Article

Short-Term Diet Restriction but Not Alternate Day

Fasting Prevents Cisplatin-Induced
Nephrotoxicity in Mice
Evrin Gunebakan 1, Esra Yalcin 1, Esra Cikler Dulger 2, Ahmet Yigitbasi 3, Nilay Ates 4,
Aysun Caglayan 1, Mustafa C. Beker 1, Kazim Sahin 5, Hasan Korkaya 6 and Ertugrul Kilic 1,*
1 Department of Physiology, School of Medicine, Istanbul Medipol University, Istanbul 34810, Turkey;
[email protected] (E.G.); [email protected] (E.Y.); [email protected] (A.C.);
[email protected] (M.C.B.)
2 Department of Histology and Embryology, Hamidiye Medical School, University of Health Sciences,

Istanbul 34668, Turkey; [email protected]

3 Department of Internal Medicine, School of Medicine, Trakya University, Edirne 22030, Turkey;

[email protected]
4 Department of Pharmacology, School of Medicine, Istanbul Medipol University, Istanbul 34810, Turkey;

[email protected]
5 Animal Nutrition Department, School of Veterinary Medicine, Firat University, Elazig 23119, Turkey;

[email protected]
6 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University,

Augusta, GA 30912, USA; [email protected]

* Correspondence: [email protected] or [email protected];
Tel.: +90-216-681 5344; Fax: +90-212-531 7555

Received: 18 December 2019; Accepted: 24 January 2020; Published: 3 February 2020

Abstract: Cisplatin (CP) is one of the most preferred platinum-containing antineoplastic drugs.
However, even in nontoxic plasma concentrations, it may cause kidney injury. To be able to increase
its effective pharmacological dose, its side effects need to be regarded. Diet restriction (DR) has been
demonstrated to improve cellular survival in a number of disorders. In this context, we investigated
the role of DR in CP-induced nephrotoxicity (CPN). Besides alternate DR, animals were exposed to
DR for 3 days prior or after CP treatment. Here, we observed that both 3 days of DR reverses the
nephrotoxic effect of CP, which was associated with improved physiological outcomes, such as
serum creatine, blood-urea nitrogen and urea. These treatments significantly increased phosphorylation
of survival kinases PI3K/Akt and ERK-1/2 and decreased the level of stress kinase JNK were noted.
In addition, the activation level of signal transduction mediator p38 MAPK phosphorylation was
higher particularly in both three-day DR groups. Next, animals were fed with carbohydrate-,
protein- or fat-enriched diets in the presence of CP. Results indicated that not only fasting but also
dietary content itself may play a determinant role in the severity of CPN. Our data suggest that DR
is a promising approach to reduce CPN by regulating metabolism and cell signaling pathways.

Keywords: cisplatin; fasting; renal protection; acute kidney injury; nephrotoxicity; MAPK pathway

1. Introduction
Cisplatin (Cis-diaminedichloroplatinum, CDDP or CP) is the most commonly used platinum-
containing antineoplastic drug for the treatments of various solid tumors, ranging from bladder [1]
to breast [2] to non-small cell lung cancer [3]. It may cause serious parenchymal toxicity due to its
accumulation in the kidney tissue, as it is usually excreted in the urine. Even though cisplatin-induced
nephrotoxicity (CPN) could be alleviated by diuretics, prevalence of CPN is still high [4].
Approximately in 60% of CP-treated patients, acute kidney injury (AKI) is developed, in which renal
Biomedicines 2020, 8, 23; doi:10.3390/biomedicines8020023 www.mdpi.com/journal/biomedicines
Biomedicines 2020, 8, 23 2 of 13

