Protective effect of C-peptide on experimentally induced diabetic nephropathy and the possible link between C-peptide and Nitric Oxide

Abstract

Diabetic nephropathy one of the major microvascular diabetic complications. Besides hyperglycemia, other factors contribute to the development of diabetic complications as the proinsulin connecting peptide, C-peptide. We described the role of C-peptide replacement therapy on experimentally induced diabetic nephropathy, and its potential mechanisms of action by studying the role of Nitric oxide (NO) as a mediator of C-peptide effects by in vivo modulating its production by NG-nitro-L-arginine methyl ester (L-NAME). Renal injury markers measured were serum urea, creatinine, TNF-α and Angiotensin II and renal tissue malondialdehyde, total antioxidant, Bcl-2 and NO. Conclusion, diabetic induction resulted in islet degenerations and decreased insulin secretion with its metabolic consequences, and subsequent renal complications. C-peptide deficiencies in diabetes might contributed to the metabolic and renal error, since C-peptide treatment to the diabetic rats corrected completely these errors. The beneficial effects of C-peptide partially antagonized by L-NAME co-administration indicating that NO partially mediates C-peptide effects.

Full text

ARTICLE
Protective effect of C-peptide on experimentally induced
diabetic nephropathy and the possible link between C-peptide
and nitric oxide
Eman A. Elbassuoni, Neven M. Aziz, and Nashwa F. El-Tahawy
Abstract: Diabetic nephropathy one of the major microvascular diabetic complications. Besides hyperglycemia, other factors
contribute to the development of diabetic complications as the proinsulin connecting peptide, C-peptide. We described the role
of C-peptide replacement therapy on experimentally induced diabetic nephropathy, and its potential mechanisms of action by
studying the role of nitric oxide (NO) as a mediator of C-peptide effects by in vivo modulating its production by N
G
-nitro-L-arginine
methyl ester (L-NAME). Renal injury markers measured were serum urea, creatinine, tumor necrosis factor alpha, and angio-
tensin II, and malondialdehyde, total antioxidant, Bcl-2, and NO in renal tissue. In conclusion, diabetic induction resulted in islet
degenerations and decreased insulin secretion with its metabolic consequences and subsequent renal complications. C-Peptide
deficiencies in diabetes might have contributed to the metabolic and renal error, since C-peptide treatment to the diabetic rats
completely corrected these errors. The beneficial effects of C-peptide are partially antagonized by L-NAME coadministration,
indicating that NO partially mediates C-peptide effects.
Key words: C-peptide, diabetic nephropathy, Nitric Oxide.
Résumé : La néphropathie diabétique est une des complications microvasculaires majeures du diabète. D’autres facteurs en plus
de l’hyperglycémie contribuent au développement des complications diabétiques comme le peptide de connexion incorporé
dans la proinsuline (peptide C). Nous décrivons le rôle du traitement de substitution du peptide C dans la néphropathie
diabétique induite expérimentalement; de plus, nous présentons un mécanisme potentiel d’action en étudiant les effets de
l’oxyde nitrique (« NO ») en tant que médiateur des effets du peptide C en modulant in vivo sa production au moyen du L-NAME
(N
G
-nitro-L-arginine méthylester). Les marqueurs mesurés des lésions rénales sont l’urée, la créatinine, le facteur de nécrose
tumorale alpha et l’angiotensine II du sérum et le malonaldéhyde, la capacité totale d’antioxydation, Bcl-2 et NO dans le tissu
rénal. En conclusion, l’induction du diabète engendre la dégénérescence des îlots et la diminution de la sécrétion de l’insuline
accompagnée des conséquences métaboliques et des complications rénales subséquentes. La carence en peptide C dans le
diabète pourrait contribuer aux troubles du métabolisme et du rein étant donné que le traitement au peptide C appliqué à des
rats diabétiques corrige entièrement ces troubles. Les effets bénéfiques du peptide C sont partiellement contrés par la coadmin-
istration du L-NAME suggérant ainsi que NO intervient partiellement dans les effets du peptide C. [Traduit par la Rédaction]
Mots-clés : peptide C, néphropathie diabétique, oxyde nitrique.
Introduction
Diabetic nephropathy, as one of the microvascular diabetic
complications, is clinically defined as the progressive develop-
ment of renal insufficiency with hyperglycemia. This disease is
now the major single cause of end-stage renal failure in various
countries (Afkarian et al. 2013).
There are several theories for the pathogenic mechanisms that
result in the development of diabetes-induced renal dysfunction.
Early in the course of type 1 diabetes mellitus (T1DM), specific
organs’ function deteriorates and tissue abnormalities arise,
while hyperglycemia results in abnormal homeostasis in blood
flow and vascular permeability in the glomerulus. The increased
blood flow and intra-capillary pressure is assumed to reveal
hyperglycemia-induced decreased nitric oxide production on the
efferent side of renal capillaries and eventually an increased sen-
sitivity to angiotensin II (Ang II). As a consequence of increased
intra-capillary pressure and endothelial cell dysfunction, glomer-
ular capillaries have a higher albumin excretion rate. By this early
stage, the increased permeability is still reversible, but below the
continuing triggering effect of hyperglycemia, the lesions become
irreversible (Papadopoulou-Marketou et al. 2017).
