Keywords
acute kidney injury - gentamicin - moderate-intensity aerobic exercise - proteinuria
- Renin-Angiotensin System - Kallikrein-Kinin System
Introduction
Acute kidney injury (AKI) is a leading cause of morbidity and mortality worldwide
[1]
[2]. AKI episodes are characterized by
transient kidney dysfunction caused by different etiologies [3]
[4]
[5]
[6]. The use of aminoglycosides (AG), such
as gentamicin, to treat life-threatening infections is an important etiological
factor of AKI [3]
[7]
[8]
[9]. It has been shown that
5–10% of adult patients and 20–33% of non-critical ill children treated with AGs
develop AKI [3]
[7]
[8]
[9]. AG enters proximal
tubule epithelial cells (PTECs) through receptor-mediated endocytosis, accumulating
in high concentrations in its intact form, which in turn leads to PTECs death and
tubular injury [7]
[10]
[11]. However, the mechanisms underlying the gentamicin-induced PTEC
injury are still poorly understood.
It is known that PTEC injury correlates with the development of acute tubular
necrosis (ATN), tubular dysfunction, and proteinuria in AKI [12]
[13]. Additionally, there is an association between these factors and the
abnormal activation of components from the renin-angiotensin system (RAS) and/or
kallikrein-kinin system (KKS) in different forms of AKI [14]
[15]
[16]
[17]. Ali and Bashir [18] showed increased plasma ACE activity
in gentamicin-induced AKI. Furthermore, Heeba [19] showed that blocking the angiotensin II receptor (AT1R)
with losartan (a well-known angiotensin receptor blocker - ARB) ameliorated ATN and
albuminuria in gentamicin-induced AKI. On the other hand, Bledsoe et al. [20] showed that the infusion of tissue
kallikrein ameliorated tubular injury and cell apoptosis observed in
gentamicin-induced AKI through the activation of bradykinin B2 receptor
(B2R). These data indicate the potential involvement of RAS and KKS
in the development of gentamicin-induced AKI. However, the therapeutical use of ARBs
or ACE inhibitors is not recommended during an AKI episode because of their adverse
effects on renal hemodynamics, further disturbing glomerular flow rate (GFR) levels
[14]. Therefore, it becomes
attractive to use alternative therapeutic strategies to modulate intrarenal RAS
and/or KKS as well as the kidney’s responses prior to an AKI episode, which in turn
would prevent the development of gentamicin-induced AKI.
Since the levels of renal injuries observed during AKI onset depend on the background
susceptibility and the state of the renal microenvironment [12]
[13], systemic adaptations following preconditioning by aerobic exercise
appear to be a promising candidate. Beyond the known effects of skeletal muscles,
regular physical activity, such as aerobic exercise, also promotes several positive
systemic adaptations in blood flow, cell metabolism, and immunological responses in
different systems, including the kidneys [21]. It has been shown that physical exercise acts as an adjuvant in the
treatment of many disorders [22]
[23]
[24]
[25]. Some studies have
shown that moderate-intensity aerobic exercise (MIAE) promotes protective effects
in
AKI and CKD animal models [26]
[27]
[28]
[29]. De Lima et al. [27] demonstrated that ATN and increased
active caspase-3 expression were prevented following preconditioning by MIAE in
ischemia/reperfusion (I/R)-induced AKI. Additionally, Francescato et al. [28] showed that tubular injury,
inflammation, and fibrosis were reduced due to an increase in renal nitric oxide
(NO) after preconditioning by MIAE in cisplatin-induced AKI. Furthermore, previous
work showed that MIAE modulates RAS and KKS components in skeletal muscle, lung, and
liver [30]
[31]
[32]
[33]. Based on the above,
we hypothesized that preconditioning by MIAE could modulate the components of
intrarenal RAS and KKS, which in turn could prevent the development of the tubular
injury and proteinuria observed in gentamicin-induced AKI.
In the present work, we aimed to study the potential protective effect of
preconditioning by MIAE on tubular injury observed gentamicin-induced AKI and verify
its possible correlation with changes in intrarenal RAS and KKS. To this purpose,
we
used male Wistar rats subjected or not to an MIAE protocol for 8 consecutive weeks.
Then, AKI was induced by daily subcutaneous injections of gentamicin (80 mg/kg/day)
for 5 consecutive days. Our results showed that preconditioning by MIAE attenuated
the tubular injury and proteinuria observed in gentamicin-induced AKI. These
protective effects correlated with the inhibition of renal ACE and B1R.
These findings shed new light on the potential reno-protective effects of aerobic
exercise under pathological conditions.
Materials and Methods
Animals
Male Wistar rats (250–300 g) were obtained from the Central Animal Facility of
Federal University of Minas Gerais. All procedures were conducted according to
protocols approved by the Institutional Ethics Committee
(CEUA-UFMG#59/2015).