vasoconstriction, decrease in glomerular filtration rate and increase in serum creatinine levels were
observed [5]. This dose-limiting side effect of CP renders dose augmentation almost impossible in
patients requiring higher therapeutic efficacy.
Fasting or diet restriction (DR) is described as food deprivation from the system for certain
periods of time [6]. Several lines of clinical and nonclinical studies suggested that DR prior to
chemotherapy potentiates the effect of the treatment by sensitizing cancer cells to chemotherapy,
through a decrease in circulating growth factors and by supporting healthy cells to become more
resistance to oxidative stress [7–10]. Alternate day fasting (AF) is one of the experimental DR models
in which 24 hour long cycles of feeding and fasting are applied. In AF, food is provided ad libitum
for 24 h, followed by a 24 h long period of fasting (no food or reduced food) [11]. Studies have shown
that AF has antineoplastic effects, such as inhibition of tumor growth and tumor cell proliferation,
and decreased insulin-like growth factor-1 (IGF-1) levels [12–17]. In addition, prolonged fasting (PF)
lasting for 48–120 h has been studied in terms of cellular survival and antineoplastic benefits. The full
switch to fat and ketone bodies in prolonged fasting conditions enhances tumor cells’ resistance to
toxicities in both mice and humans and inhibits tumor growth [18,19]. It has also been shown that PF
can play a role in chemoprotection and promote hematopoietic stem-cell self-renewal and
reconstitution [18].
However, the cell-protective effect of DR on CPN is largely unknown. In this study, we
investigated the effect of AF or fasting for 72 h prior to or after CP-treatment on CPN and analyzed
renal functions and cellular survival using histological examinations. Next, we evaluated the
activations of signaling pathways, including survival and stress kinases. Finally, to further identify
the effect of dietary contents, we compared the nephrotic histopathologies in ad libitum feeding and
protein-, carbohydrate- or lipid-rich diet groups.

2. Materials and Methods

2.1. Animals
Adult male Balb/c mice (n = 60, 14–17 weeks old) weighing 30 ± 4 g were kept in an
environmentally controlled room with a constant temperature and humidity. Animals were
maintained into a 12:12 h long light–dark cycle and had free access to water and standard rodent diet
ad libitum. All experimental procedures were carried out with local governmental approval (Istanbul
Medipol University, 29/8/2014), according to NIH guidelines for the care and use of laboratory
animals

2.2. Experimental Design

In the first set of experiments, animals were randomly assigned to one of 4 groups (n = 8/ group/
each), with continuous water accessibility. Then, 7 mg/kg of cisplatin (CP) (Sigma Chemical Co, MO,
USA) dissolved in 0.9% saline (1 mL/100 mg) [20,21] was administered, with a single intraperitoneal
(i.p.) injection for the induction of CP- induced nephrotoxicity (CPN). (i) Control group with ad
libitum feeding; (ii) alternate day fasting (AF) group were maintained in a eating-fasting cycle for 14
days with 24 h ad libitum feeding, followed by 24 h fasting period with CP injection being done on
day 7; (iii) 72P group fasted for 72 h before CP injection; (iv) 72A group fasted for 72 after CP injection.
All mice were sacrificed by using high dose chloralhydrate anesthesia one week after CP
administration (day 14). Blood was collected, and kidneys were removed and shock-frozen on dry
ice.
In the second set of experiments, animals were divided into 4 groups (n = 7/group/ each) and
treated with isocaloric diets. (i) Control group received ad libitum feeding (Altromin); (ii)
carbohydrate group was fed with 79.6 % carbohydrates, 10.6% fat and 9.8% protein; (iii) protein
group was fed with 76.9% protein, 11.7% carbohydrates and 11.3% fat; (iv) fat group was fed with
78.4% fat, 11.2% carbohydrates and 10.4% protein. Feeding started on day 0, cisplatin (7 mg/kg) was
administered on day 7 and animals were sacrificed on day 10.
Biomedicines 2020, 8, 23 3 of 13

2.3. Histopathological Analysis

Two sets of 6 µm thick frozen cryostat (LEICA CM 1950, Wetzlar, Germany) sections from each
animal were prepared for histopathological examinations. Both sets were fixed in 70% alcohol and
stained according to standard protocols. One set was stained with hematoxylin and eosin (H&E) for
the general morphologic examinations, and the others were stained with periodic acid–Schiff (PAS)
for the examination of glomerular constitution, brush border of the differentiated apical surface of
proximal tubular epithelial cells and basal membranes. Histomorphometric evaluation of H&E
stained cortical and cortico-medullary proximal tubules was conducted based on acute tubular
necrosis criteria, including tubular epithelia loss, intercellular cavities, brush-border loss, swollen
apical membranes [22,23] and acute tubular damage criteria, including epithelia loss, pallid/pale
stained cytoplasm and vacuolization, brush border loss and luminal dilatation caused by thinning
tubule epithelia [22].
For each kidney section, 5 light microscopic fields at a magnitude of ×200 were examined.
Distribution and existence of lesions mentioned above were evaluated on a scale of 0–4. Microscopic
areas showing no damage were scored 0; microscopic areas showing damage less than 10% were
scored 1; between 10–25% were scored 2; between 26–75% were scored 3; and between 76–100% were
scored 4 [23,24].