Besides hyperglycemia, other causal factors appear to contrib-
ute to the development of diabetic complications. One such factor
is the proinsulin connecting peptide, C-peptide. C-Peptide is a
cleavage product of insulin production formed in the pancreas as
part of insulin production, and is released into the circulation
with insulin. When insulin synthesis is impaired, as in T1DM
and late type 2 diabetes, synthesis of C-peptide is also impaired
(Samnegard and Brundin 2001). Moreover, (Luppi and Drain 2014,
2017) reported that C-peptide has an anti-oxidant effect on the
␤-cells producing it, which limit ␤-cell dysfunction and loss con-
tributing to diabetes and suggesting a positive feedback that
might potentiate the endogenous and exogenous C-peptide bene-
fits on the kidney.
Received 19 September 2017. Accepted 19 December 2017.
E.A. Elbassuoni and N.M. Aziz. Physiology Department, Minia University Faculty of Medicine, Minia 61111, Egypt.
N.F. El-Tahawy. Histology and Cell Biology Department, Minia University Faculty of Medicine, Minia 61111, Egypt.
Corresponding author: Eman A. Elbassuoni (email: emanelbassuoni@yahoo.com).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.
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Besides the 2 major factors (insulin deficiency and subsequent
hyperglycemia) that contribute to the development of diabetic
complications, C-peptide deficiency is suggested to be the third
major factor because of beneficial effects of C-peptide against
diabetic complications (Wahren et al. 2012). Thus, therapeutic ap-
proaches to hyperglycemic control only have been ineffective in
preventing diabetic complications, and alternative therapeutic
approaches are necessary to target both hyperglycemia and dia-
betic complications.
Numerous clinical and experimental studies demonstrated that
C-peptide treatment alone or in combination with insulin has
physiological functions and might be beneficial in preventing di-
abetic complications (Lachin et al. 2014;Yosten et al. 2014).
In this work, we describe the role of C-peptide replacement
therapy in diabetic nephropathy as one of the most serious dia-
betic complications, and its potential mechanisms of action.
To elucidate this concept, this study was planned to: (i) induce
experimental type 1 diabetes mellitus by streptozotocin (STZ) in
adult male albino rats, (ii) study the effects of C-peptide treatment
on the developed renal injury, and (iii) study the role of nitric
oxide (NO) as a mediator of C-peptide effects by modulating its
production in vivo by blocking its synthesis using the nitric oxide
synthase (NOS) inhibitor: N
G
-nitro-L-arginine methyl ester (L-NAME).
Materials and methods
I. Animals
Fifty adult male albino rats (Sprague–Dawley strain) that were
of average weight (150–200 g) and were approximately 4 months
old were used in the present study. They were purchased from the
National Research Center, Cairo, Egypt. They were housed in
groups of 6 in stainless steel cages that offered adequate space for
free movement and wandering (40 cm × 40 cm × 25 cm) at room
temperature with natural dark/light cycles, and allowed free ac-
cess to water and commercial rat diet (Nile Company, Egypt) for
2 weeks for acclimatization. All experimental protocols were ap-
proved by the animal care committee of Minia University, which
coincides with international guidelines. All applicable interna-
tional, national, and (or) institutional guidelines for the care and
use of animals were followed. Rats were classified randomly into
the following groups (10 rats each):
1. Control (C) group: in which 10 rats were fed a commercial rat
diet and received citrate buffer only.
2. STZ treated (T1DM) group: in which 10 rats were injected by a
single intraperitoneal injection of STZ (SigmaMillipore, Egypt)
at a dose level of 55 mg/kg of body weight (Al-Trad et al. 2015)
and were left for 4 weeks untreated to induce early diabetic
nephropathy (Honore et al. 2012).
3. STZ+C-peptide (T1DM+CP) treated group: in which 10 rats were
injected by a single intraperitoneal injection of STZ as above.
Three days later (diabetes was verified) these rats received
50 nmol/(kg·day) C-peptide (Biorbyte, UK) by intraperitoneal
injection for 4 weeks (Samnegard et al. 2005).
4. STZ+L-NAME (T1DM+L-NAME) treated group: in which 10 rats
were injected by a single intraperitoneal injection of STZ as
above. Three days later (diabetes was verified) these rats re-
ceived 20 mg/(kg·day) L-NAME (SigmaMillipore, USA) via drink-
ing water for 4 weeks (Regadas et al. 2014).
5. STZ+L-NAME+C-peptide (T1DM+L-NAME+CP) treated group: in
which 10 rats were injected by STZ then received C-peptide and
L-NAME as above for 4 weeks.