Preconditioning by moderate-intensity aerobic exercise (MIAE)
Rats were trained on a treadmill (Gaustec Magnetismo, MG, Brazil) according to a
training protocol described by Previero et al. [34]. The efficacy of the training
protocol was evaluated by applying three maximum progressive effort tests (MPET)
[35]. The progression of the
training protocol is summarized in [Table
1]. MPET allows for the evaluation of total time of exercise (TTE),
maximum running speed (MRS), distance (D), and workload (W), which is
calculated as W (k x gm)=body weight (kg) x TTE (min) x speed on the
treadmill when fatigue is reached (m/min) x grade of treadmill inclination [36].
Table 1 Protocol of moderate intensity aerobic
training.
|
Training Progression
|
|
MEPT
|
1st week
|
2nd week
|
3rd week
|
4th week
|
|
1st
|
28% MRS/30–40 min
|
44% MRS/45 min
|
65% MRS/50 min
|
65% MRS 60 min
|
|
5–8th week
|
|
2nd
|
65% of MRS*/60 min
|
|
3rd
|
65% of MRS*/60 min
|
The training protocol was adapted from Priviero et al. [34]. *Maximal run speed (MRS)
was adjusted according to running performance achieved at 2nd
MPET.
Induction of gentamicin-induced AKI after preconditioning by MIAE
After completing the 8-week MIAE training protocol, rats were housed individually
in metabolic cages with free access to water and chow. Four experimental groups
were generated: 1) non-trained injected with saline (NT+SAL); 2) non-trained
injected with gentamicin (NT+AKI); 3) trained injected with saline (T+SAL); and
4) trained injected with gentamicin (T+AKI). Gentamicin-treated groups (NT+AKI
and T+AKI) received gentamicin (Gentotec, 80 mg/kg/ day, 0.1 ml/100 g body
weight, subcutaneously) for 5 days. Saline (0.9% NaCl) was used as a vehicle.
After treatment, 24-hour urine was collected for further analysis. Rats were
anesthetized (15 mg/kg ketamine and 7.5 mg/kg xylazine, intraperitoneally) and a
blood sample was collected from the inferior vena cava. Under anesthesia, rats
were euthanized through diaphragm perforation. Soleus muscles, kidneys, plasma,
and urine were stored at − 80°C until use.
Cardiovascular parameters
Systolic, diastolic, and mean arterial pressures (SAP, DAP, and MAP,
respectively) and heart rate were measured using tail-cuff plethysmography (CODA
Noninvasive Blood Pressure Monitor - Kent Scientific Corporation, Connecticut,
USA).
Assessment of renal function
Urine flow was determined volumetrically, and GFR was estimated by creatinine
clearance (CCr) [17]
[37]. Creatinine concentrations were
determined colorimetrically using a commercial kit (Bioclin/Quibasa, MG,
Brazil). Proteinuria and γ-glutamyl transferase enzyme activity (γ-GT) were
measured using commercial kits (Labtest, MG, Brazil). Sodium and potassium
concentrations were measured by flame photometry (CELM, FC 280, MG, Brazil).
Osmolality was determined by freezing point osmometry (Micro-osmette, Precision
System Inc, MA, USA).
Measurement of citrate synthase activity
Citrate Synthase (CS) activity in soleus muscle homogenates was measured as
previously described [38]. The CS
assay consisted of the incubation of homogenate (5 µl) with a buffered solution
(50 mM Tris-HCl containing 5 mM EDTA, 0.05% Triton X-100, 30 mM Acetil-CoA,
10 mM DTNB, pH 8.1).
Histological analysis
Kidneys were fixed in 10% buffered formaldehyde and embedded in paraffin. 4 μm
kidney sections were used for Hematoxylin-Eosin staining. Analysis was carried
out in a microscope (Axiolab, Carl Zeiss, Oberkochen, Germany) at a
magnification of×40 under blinded conditions. Morphometric analysis was
performed using NIH ImageJ software (version 1.6.0). The total glomerular area
(area by glomerulus), glomerular tuft area (% of total glomerular area) and
Bowman’s space area (% of total glomerular area), as well as glomerular
cellularity (%), number of tubular cells (cells per field), vacuolization rate
(vacuolization area normalized by tubular cells), tubular pyknotic cells (%) and
accumulation of interstitial cells (%) were analyzed. It is important to mention
that all morphometric analyses were performed in a double-blind manner.