2.4. Biochemical Analysis

Blood samples were collected and centrifuged at 10,000 rpm for 10 min at room temperature (22
°C), and sera were collected and stored at −80 °C for subsequent measurements of renal functions.
Serum creatine, blood urea nitrogen and urea levels were measured with the Roche (Basel,
Switzerland) C501 instrument, photometrically with the instruction kits, according to the
manufacturer’s protocol.

2.5. Western Blot

Tissue samples belonging to the same group were pooled, homogenized and treated with
protease and phosphatase inhibitor cocktails. Protein concentrations were measured with Qubit 2.0
Fluorimeter, according to the manufacturer’s protocol (Invitrogen, CA, USA). Equal amounts of
protein (20 µg) were loaded on Any kD™ Mini-PROTEAN gels and run at 100 V for 2 h. Size-
fractioned proteins were then transferred to polyvinylidene fluoride membrane (PVDF), using a
semidry Turbo Transfer System (Bio-Rad, CA, USA). Membranes were blocked in 5% nonfat milk in
50 mM of Tris-buffered saline containing 0.1% Tween (blocking solution), for 1 h, washed in Tris-
buffered saline containing 0.1% Tween (TBS-T) and incubated overnight with rabbit anti-phospho
Akt (4060; Cell Signaling Technology, MA, USA), rabbit anti-total Akt (9272; Cell Signaling), rabbit
anti-phospho ERK-1/-2 (9101; Cell Signaling), rabbit anti-total ERK-1/2 (9102; Cell Signaling), mouse
anti-phospho JNK-1/2 (9255; Cell Signaling), rabbit anti-total JNK-1/2 (9252; Cell Signaling), rabbit
anti-phospho p38 (9211; Cell Signaling) and rabbit anti-total p38 (9212; Cell Signaling). Each antibody
was diluted 1:1000 directly in blocking solution. On the following day, membranes were incubated
with peroxidase-conjugated goat anti-rabbit (Amersham, GE Health Care, MA, USA) or goat anti-
mouse (7076; Cell Signaling) antibodies. Protein loading was controlled by stripping and re-probing
the blots with total antibodies for each phospho antibody of interest. Blots were developed with Bio
Rad ECL kit (Clarity™ Western ECL Substrate, #1705060) and visualized by the ChemiDoc MP
(Biorad, CA, USA) system. The intensity of each signal was measured on a total of three digitized
blots each, using the ImageJ software program. Protein levels were analyzed densitometrically and
corrected with their total proteins each and expressed as a percent of total protein.

2.6. Statistical Analysis

Statistical analysis for the histologic score values was performed by using the Kruskal–Wallis
test and Dunn′s multiple comparison test for the histopathological score values and group
comparison, respectively. Data comparisons of Western blot and, biochemical analyses of serum
Biomedicines 2020, 8, 23 4 of 13

samples were analyzed with a standard software package (SPSS 18 for Windows; SPSS Inc., Chicago,
IL, USA). Differences between groups were analyzed by one-way ANOVA, followed by LSD test. All
values were given as mean ± S.D., with n values, indicating the number of animals/each group
analyzed. The p-values < 0.05 are considered significant.

3. Results

3.1. Prolonged Starvation Resulted in Conserved Morphology in Nephrotic Structures

In order to examine the effect of varying fasting regimes on CPN, three DR regimes chosen, and
compared with control animals which were also treated with CP but had ad libitum access to water
and food. Parameters of (i) epithelial loss on the tubules, (ii) brush border loss of the remaining
epithelial cells, (iii) tissue loss within intratubular space and (iv) loss of cell height were evaluated as
indicators of the extent of CPN. The most extensive and severe damage was observed in the control
group when compared with DR groups (Figure 1A).
The tissue in intertubular space was more damaged and tubules were more shriveled in the
control group when compared with DR groups (Figure 1A). Moreover, PAS (+) areas were decreased
in the basal membranes of the deformed tubules (Figure 1A). In the AF group, this decrease, along
with the severity and extent of the damage, was slightly but not significantly recovered compared to
the control group (Figure 1A, B). Decreased cell height, increased brush border loss and widened
intertubular spaces were detected in the AF group, together with hypertrophic but less severe
glomerular degeneration (Figure 1A). Interestingly, the tubular damage inflicted by CP
administration was significantly decreased in both the 72P and 72A groups compared to the control
group (Figure 1A, B). Moreover, in the 72A group, epithelial cells lining the inner part of the tubules
were more intact as compared with other groups. Also, the epithelial cell height was normal, and the
apical brush border was present. Normal tubule appearance and diminished gaps between the
tubules were also observed (Figure 1A). In the 72P group, tissue damage was decreased, and tissue
integrity remained relatively intact compared to the other groups (Figure 1A). The comparison of
72A and 72P revealed decreased tissue damage in the 72P group. However, there was no statistically
significant difference between the 72A and 72P groups (Figure 1B).
Biomedicines 2020, 8, 23 5 of 13