II. Induction of T1DM
To induce experimental T1DM, rats were injected by a single
intraperitoneal injection of freshly prepared STZ 55 mg/kg, dis-
solved in 0.1 mol/L citric acid buffer, pH (4.5) after an overnight
fast (Al-Trad et al. 2015). The STZ-treated animals were allowed to
drink 20% glucose solution for 24 h to overcome initial drug-
induced hypoglycemic mortality. Diabetes was verified 3 days
later by evaluating blood glucose levels with the use of glucose-
oxidase reagent strips (Accu-Chek; Roche Inc., Indianapolis, Ind.,
USA). Rats having blood glucose level of 200 mg/dL or greater were
considered to be diabetic and selected for the study. At the end
of study, histological examination of pancreas will confirm our
finding.
III. Experimental protocol
By the termination of experimental procedure, rats were sacri-
ficed by decapitation under light halothane anesthesia and blood
samples were collected from the jugular vein, allowed to clot, and
centrifuged and supernatant serum was collected in Eppendorf
tubes and stored at –20 °C till the time for biochemical assay. The
pancreas and kidneys were rapidly removed, weighed, and di-
vided; some specimens were fixed for paraffin embedding and
some renal specimens were stored at –80 °C for renal tissue assay
of malondialdehyde (MDA), total antioxidant capacities (TAC),
B-cell leukemia/lymphoma-2 (Bcl-2), and NO levels.
IV. Biochemical analyses
Sera were used for estimation of urea by Berthelot enzymatic
colorimetric method, creatinine by Jaffé colorimetric-endpoint
method, tumor necrosis factor alpha (TNF-␣) by enzyme-linked
immunosorbent assay (ELISA) kit (ALPCO Diagnostic) and Ang II
by ELISA kit (ALPCO Diagnostic) according to the manufacturer’s
instructions.
V. Preparation of tissue homogenates
Kidney specimens were weighed and homogenized separately
in potassium phosphate buffer 10 mmol/L; pH (7.4). The ratio of
tissue weight to homogenization buffer was 1:10. The homoge-
nates were centrifuged at 2795gfor 10 min at 4 °C. The resulting
supernatant was used for determination of MDA according to the
method of Ohkawa et al. (1979), TAC using a colorimetric assay kit,
NO by enzymatic colorimetric methods using commercial kits
(Biodiagnostic, Egypt), and Bcl-2 by ELISA kit (Calbiotech, USA)
according to the manufacturer’s instructions.
VI. Histological examination
Pancreases (the tail of pancreas) and kidney specimens from all
groups were fixed in 10% neutral-buffered formalin, dehydrated in
a graded ethanol series, cleared in xylene embedded in paraffin
wax, and sectioned into 6–7 ␮m sections. Sections were stained
with hematoxylin and eosin (H&E).
Image capture
Tissue sections were examined and images were digitally cap-
tured using a digital camera mounted on an Olympus microscope
and connected to a computer.
Morphometry
For histological evaluation of the severity of renal lesions, a
semiquantitative analysis of sections were done (Houghton et al.
1978): Score –: assigned normal; Score +: in between normal and
mild level; Score ++: mild level, <25% of the total fields examined
revealed alterations; Score +++: moderate level, <50% of the total
fields examined revealed alterations; and Score ++++: severe
level, <75% of the total fields examined revealed histopathological
alterations.
VII. Statistical analysis
Data were represented as means ± SE. Statistical analysis was
performed using Graph pad Prism 5 software and significant dif-
ference between groups was done by 1-way ANOVA followed by
Tukey–Kramer post hoc test for multiple comparisons with a
value of P≤ 0.05 considered statistically significant.
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Results
A. Metabolic and oxidative parameters
As shown in Table 1, the diabetic nontreated group showed
significantly higher fasting serum glucose and lower insulin levels
as compared with rats of the control group. These effects were
completely reversed with C-peptide treatment. Administration of
L-NAME to diabetic rats significantly decreased the insulin level as
compared with the diabetic group, and the hyperglycemia of dia-
betes was worsened as indicated by a significantly higher blood
glucose level. When L-NAME was coadministered with C-peptide it
partially but significantly antagonized the hypoglycemic and the
insulin increasing effects of C-peptide.
In Table 2, the levels of serum urea, serum creatinine, and renal
MDA significantly increased with significantly decreased in renal
TAC level (renal injury parameters) in the T1DM group as com-
pared with the C group. However, compared with T1DM group,
these levels significantly decreased in the T1DM+CP group. Ad-
ministration of L-NAME was injurious as it markedly increased
the renal injury parameters as compared with the T1DM group.
On the other hand, treatment of the diabetic group by both
L-NAME and C-peptide significantly antagonized the positive re-
nal effect of C-peptide.