Extraction of renal mRNA and analysis of gene expression
Renal gene expression of angiotensin-converting enzyme (ACE) and
angiotensin-converting enzyme 2 (ACE2) enzymes, angiotensin II type 1 receptor
(AT1R), Mas receptor (MasR), bradykinin B1 receptor
(B1R), and B2 receptor (B2R) were analyzed by
real-time PCR. Renal mRNA was extracted using Trizol reagent (Life Technologies,
Carlsbad, CA, USA) according to the manufacturer’s protocol, and mRNA
concentration was determined in a NanoVue Plus Spectrophotometer (GE Healthcare,
Piscataway, NJ, USA). Reverse transcription was performed using 1 µg of total
pure mRNA, 200 units of reverse transcriptase, 100 mM DTT (dithiothreitol,
1.0 µL), 5x reverse transcription buffer (2.5 µL), 10 mM dNTPs (1.8 µL), 10000
units of RNAsin ( 0.2 µL) and 50 ng/ml of oligo (dT) (1.0 µL). The resultant
cDNA was used for real-time PCR. Specific sense and anti-sense primers’
sequences were: ACE (5’-CTTCACTGACCAAAAGCTGCG-3’ and
5’-CCTAGGGTCTGTACGGATCCG-3’); ACE2: 5’-GTGGAGGTGGATGGTCTTTCA-3’ and
5’-TTGGTCCACTGTTCTCTGGGA-3’); AT1R: 5’-TCTCAGCATCGATCGCTACCT-3’ and
5’-AGGCGAGACTTCATTGGGTG-3’); Mas: 5’-TGACCATTGAACAGATTGCCA-3’ and
5’-TGTAGTTTGTGACGGCTGGTG-3’); B1R (5’-AACATCGGGAACCGTTTCAAC-3’ and
5’-CACCCGGCAGAGGTCAGTT-3’); and B2R (5’-GCCTCCCTTCCGGCATATT-3’ and
5’-TCATAAAAGGCAGACCATTTGG-3’). Real-time PCR was carried out on an ABI PRISM
7000 Sequence Detection System (Applied Biosystems, California, USA) with SYBR
Green PCR Master Mix (Applied Biosystems, California, USA). The relative levels
of gene expression were determined using the comparative threshold cycle method
and normalized to GAPDH expression.
Statistical analysis
Results are expressed as the means±SEM. After confirmation of normal distribution
by the Kolmogorow-Smirnov test, the data were analyzed by two-way ANOVA followed
by the Bonferroni test. Statistical analyses were performed using GraphPad
Prism, version 8 for Windows (GraphPad Software, San Diego, CA).
Results
Protective effect of preconditioning by moderate-intensity aerobic exercise
on the glomerular and tubular dysfunctions observed in gentamicin-induced acute
kidney injury
Initially, we randomly divided the rats into two distinct groups: 1) non-trained
rats, sedentary animals; and 2) trained rats, animals submitted to MIAE protocol
for 8 consecutive weeks ([Table
1]). At this point, MPETs were assessed ([Fig. 1]). TTE, MRS, D, and W
were significantly increased after the 8th week of applied training
protocol ([Fig. 1a-d]). In
addition, we observed a reduction in heart rate without any changes in SAP, DAP,
or MAP ([Fig. 2a-d]).
Fig. 1 The effect of preconditioning by moderate-intensity aerobic
exercise on maximum progressive effort tests. Male Wistar rats (8–10
weeks) were submitted to two aerobic training protocols (using a
treadmill) for 8 consecutive weeks: 1) non-trained group (5 m/min,
5 min/day, 5 days/week, n=13), and 2) trained group (28–65% of maximum
running speed, 60 min/day, 5 days/week, n=13). (a-d) Assessment
of different parameters of maximum progressive effort tests (MPETs).
(a) Time of training. (b) Distance. (c) Maximal
running speed. (d) Workload. In Graph a-d: the black circles
denote the 1st MPET (period before the application of the
training protocol); the black triangles denote the 2nd MPET;
and black squares denote the 3rd MPET (period after the
application of the training protocol). Data were presented in the
mean±standard error of the mean (SEM).
Fig. 2 The effect of preconditioning by moderate-intensity aerobic
exercise on cardiovascular parameters. Male Wistar rats (8–10 weeks)
were submitted to two aerobic training protocols (using a treadmill) for
8 consecutive weeks: 1) non-trained group (5 m/min, 5 min/day, 5
days/week, n=13), and 2) trained group (28–65% of maximum running speed,
60 min/day, 5 days/week, n=13). In Graph a-d, the black circles denote
the period before the application of the training protocol, while the
black squares denote the period after the application of the training
protocol. (a-d) Measurement of cardiovascular parameters. (a)
Heart rate. (b) Systolic arterial pressure. (c) Diastolic
arterial pressure. (d) Mean arterial pressure. The cardiovascular
parameters were assessed by tail plethysmography. Data were presented in
the mean±standard error of the mean (SEM).
[Fig. 3a] shows the AKI development
protocol in non-trained and trained groups. We found that the trained rats
presented higher CS activity (a marker of physical training) in soleus muscle
extracts ([Fig. 3b]), indicating
that the influence of preconditioning was maintained.