Figure 1. Seventy-two hours of fasting improves the histological parameters for CP induced
nephrotoxicity. (A) Microphotographs of H&E and PAS staining. (a,b) Kidney tissue of the control
group was shown. (a) Damaged tubules and staining difference (arrow), glomerular degeneration
(arrowhead), loss of epithelial cells lining inner part of tubule (double arrow) and small gaps between
the tubules (*); and (b) basal membrane degeneration of dysmorphic tubules (arrowhead) and
degeneration in epithelial cells lining the inner part of the tubules (arrow) were demonstrated. (c,d)
Kidney tissue of the alternate fasting (AF) group was shown. (c) Damaged tubular shape/appearance
and staining difference (arrow), glomerular degeneration (arrowhead), ripped off-like gaps between
the tubules (*) and (d) degeneration of the basal membrane (arrowhead) were demonstrated. (e,f)
Kidney tissue of fasting 72 h prior CP (72P) group was shown. (e) Small damage and widening
between the tubules (*) and tubule shape (arrow), decrease in glomerular size and (f) brush border at
the apical surface of the epithelial cells lining the inner part of the tubule (arrow) and PAS (+) reaction
of basal membranes (arrowhead). No PAS (+) reaction of basal membranes was observed at separated
Biomedicines 2020, 8, 23 6 of 13

tubules (*) were demonstrated. (g,h) Kidney tissue of fasting 72 h after CP (72A) group was shown.
(g) Little misshaped tubules (arrow) and gaps between neighboring tubules (*) and (h) brush border
of tubular epithelial cells (arrow) and PAS (+) reaction of basal membranes were demonstrated
(arrowhead. a, c, e, g: H&E dye, 200× magnification; b, d, f, h: PAS dye, 400× magnification. (B)
Histological score of CP treated mice kidney in control, AF, 72P and 72A groups. Data are given as
mean ± S.D. ** p < 0.01, and *** p < 0.001.

3.2. Biochemical Tests and Analysis of DNA Fragmentation Reveal that 72A Group Is Protected from CP-
Induced Oxidative Stress
To determine the CP-induced kidney functions, serum creatinine, urea and blood urea nitrogen
(BUN) were assessed. In all fasting groups (AF, 72P and 72A), serum creatinine was significantly
decreased (p < 0.05) and was highest in the control group (Figure 2A). BUN and urea levels were
significantly lower in both 72 h fasting groups (72A and 72P) compared to the control (Figure 2B, C).

Figure 2. Biochemical analysis of mice serum exhibits recovery in CP induced oxidative stress and
kidney function. Serum levels of (A) Creatinine, (B) BUN, (C) Urea were presented. Data are given as
mean ± S.D. * p < 0.05.

3.3. Seventy-Two Hours of Fasting Improves Cellular Survival via Regulating MAPK Pathway Elements in
Kidney
Biomedicines 2020, 8, 23 7 of 13