B. Serum TNF-␣and Ang II levels
Both serum levels of TNF-␣(Fig. 1) and Ang II (Fig. 2) were found
to be significantly increased in T1DM group as compared with the
C group. In addition, administration of L-NAME significantly pro-
duced higher serum levels of TNF-␣(Fig. 1) and Ang II as compared
with the T1DM group. On the other hand, C-peptide administra-
tion significantly lowered these levels as compared with the T1DM
group, while its coadministration with L-NAME significantly in-
creased these levels as compared with the T1DM+CP group, which
was still significantly lower than the T1DM+L-NAME group.
C. Kidney Bcl-2 and NO levels
As shown in Table 3, the T1DM+CP group recorded the highest
renal NO level amongst all studied groups, while the T1DM+
L-NAME group recorded the lowest renal NO level. On the other
hand, treatment of the diabetic group by both L-NAME and
C-peptide produced a significant lower renal NO level as com-
pared with the T1DM+CP group. In addition, renal NO level was
found to be lower in the T1DM group as compared with the
C group.
Regarding the renal Bcl-2 level, the lowest mean value was
recorded in the T1DM+L-NAME group. However, C-peptide treat-
ment alone significantly elevated the renal Bcl-2 level as com-
pared with all studied group but failed to produce any significant
change when compared with the C group. While treatment of the
diabetic group by both L-NAME and C-peptide significantly low-
Table 1. Changes in fasting serum glucose and insulin in the different studied groups.
Group
Parameter C T1DM T1DM+CP
T1DM+
L-NAME
T1DM+
L-NAME+CP
Glucose (mg/dL) 80.6±1.2 224.6±3.5a 85.7±1.4b 242.5±4.7abc 161.3±2.4acd
Insulin (␮IU/mL) 7.9±0.4 5.5±0.3a 7.3±0.5b 4.2±0.4abc 6.9±0.7acd
Note: Data are expressed as means ± SE of 10 rats in each group. a, Significant from C group; b, significant from
T1DM group; c, significant from T1DM+CP group; d, significant from T1DM+L-NAME treated group; P≤ 0.05.
C, control group; T1DM, STZ treated group; T1DM+CP, STZ+C-peptide treated group; T1DM+L-NAME, STZ+L-NAME
treated group; T1DM+L-NAME+CP, STZ+L-NAME+C-peptide treated group.
Table 2. Change in the renal injury parameters in the different studied groups.
Renal injury parameter
Group
C T1DM T1DM+CP
T1DM+
L-NAME
T1DM+
L-NAME+CP
In blood
Urea (mmol/L) 23.4±1.2 87.6±4.7a 26.4±1.85b 99.9±1.7abc 76.4±3.1acd
Creatinine (␮mol/L) 0.5±0.09 1.7±0.09a 0.6±0.02b 2.6±0.1abc 1.5±0.1acd
In renal tissue
Renal MDA (pg/mg tissue) 41.3±1.8 79.09±3.4a 40±2.7b 89.5±2.1abc 69.5±3.5acd
Renal TAC (␮mol/(L·mg) tissue) 79.5±3.2 54.8±2.9a 80.9±4.5b 42.1±2.2abc 61±2.1acd
Note: Data are expressed as means ± SE of 10 rats in each group. a, Significant from C group; b, significant from
T1DM group; c, significant from T1DM+CP group; d, significant from T1DM+L-NAME group; P≤ 0.05. C, control
group; T1DM, STZ treated group; T1DM+CP, STZ+C-peptide treated group; T1DM+L-NAME: STZ+L-NAME treated
group; T1DM+L-NAME+CP, STZ+L-NAME+C-peptide treated group; MDA, malondialdehyde; TAC, total antioxidant
capacity.
Fig. 1. Tumor necrosis factor alpha (TNF-␣) levels in serum samples
of the different studied groups. Data are expressed as means ± SE
of 10 rats in each group. a, Significant from control group;
b, significant from STZ treated group; c, significant from STZ+
C-peptide treated group; d, significant from STZ+L-NAME treated
group; P≤ 0.05. STZ, streptozotocin. C, control group; T1DM, STZ
treated group; T1DM+CP, STZ+ C-peptide treated group; T1DM+
L-NAME, STZ+L-NAME treated group; T1DM+L-NAME+CP, STZ+
L-NAME+C-peptide treated group. [Color online.]
Elbassuoni et al. 619
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ered the renal Bcl-2 level as compared with the T1DM+CP group
and was still insignificant compared with the T1DM group.
D. Histological study of kidney and pancreas using H&E
H&E stained sections of pancreas from the C group showed a
normal lobular architecture with delicate interlobular connective
tissue. Numerous islets of Langerhans surrounded by the pancre-
atic acini. The islets appeared lightly stained and consisted of
cords of polygonal cells separated by blood capillaries. The acinar
cells were characterized by its basal basophilia and apical acido-
philia (Figs. 3aand 3b). The pancreatic sections of the T1DM group
showed marked morphological changes. Some islets were com-
pletely destroyed leaving empty spaces. Other islets showed de-
generations as some cells appeared with nuclear pyknosis and
fragmentation and dense acidophilic cytoplasm; apoptotic fig-
ures, while other islet’s cells showed vacuolated cytoplasm and
ghosts of nuclei. The degenerated cells surrounded by empty
spaces that filled with amyloid-like material (Figs. 3cand 3d).
