steffes MW, Mauer SM

(1989) Glomerular lesions and urinary albumin excretion rate in

type 1 diabetic patients without overt proteinuria. N Engl J Med

320:966–970

22. Wang X, Shaw S, Amiri F, Eaton DC, Marrero MB (2002)

Inhibition of the Jak/STAT signaling pathway prevents the high

glucose induced increase in tgf-beta and fibronectin synthesis in

mesangial cells. Diabetes 51:3505–3509

23. Schnaper HW, Kopp JB, Poncelet AC et al (1996) Increased

expression of extracellular matrix proteins and decreased

expression of matrix proteases after serial passage of glomerular

mesangial cells. J Cell Sci 109:2521–2528

24. Ulusu NN, Turan B (2005) Beneficial effects of selenium on

some enzymes of diabetic rat heart. Biol Trace Elem Res

103(3):207–216

25. Hafeman DG, Sunde RA, Hoekstra WG (1974) Effect of dietary

selenium on erythrocyte and liver glutathione peroxidase in the

rat. J Nutr 104(5):580–587

26. Ayaz M, Ozdemir S, Ugur M, Vassort G, Turan B (2004) Effects

of selenium on altered mechanical and electrical cardiac activities

of diabetic rat. Arch Biochem Biophys 426(1):83–90

27. Bilginoglu A, Kandilci B, Turan B (2013) Intracellular levels of

Na(?) and TTX-sensitive Na(?) channel current in diabetic rat

ventricular cardiomyocytes. Cardiovasc Toxicol 13(2):138–147.

doi:10.1007/s12012-012-9192-9

28. Cam M, Yavuz O, Guven A, Ercan F, Bukan N, Ustundag N

(2003) Protective effects of chronic melatonin treatment against

48 Mol Cell Biochem (2014) 390:41–49

123

renal injury in streptozotocin-induced diabetic rats. J Pineal Res

35(3):212–220

29. Monnier VM, Miyata S, Nagaraj RH, Sell DR (1993) Relevance

of the early and advanced Maillard reaction in diabetic neurop-

athy. Diabet Med 10(Suppl 2):103S–106S

30. Ha H, Kim KH (1999) Pathogenesis of diabetic nephropathy: the

role of oxidative stress and protein kinase C. Diabetes Res Clin

Pract 45(2–3):147–151

31. Mauer SM, Steffes MW, Ellis EN, Sutherland DE, Brown DM,

Goetz FC (1984) Structural-functional relationships in diabetic

nephropathy. J Clin Invest 74(4):1143–1155

32. Aydemir-Koksoy A, Bilginoglu A, Sariahmetoglu M, Schulz R,

Turan B (2010) Antioxidant treatment protects diabetic rats from

cardiac dysfunction by preserving contractile protein targets of

oxidative stress. J Nutr Biochem 21(9):827–833

33. Suziki D, Miyata T, Saotome N, Horie K, Inagi R, Yasuda Y,

Uchida K, Izuhara Y, Yagame M, Sakai H, Kurokawa K (1999)

Immunohistochemical evidence for an increased oxidative stres

and carbonyl modification of proteins in diabetic glomeruler

lesions. J Am Soc Nephrol 10:822–832

34. Doi T, Mima A, Matsubara T, Tominaga T, Arai H, Abe H (2008)

The current clinical problems for early phase of diabetic

nephropathy and nephropathy and approach for pathogenesis of

diabetic nephropathy. Diabetes Res Clin Pract 8:21–24

35. Reeves WB, Andreoli TE (2000) Transforming growth factor bcontributes to progressive diabetic nephropathy. PNAS 97:7667–7669

36. Bangstad HJ, Osterby R, Dahl-Jørgensen K, Berg KJ, Hartmann

A, Hanssen KF (1994) Improvement of blood glucose control in

IDDM patients retards the progression of morphological changes

in early diabetic nephropathy. Diabetologia 37(5):483–490

37. Makino H, Yamasaki Y, Haramoto T, Shikata K, Hironaka K,

Ota Z, Kanwar YS (1993) Ultrastructural changes of extracellular

matrices in diabetic nephropathy revealed by high resolution

scanning and immunoelectron microscopy. Lab Invest 68(1):

45–55

38. Tsuchitani M, Kuroda J, Nagatani M, Miura K, Katoh T (1990)

Glycogen accumulation in the renal tubular cells of spontane-

ously occurring diabetic WBN/Kob rats. J Comp Pathol 102:

179–190

39. Mauer SM, Steffes MW, Michael AF, Brown DM (1976) Studies

of diabetic nephropathy in animal and man. Diabetes 25:850–857

40. McCane KL, Huether S (1994) Pathophysiology the biologic

basis for disease in adults and children. In: McCane KL (ed)

Altered cellular and tissue biology. Mossby, Maryland Heights,

pp 73–78

41. Kang J, Dai XS, Yu TB, Wen B, Yang Z (2005) Glycogen

accumulation in renal tubules, a key morphological change in the

diabetic rat kidney. Acta Diabetol 42:110–116

42. Wang Z, Jiang T, Li J, Proctor G, McManaman JL, Lucia S, Chua

S, Levi M (2005) Regulation of renal lipid metabolism, lipid

accumulation, and glomerulosclerosis in FVB mice with type 2

diabetes. Diabetes 54:2328–2335

43. Takeji M, Moriyama T, Oseto S, Kawada N, Hori M, Imai E,

Miwa T (2006) Smooth muscle a-actin deficiency in myofibro-

blasts leads to enhanced renal tissue fibrosis. J Biol Chem

281:40193–40200

44. Chebotareva NV, Proppe D, Rudolf P, Kozlovskaia LV (2002)

Clinical significance of expression of the smooth muscle actin-

alpha and CD34 antigen in mesangial cells in glomerulonephritis.

Ter Arkh 74:27–31

45. Essawy M, Soylemezoglu O, Muchaneta C, Shortland J, Brown

B, Nahas AM (1997) Myofibroblasts and the progression of

diabetic nephropathy. Nephrol Dial Transplant 12:43–50

Mol Cell Biochem (2014) 390:41–49 49

123