To assess the changes in survival kinases in different dietary groups, Western blot analysis of
the kidney tissues was performed. As compared with control and AF, the level of phosphorylated
Akt (p-Akt), an indicator of cellular survival in kidney tissue was significantly upregulated in both
72 h DR regimes (p < 0.01), with no significant difference between them. Interestingly, only AF group
decreased the p-Akt level compared with the control group (p < 0.01) (Figure 3A). In terms of another
survival kinase p-ERK1/2 levels, AF group decreased p-ERK1/2 levels compared with control group,
whereas both 72 h fasting groups showed an increase in p-ERK1/2 compared with AF or control (p <
0.01) (Figure 3B). In addition, CP administration caused a significant decrease in the phosphorylation
level of c-Jun NH2-terminal kinase (JNK) in all fasting groups, compared with the control group (p <
0.01). On the other hand, 72P and 72A groups had significantly higher p-JNK compared to AF (p <
0.05 and p < 0.01, respectively) and compared to the other two fasting regimes, 72A group showed
the highest JNK phosphorylation (both p < 0.01) (Figure 3C). Finally, we analyzed the
phosphorylation levels of another MAPK stress signaling protein, p38 (p-p38). Results showed that,
in both 72 h fasting groups, the level of p-p38 was significantly upregulated when compared with the
control or AF groups (p < 0.05 for 72A; p < 0.01 for 72P) (Figure 3D). However, 72P showed a more
pronounced increase in the level of p-p38 than 72A compared to control (p < 0.01). Finally, p-p38 level
was similar to that of the control group in the AF group, following CP-induced CPN.

Figure 3. Seventy-two-hour fasting rescues kidney morphology and function through MAPK
pathway. Western blot analysis of pooled tissue samples. Changes in protein levels of (A) p-JNK, (B)
p-AKT, (C) p-ERK-1/-2 and (D) p-p38. * p < 0.05, ** p < 0.01 compared with control; $ p < 0.5, $$ p < 0.01
compared with alternate fasting; # p < 0.05, ## p < 0.01 compared with 72 h before.

3.4. Dietary Content Is Also a Rate-Limiting Factor in Expansion of AKI Followed by CP Administration
In general, glomerular destruction and proximal- and distal-tubular cell loss was observed in all
dietary groups studied dur to the injection of CP (Figure 4A, B). Nuclear dilatation and loss of
microvilli on the proximal tubular cells were observed in the remaining tubular epithelial cells. In
addition, dilated tubules were presented with an altered intensity in gaps and infiltration of
inflammatory cells. Histopathological analysis of the study groups revealed partial epithelial loss,
while some epithelial cells remained intact in distal tubules (Figure 4A, left panel). However, the
Biomedicines 2020, 8, 23 8 of 13

basal membrane was continuous (Figure 4A, right panel). Moreover, inflammation was reduced in
the control group compared to carbohydrate-, protein- and lipid-rich dietary groups.
In the carbohydrate group, glomeruli covering the urinary space were distinguishable despite
being swelled. Inflammatory cell infiltration was present, through basal membranes of some tubules
indicated continuity (Figure 4A).
Although intertubular gaps were observed in the control, carbohydrate and fat groups, the most
enlarged gaps were detected in the protein group. There was an increase in the number of
inflammatory cells and a loss in epithelial cells that form tubules and microvilli, and remaining viable
cells indicated scattered and abnormal nuclei (Figure 4A). Glomerular structures were swelled
(Figure 4A), and the expected PAS reaction of the basal membrane around the tubules was not
observed in the protein group (Figure 4A).
The fat group displayed intensive inflammation, destroyed basal membrane of tubules, loss of
tubular epithelium and enlarged nucleus, especially in the proximal tubules (Figure 4A).
Overall, these data indicated that both protein and fat groups exhibited the most severe damage
while the difference that the carbohydrate diet made in renal parenchyma was not significant when
compared with the control group(Figure 4B).

Figure 4. Dietary content determines the extend of CP-induced nephrotoxicity. Tissue of null diet
kidney. (A) Microphotographs of H&E and PAS staining. (a,b) Kidney tissue of control group was
shown. (a) Damage of tubular shape and loss of epithelial cells lining inner part of tubule (doble
arrow), gaps in between the tubules (*) and inflammation (arrow), and (b) intact basal membrane
(arrow) were demonstrated. (c,d) Kidney tissue of carbohydrate group was shown. (c) Dysmorphic
Biomedicines 2020, 8, 23 9 of 13

tubules and cell loss (double arrowhead), hypertrophic glomeruli (arrowhead) and small gaps in
intertubular space (*), and (d) basal membrane (arrow) were demonstrated. (e,f) Kidney tissue of
protein group was shown. (e) Large separations and destructions in between the tubules (*) and loss
of morphology (arrowhead) and (f) microvilli loss at the apical surfaces of the tubules (double
arrowhead) and destruction of tubular basal membrane morphology (arrow). (g,h) Kidney tissue of
fat group was shown. (g) Morphological disturbances and epithelial cell loss (arrow), gaps (*) and
inflammation (double arrow) in between neighboring tubules and (h) basal membrane loss around
the tubules (arrow) and glomerular dilatation (arrowhead) were demonstrated. (a,c,e,g) H&E dye,
200× magnification; (b,d,f,h) PAS dye, 400× magnification. (B) Histological score of CP-treated mice
kidney in carbohydrate, protein, fat, or null (Control) groups were given. Data are given as mean ±
SD. * p < 0.05.