In the T1DM+CP group, C-peptide administration showed im-
provement in the previous morphological changes. Many islets
were observed and showed an increase in the cellular density.
Appearance of small newly formed islets was also observed
(Figs. 3eand 3f).
In T1DM+L-NAME group, L-NAME administration worsened the
condition compared with the T1DM group, where more degener-
ations were observed with aggregation of lymphocyte infiltration
(Figs. 3gand 3h). While treatment of the diabetic group by L-NAME
and C-peptide in the T1DM+L-NAME+CP group decreased this pic-
ture (Figs. 3iand 3j) where islets of variable sizes were observed
with few degenerated cells compared with T1DM+L-NAME group.
H&E stained sections of the rat renal cortical tissue of control
group showed normal architecture, comprising numerous renal
corpuscles (RCs), proximal convoluted tubules (PCTs), and distal
convoluted tubules (DCTs). The RC contained the glomeruli that
surrounded by the Bowman’s capsules with urinary spaces in be-
tween. The parietal layers were lined by simple squamous epithe-
lium. The PCTs were lined with thick large cubical epithelium
with acidophilic cytoplasm. The DCTs showed considerably lower
cubical epithelium surrounding relatively larger regular distinct
lumens (Fig. 4a).
The histological changes in the T1DM group were variable and
patchy, it showed slightly congested vascular glomerulus with
few cytoplasmic vacuolations of some PCT and DCT cells (Fig. 4b).
In the T1DM+CP group, apparent normal renal cortical tissues
were observed where apparent RCs, PCTs, and DCTs were seen
(Fig. 4c).
In the T1DM+L-NAME group, marked distortion of the cortical
architecture were observed in the form of markedly congested
glomerulus with obliterated Bowman’s space, severe tubular dil-
atation and disturbed morphology of the convoluted tubules with
marked cytoplasmic vacuolations, peritubular capillaries dilata-
tion and congestion, and appearance of epithelial cast in the tu-
bular lumen (Fig. 4d).
Treatment of the diabetic group by both L-NAME and C-peptide
in the T1DM+L-NAME+CP group ameliorated the damaging ef-
fects. Less tubular distortion and few cytoplasmic vacillations of
some tubular cells compared with the T1DM+L-NAME group with
minimal congestion. Some tubules showed casts in the lumen
(Fig. 3e).
Morphometric results
The severity of the morphological changes was assessed semi-
quantitatively; the groups exposed to L-NAME showed increase in
the glomerular and tubular morphological changes at the light
microscopic levels when compared with the diabetic group. These
changes were suppressed by the administration of C-peptide
(Table 4).
Discussion
STZ is broadly used as a chemical inducer to set up the model of
T1DM (Kasono et al. 2004). The results of the present study showed
that STZ injection resulted in a significant increase in fasting
serum glucose to the diabetic levels, with a significant decrease in
serum insulin level. The histological finding of this study showed
that STZ selectively destroys pancreatic ␤-cells, which could ex-
plain high fasting serum glucose levels in the T1DM group. This
was in agreement with Al-Trad et al. (2015). These effects were
completely reversed with C-peptide treatment. Administration of
L-NAME to diabetic rats worsened the condition more as indicated
by the greater increase in blood glucose level and the greater
decrease in blood insulin level. When L-NAME was coadminis-
tered with C-peptide it partially but significantly antagonized the
hypoglycemic effects of C-peptide.
These results are compatible with previous studies that had
shown that C-peptide is much more than a byproduct of insulin
synthesis and has biological role in metabolism and its deficiency
with insulin in diabetes predisposes to the metabolic error.
Through binding to insulin by charge interaction, C-peptide pre-
vents insulin aggregation to form polymeric inactive forms, there-
fore keeping the monomeric biologically active insulin (Ghorbani
and Shafiee-Nick 2015). C-Peptide increases muscles and periph-
eral tissues glucose utilization by increasing translocation of glu-
cose transporter 4 to cell membranes to facilitate glucose uptake.
Moreover, by activating tyrosine kinase and phosphorylating in-
sulin receptor substrate, C-peptide can activate insulin signaling
pathways, thus increasing its sensitivity. These effects can explain
the normalizing effects of C-peptide on blood glucose level seen in
the present study, which agrees with Wu et al. (2012).
The partially antagonizing effect of L-NAME on the positive
metabolic effect induced by C-peptide is in line with Wu et al.
(2012), who reported that L-NAME has the ability to block about
85% of the C-peptide–induced increase in glucose disposal rates,
suggesting the mediating role of NO in C-peptide stimulation of
glucose utilization. A G-protein–coupled C-peptide receptor has
been recognized and its stimulation induces NOS activation;
C-peptide can produce vasodilatation of pancreatic vessels with
enhanced function of ␤-cells and increased insulin secretion
through increased NO production. In the present study, the
higher levels of insulin in the diabetic group treated with
C-peptide and the lower levels found with L-NAME treatment sup-
ports this role of NO according to Bhatt et al. (2014).
Fig. 2. Angiotensin II (Ang II) levels in serum samples of the different
studied groups. Data are expressed as means ± SE of 10 rats in each group.
a, Significant from control group; b, significant from STZ treated group; c,
significant from STZ+C-peptide treated group; d, significant from STZ+
L-NAME treated group; P≤ 0.05. See Fig. 1 for definitions of terms. [Color
online.]
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In the present study, diabetes induction of nephrotoxicity was
confirmed by renal histopathological changes that showed mor-
phological damage of the renal tissue. This was in agreement with
Patschan and Muller (2016).
Serum urea and creatinine levels are considered to be diagnos-
tic markers of renal cell injury (Zuo et al. 2008). The results of the
present study show that the levels of both serum urea and creat-
inine significantly increased in diabetic group as compared with
the C group, and these results are compatible with previous stud-
ies, such as Maheshwari et al. (2017), which was confirmed by the
histological changes observed in the T1DM group, owing to the
evidenced occurrence of oxidative stress and release of reactive
oxygen species (ROS). L-Arginine (Arg), the main source for the
generation of NO via NOS, the metabolism and the enzymes that
participate in its synthesis are downregulated in diabetes, thus
contributing to the resulting NO deficiency (Ortiz et al. 2014).
Moreover, diabetic animals have increased hepatic Arg degrada-
tion leading to decrease in plasma Arg levels, which limit the
renal NO levels (Palm et al. 2008). On the other hand, serum levels
of asymmetric dimethylarginine, an endogenous inhibitor of
NOS, are increased in both type 1 and type 2 diabetes, leading to a
decrease in NO production, as proved in our study, and therefore
contributes to diabetic complications as nephropathy through
renal hypoxia induction and retinopathy (Altinova et al. 2007).
Oxidative stress plays a significant role in the pathogenesis and
progression of renal disease (Jha et al. 2016). The data presented in
this study showed increased lipid peroxidation expressed by in-
creasing level of MDA, and decreased TAC in renal tissue with
diabetic induction, and these results are in line with previous
experimental (Huang et al. 2012) and clinical (Chang et al. 2012;
Whaley-Connell and Sowers 2012) studies.
In diabetes mellitus, oxidative stress is present because the
stimulatory effect of glucose on mitochondrial nicotinamide ade-
nine dinucleotide phosphate oxidase leading to generation of ex-
cess superoxide ions (Palm et al. 2003). In addition, in endothelial
cells, hyperglycemia inhibits endothelial NOS (eNOS) activity,
leading to reduced NO and increased ROS production (Böger
2003). On the other hand, inducible NOS is stimulated by inflam-
matory cytokines, such as interleukin (IL)-1, IL-6, and TNF-␣, result-
ing in excess NO formation, which reacts with superoxide ions to
form the highly toxic peroxinitrite, and thus decrease the bio-
availability of NO. Reactive oxygen and nitrogen species (ROS/RNS)
further destroy metabolic enzymes, membrane phospholipids, and
insulin receptors, decreasing both insulin release and sensitivity.
ROS/RNS could also oxidize and nitrosylate NOS, leading to de-
crease its activity and producing a vicious cycle of reduced NO,
reduced blood flow, ischemia, and more ROS generation. This can
contribute to both diabetic pathogenesis and complications (Palm
et al. 2005).
Many studies suggested the role of inflammatory cytokines in
the development of diabetic nephropathy. In this study, serum
TNF-␣is one of the pro-inflammatory cytokines that was signifi-
cantly increased with diabetic induction. This result is in line with
previous studies (Donate-Correa et al. 2015). They added that in-
creased oxidative stress, as we found in this study, can increase
the production of inflammatory cytokines and stimulate the pro-
duction of free radicals.
Regarding serum Ang II level, we found that induction of dia-
betes produced significant increase in its level. There have been
contradictory reports on the activity of the renin-angiotensin sys-
tem (RAS) in diabetes mellitus and numerous abnormalities have
been described (Chawla et al. 2010). These studies strongly con-
cerned the RAS as a mediator of diabetic nephropathy. These
results may be because during inflammation, lymphocytes and
macrophages can generate reactive oxygen species and Ang II.
Diabetic nephropathy being an inflammatory condition, Ang II
levels have been found to be elevated. This rise activates immune
cells and causes production of chemokines, which lead to further
renal damage (Ruiz-Ortega et al. 2001).