4. Discussion
CP is the most commonly used chemotherapeutic compound, and it was the first approved
platinum-containing molecule by the FDA in 1978 [25]. It is stable in ambient conditions [26], and its
efficacy has been shown in different types of cancers, including sarcomas, blood vessels, ovarium,
testis or different kinds of solid tumors [27]. It crosslinks with the purine bases on the DNA, interferes
with DNA repair mechanisms and consequently induces DNA damage in cancer cells [14].
Numerous studies have been conducted to address dose-limiting side effect of CP. It faces restrictions
in clinical applications due to its side effects, such as nephrotoxicity. Even when it is applied below
therapeutic concentrations, it may accumulate in the kidney tissue and cause acute irreversible
kidney injury (AKI) [28].
Fasting is an attractive therapeutic approach to extend the life span of different organs and can
be used as an adjuvant in the treatment of several diseases, including cancer [10]. It is estimated that
healthy cells and cancer cells react to fasting differentially [19]. Nutrient deprivation inactivates
proliferative signals in the cell, while it conserves protective mechanisms in turn. It has been
proposed that fasting causes changes in gene expression and metabolism so that healthy cells of the
body use alternate resistance pathways to prevent tissue injury. Since cancerous cells cannot exhibit
such properties, chemotherapy, followed by fasting, makes those cells more vulnerable to drugs than
healthy cells, possibly because of their lower ability to adapt to restricted energy supply [9,19,29].
Therefore, short-term fasting has emerged as a critical intervention for the attenuation of the side
effect of chemotherapeutic compounds. In particular, 72 h short-term fasting was found feasible and
was well-tolerated in a cancer patient, when DR was applied during chemotherapy. However, the
role of DR in the CP-induced nephrotoxicity was still unknown [30]. Notably, it may also be a
consequence of the excretion function of kidneys. In addition to the waste products of our cellular
metabolism, drugs and/or their metabolites are also removed by the kidneys. In this excretion
process, renal cells are exposed to such toxic molecules more than other cells in our body. In this
context, here, we studied the role of short-term DR in the development of CP-induced nephrotoxicity.
By using alternate day fasting (AF), 72 h fasting prior (72P) and 72 h fasting after (72A) CP treatment,
we analyzed the effect of DR on CP-induced nephrotoxicity. General morphologic examination with
H&E staining revealed that control group had significantly more tubular damage compared to the
other groups. On the one hand, compared to the control, even with continuing hypertrophy, AF
resulted in higher glomerulus integrity. On the other hand, there was a substantial decrease in
structural damage in the 72P and 72A groups. Moreover, in terms of CP-induced damage extensity
and severity, the control group was the most affected group. As compared with control animals, AF
decreased tissue damage moderately, while 72P and 72A significantly decreased both damage
extensity and severity. In general, these results indicate that fasting either 72 h prior to or after CP
treatment significantly ameliorates histopathological outcomes of CP-induced nephrotoxicity.
We further analyzed the physiological outcomes of DR treatment after CP toxicity. Biochemical
analysis of renal activity showed that AF significantly decreased serum creatinine levels. However,
both 72 h DR regimes decreased serum creatinine, as well as BUN and urea levels. These are
metabolic byproducts, and removed from the body by the kidneys. Increased levels of these
molecules indicate functional failure of the kidney. In addition, these accumulated byproducts
Biomedicines 2020, 8, 23 10 of 13