Apoptosis, a form of programmed cell death, can be induced
by various stimuli. The anti-apoptotic protein Bcl-2 plays a main
role in apoptosis regulation, both in physiological and pathologi-
cal conditions (Zhang et al. 2008), which explained increased
apoptotic-like features observed in the islets of the T1DM group
and epithelial degeneration and epithelial casts in the tubular
lumen. Bugliani et al. (2007) reported that exposure to C-peptide
reduced human islet cell apoptosis, which was accompanied by
increased expression (both at the gene and protein levels) of the
anti-apoptotic molecule Bcl-2. In our study we found significant
decrease in renal Bcl-2 level in diabetic group compared with the
C group. This result is in line with previous studies, such as
Verzola et al. (2004), who found that high-glucose concentration
promotes apoptosis in variety of cell types, including proximal
tubular epithelial cells. The mechanism by which hyperglycemia
leads to apoptosis is not completely understood but it may be
because of increase oxidative and nitrosative stress. It was also
found that there is a decrease in Bcl-2 gene expression with dia-
betes (da Silva Faria et al. 2015).
As reported previously, exogenous C-peptide administration in
T1DM has been shown to exert beneficial effects in many tissues
affected by microvascular complications (Nordquist and Wahren
2009). In this study, C-peptide administration to the diabetic rats
enhanced both morphological changes occurred in the islets and
renal structures.
The renal injury parameters, either in the blood or the kidney,
was improved; serum urea, creatinine, TNF-␣, and Ang II de-
creased, and renal tissue level of TAC, NO, and Bcl-2 increased
with decreasing renal MDA. In addition, the pancreatic and renal
histopathological changes induced by diabetes were improved
by C-peptide treatment. These results were in agreement with
Samnegard and Brundin (2001) and Sun et al. (2010), who detected
a protective effect of C-peptide on diabetes induced nephrotoxic-
ity in rats. However, other studies, such as Bhatt et al. 2014, have
reported C-peptide hazardous effects in diabetes mellitus, with
increased recruitment of inflammatory cells in subendothelial
layer of blood vessels and stimulation of smooth muscle prolifer-
ation that predispose to atherosclerosis. Differences in doses, du-
ration of treatment, animal species, and experimental protocol
can explain such variations in responses.
Table 3. Kidney NO and Bcl-2 in the different studied groups.
Group
Parameter C T1DM T1DM+CP
T1DM+
L-NAME
T1DM+
L-NAME+CP
NO (pmol/mg wet tissue) 8.5±0.4 4.2±0.1a 14.6±0.7ab 2.4±0.09abc 3.9±0.12acd
Bcl-2 (ng/mg wet tissue) 1.5±0.09 0.7±0.05a 1.4±0.04b 0.5±0.02abc 0.8±0.07acd
Note: Data are expressed as means ± SE of 10 rats in each group. a, Significant from C group; b, significant from
STZ treated group; c, significant from STZ+C-peptide treated group; d, significant from STZ+L-NAME treated group;
P≤ 0.05. C, control group; T1DM, STZ treated group; T1DM+CP, STZ+C-peptide treated group; T1DM+L-NAME,
STZ+L-NAME treated group; T1DM+L-NAME+CP, STZ+L-NAME+C-peptide treated group; Bcl-2, B-cell leukemia/
lymphoma-2; NO, nitric oxide.
Elbassuoni et al. 621
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C-Peptide can affect regional blood flow of the kidney by stim-
ulating eNOS and eNOS gene transcription with increasing NO
and vasodilatation (Bhatt et al. 2014). It also decrease intracellular
ROS generation by improving mitochondrial respiration or by
reducing or inhibiting activity of the plasma membrane NAD(P)H
oxidase enzyme or by lowering ROS sources in the cell (Cifarelli
et al. 2011). However, until now the mechanism underlying
C-peptide–mediated inhibition of intracellular ROS production
and subsequent apoptosis is not clear. Nuclear factor kappa B
(NF-␬B) is a protein complex that controls cytokine production,
transcription of DNA, and cell survival. In renal cells, C-peptide
protects against TNF-␣–mediated apoptosis through an increasing
NF-␬B–mediated mechanism. (Cifarelli et al. 2008).
From our results, the mechanism underlying the beneficial ef-
fect of C-peptide on renal function in diabetes is clear to some
extent. C-Peptide, through its hypoglycemic effect, may indirectly
improve renal function owing to the injurious effect of hypergly-
cemia on the renal tissue, as discussed previously. However, it is
also possible that C-peptide may have exerted a direct effect on
the renal tissue.
Trying to investigate the role of NO in mediating C-peptide
renal effect, L-NAME, the nonselective blocker of NOS, was given
either alone or combined with C-peptide to diabetic rats. Admin-
istration of L-NAME alone to the diabetic rats was injurious as it
worsened the renal error produced by diabetes. On the other
hand, when combined with C-peptide, L-NAME partially antago-
nized its renal-correcting effect. Our renal histopathological and
morphometrical analysis showed increased severity of tissue in-
Fig. 3. Photomicrographs of rat pancreatic tissue: hematoxylin and
eosin – a,c,e,g,i×100; b,d,f,h,j, and insets ×1000. (a) Control group
showing normal lobular architecture with delicate interlobular
connective tissue. Islets of Langerhans (stars) surrounded by the
pancreatic acini (PA). (b) Islet of Langerhans consisting of cords of
cells (arrows) separated by blood capillaries (red arrows). Notice
acinar cells with an apical cytoplasm packed with acidophilic
cytoplasmic granules (*) and basal nuclei (arrowheads). (c) The T1DM
group showing completely destroyed islets leaving empty spaces
(stars), and widening of interlobular spaces (arrow). (d) Degenerated
islet with vacuolated cells and pale nuclei (black arrows), others
with deep acidophilic cytoplasm and nuclear pyknosis (white
arrow). Notice the spaces leaved empty (stars) after cell degeneration
or filled with amyloid-like material (A). (e) Group T1DM+CP showing
many islets (black arrows). (f) Increase islet cells (arrows) with
vesicular nuclei resembling normal. The inset shows small newly
formed (star) islet. (g) Group T1DM+L-NAME showing degenerated
islet (IS). Notice aggregation of lymphocyte infiltration (arrow). The
inset showing congested blood vessel (BV) and lymphocytic
infiltration (arrow). (h) An islet with degenerated cells (arrows).
Notice the empty spaces leaved after cell degeneration (stars). Inset
showing a completely destroyed islet leaving remnants of
degenerated cells (circle). (i) Group T1DM+L-NAME+CP showing islets
of variable sizes (arrows). (j) IS with few degenerated cells (arrows)
compared with T1DM+L-NAME+CP group. See Fig. 1 for definitions of
terms. [Color online.]
Fig. 4. Photomicrographs of renal cortex of hematoxylin and eosin
×400. (a) Control group showing renal corpuscles (RCs), proximal
convoluted tubules (PCTs) (p), and distal convoluted tubules (DCTs) (d).
The convoluted tubules have a relatively regular distinct lumen.
(b) T1DM group showing slightly congested vascular glomerulus (g).
Notice few cytoplasmic vacuolations (arrows) of some PCT (p) and
DCT (d) cells. (c) T1DM+CP group showing apparent normal RCs,
PCTs (p), and DCTs (d). (d) Group T1DM+L-NAME showing marked
distortion of the cortical architecture; markedly congested
glomerulus (g) with obliterated Bowman’s space (white arrow),
severe tubular dilatation (d) with disturbed morphology of the
convoluted tubules, marked cytoplasmic vacuolations (black
arrows), peri-tubular capillaries dilatation, and congestion (circle).
Notice the nuclei of desquamated tubular cells (green arrow) in the
tubular lumen and the epithelial cast (stars). (e) T1DM+L-NAME+CP
group showing less tubular distortion and less cytoplasmic
vacuolations (arrows) of the renal PCT (p) and DCT cells (d) than in
the T1DM+L-NAME group with minimal congestion (circle). Some
tubules show casts in the lumen (star, inset). See Fig. 1 for
definitions of terms. [Color online.]
622 Appl. Physiol. Nutr. Metab. Vol. 43, 2018
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jury induced by L-NAME, either alone or in combination with
C-peptide, on the kidney. Also L-NAME increased inflammatory
cell infiltration and vascular congestion. These results were in
agreement with Choi et al. (1999),Komers et al. (2000), and
Komers and Anderson (2003).
These results confirmed the role of NO in mediating C-peptide
hypoglycemic and renal effect. The injurious renal effect of
L-NAME either alone or in combination with C-peptide because of
the concept we discussed above shows the significant role of NO in
regulating the main functions in all tissues including the kidney.
In conclusion, STZ injection to rats induced diabetic picture
similar to T1DM in humans, resulting in islet degenerations and
decreased insulin secretion with its metabolic consequences and
subsequent renal complications. C-Peptide deficiencies in diabe-
tes might contributed to the metabolic and renal error, since
C-peptide treatment to the diabetic rats corrected completely
these errors. The beneficial effects of C-peptide were partially
antagonized by L-NAME coadministration, indicating that NO par-
tially mediates the effects of C-peptide. These results open the way
for trials of C-peptide with insulin in treating T1DM with absolute
insulin and C-peptide loss, and this should be a subject of future
research as it represents a potential therapeutic option to protect
renal tissue from detrimental effects of diabetes.
Conflict of interest statement
All authors declare that they have no conflict of interest.
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Group
Findings C T1DM T1DM+CP T1DM+L-NAME T1DM+L-NAME+CP
Renal corpuscles
Glomerular vacuolation − − + +++ −
Enlarged congested renal corpuscles − +++ + ++++ ++
PCTs
Tubular cells vacuolation − + + +++ −
Lumen widening and Distortion − ++ − ++++ +
Casts − − − ++ −
DCTs
Tubular cells vacuolation − + + +++ ++
Lumen widening and distortion − ++ − ++++ +
Casts − − − ++ +
Interstitial
Peritubular capillary dilatation and congestion − − − +++ +
Inflammatory cells − − − ++++ −
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