worsen kidney damage. When evaluated with histological findings, these physiological parameters
suggest that fasting for 72 h after or prior to CP treatment can ameliorate AKI.
In addition, we analyzed the levels of activated survival kinases ERK and PI3K/Akt, in CP-
induced nephrotoxicity. As compared with the control or AF, we observed that 72 h fasting before or
after CP increases phosphorylated ERK and Akt. Interestingly, as compared with the control, the AF
resulted in a decrease in the phosphorylation of these proteins. Akt phosphorylation by PI3K is
essential for cellular survival and proliferation [31]. The ERK pathway is mainly regulated by an EGF
Receptor/Ras/Raf signaling cascade in the oxidative-stress condition. The inhibition of ERK pathway
causes cell death after kidney injury, cerebral ischemia and retinal ganglion cell injury [32–34]. In
general, the activation of both ERK and PI3K/Akt pathways is associated with cellular survival [31].
It is interesting that signaling pathways in general or ERK and PI3K/Akt survival kinases have
been a discussion of the matter with controversial results in terms of cancer cell, cellular survival and
CP toxicity. It can be a consequence of different methodological approaches for the administration of
pathway inhibitors and can be affected by the use of different injection coordinates of these molecules
to the kidney. In a recent study, Saha et al., revealed that mangiferin ameliorates kidney injury,
induced by an oxidative stress inducer, tBHP, via the deactivation of stress kinases JNK and p38 and
the activation of PI3K/Akt pathways [35]. In addition, by using the PI3K inhibitor LY294002, it was
shown that propofol protects kidneys via the phosphorylation of PI3K/Akt pathway after kidney
injury, which was associated with the improved serum BUN level [36]. Furthermore, it was shown
that CP also activates Akt pathways via EGFRi, Src and PI3K, which was associated with the
activation of p38 [37].
The second downstream target of CP is JNK, and it is known to play a role in inflammation and
apoptosis [38,39]. In our study, we found that JNK is significantly downregulated in all fasting
groups. Recent evidence suggests that inflammation is one of the main processes that leads to
proximal tubular injury [40]. Ramesh and Reeves have shown that inhibition of p38 reduces CP-
induced functional and structural renal damage [41]. In our present study, we found that AF did not
alter p38 phosphorylation levels compared to the control, but each of the 72 h fasting regimens
increases p38 phosphorylation. Previous studies suggested that TNF-alpha inhibition by means of
blocked activation of p38 provides protection from cisplatin-related renal injury and inflammation
[41,42]. It can be concluded that 72 h fasting could enhance the inflammatory response against
hydroxyl and other radicals and protect renal cells against CP-induced injury, which in turn may
explain how 72 h fasting but not AF could ameliorate CP-induced damage in our histological findings
[43]. As previously mentioned, JNK activation in both 72 h groups was significantly higher.
Compared to 72A fasting 72 h fasting before CP treatment showed significantly higher p38 activation.
It was reported that fasting before and after chemotherapy treatment may have different outcomes
regarding injury caused by re-feeding. Long-term fasting before chemotherapy induces cell death in
cancer cells, but during re-feeding, cell proliferation is induced and may lead to DNA damage in the
presence of toxins [44].
In summary, fasting for 72 h before cisplatin treatment could prevent CP-induced kidney injury
via decreased phospho-JNK and hence decreased MAPK pathway activation and caused an increase
in cell cycle arrest. Furthermore, both 72 h fasting groups showed an increase in p38 MAPK activation
and inflammatory response. This increase did not affect the perseverance of the kidney tubules, such
that we can conclude that Akt phosphorylation may be involved in the renoprotective effects of
fasting for 72 h.
As for the second part, we studied the effect of dietary contents, rather than fasting, on CP-
induced nephrotoxicity. Based on histological examinations, it can be concluded that protein- and
fat-rich diets caused more severe renal damage standard diet. It can be speculated that serving cancer
and patients that undergo CP treatment with a low-protein and -fat diet (if not fasting) could provide
beneficial effects.
Taken together, our results indicate that 72 h of fasting both prior to and after CP treatment may
not only increase the efficacy of eliminating cancer cells in the organism but also ameliorates CP-
induced AKI. We demonstrated that fasting-mediated rescue of renal failure takes place, at least in
Biomedicines 2020, 8, 23 11 of 13

part, by improving renal tissue integrity and renal functions. Our findings can make a significant
contribution to chemotherapeutic treatment strategies of cancer patients, by establishing a method
for clinical application of CP at higher and effective doses, without causing severe AKI.
Author Contributions: Design of work, E.G., E.K., H.K. and K.S. Performed experiments, E.Y., E.C.D., E.G., A.C.,
N.A. and A.Y. Data analysis, E.Y., E.C.D., E.G. and M.C.B. Wrote paper, E.K., E.G. and E.Y. All authors have
read and agreed to the published version of the manuscript.

Funding: This research was funded by the Turkish Academy of Science (TUBA).

Conflicts of Interest: The authors declare no conflicts of interest.

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