Fig. 3 The efficiency of preconditioning by moderate-intensity
aerobic exercise after gentamicin-induced AKI. After the training
protocols were applied to rats as depicted in [Fig. 1], the animals were
separated to induce gentamicin-induced AKI generating four experimental
groups (detailed in the Methods section): 1) SAL+NT (n=6); 2) AKI+NT
(n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). (a) Experimental
design. NT, non-trained rats; T, trained rats. (b) Measurement of
citrate synthase activity in skeleton muscle after 5 days
post-gentamicin daily injections. The white bar denotes rats injected
with saline. The blue bar denotes rats injected with gentamicin. Data
were presented in the mean±standard error of the mean (SEM).
No changes were observed in water intake among the experimental groups ([Fig. 4a]). Interestingly, NT+AKI and
T+AKI groups had increased urinary volume (mL/24 h) and hydric balance (%);
however, these effects were attenuated in the T+AKI group ([Fig. 4b, c]). Furthermore, no
changes were observed in the osmolar clearance and free water clearance ([Fig. 4d, e]).
Fig. 4 The effect of preconditioning by moderate-intensity aerobic
exercise on net renal function changed by gentamicin-induced AKI. The
rats were submitted to moderate-intensity aerobic training protocols as
depicted in [Table 1].
After the exercise training the animals were treated with gentamicin to
develop AKI according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=6);
2) AKI+NT (n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). NT, non-trained
rats; T, trained rats. (a) Water intake. (b) Urinary
volume. (c) Hydric balance. (d) Osmolar clearance
(COsm). (e) Free water clearance
(CH2O). The white bar denotes rats injected with saline. The
blue bar denotes rats injected with gentamicin. Data were presented in
the mean±standard error of the mean (SEM).
Assessing the glomerular function, we observed higher levels of plasma creatinine
concentration as well as a significant drop in CCr levels in the
NT+AKI and T+AKI groups ([Fig. 5a,
b]). Interestingly, these effects were attenuated by preconditioning
with MIAE.
Fig. 5 The effect of preconditioning by moderate-intensity aerobic
exercise on glomerular dysfunction triggered by gentamicin-induced AKI.
The rats were submitted to moderate-intensity aerobic training protocols
as depicted in [Table 1].
After the exercise training the animals were treated with gentamicin to
develop AKI according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=6);
2) AKI+NT (n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). NT, non-trained
rats; T, trained rats. (a) Measurement of plasma creatinine.
(b) Determination of creatinine clearance (CCr).
The white bar denotes rats injected with saline. The blue bar denotes
rats injected with gentamicin. Data were presented in the mean±standard
error of the mean (SEM).
Assessing the tubular function by investigating the tubular handling of
electrolytes and water, we did not observe any significant changes in urinary
Na+ and K+ excretion in all experimental conditions
([Fig. 6a, b]). On the other
hand, we found that the NT+AKI group had significantly higher levels of the
fractional excretion of Na+ (FENa+), K+
(FEK+), and water (FEH2O) ([Fig. 6c-e]). However, these
modifications were attenuated in the T+AKI group.
Fig. 6 The effect of preconditioning by moderate-intensity aerobic
exercise on tubular dysfunction triggered by gentamicin-induced AKI. The
rats were submitted to moderate-intensity aerobic training protocols as
depicted in [Table 1].
After the exercise training the animals were treated with gentamicin to
develop AKI according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=6);
2) AKI+NT (n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). NT, non-trained
rats; T, trained rats. (a-b) The measurement of ext-linknary mass
of Na+ (a) and K+ (b). To determine
tubular function were measured: (c) the fractional excretion of
water (FEH2O); (d) the fractional excretion of sodium
(FENa+); and (e) the fractional excretion of
potassium (FEK+). The white bar denotes rats injected with
saline. The blue bar denotes rats injected with gentamicin. Data were
presented in the mean±standard error of the mean (SEM).
Preconditioning by moderate-intensity aerobic exercise attenuates both
glomerular and tubular injuries observed in gentamicin-induced acute kidney
injury
Next, we assessed proteinuria (a marker of renal disease) and urinary γ-GT
activity (a marker of PTEC injury). Our results showed that proteinuria and
urinary γ-GT activity were increased in both NT+AKI and T+AKI groups, but to a
lesser extent in the T+AKI group ([Fig.
7a, b]).
Fig. 7 The effect of preconditioning by moderate-intensity aerobic
exercise on the modulation of ext-linknary kidney injury markers by
gentamicin-induced AKI. The rats were submitted to moderate-intensity
aerobic training protocols as depicted in [Table 1]. After the exercise
training the animals were treated with gentamicin to develop AKI
according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=6);
2) AKI+NT (n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). NT, non-trained
rats; T, trained rats. (a) Determination of ext-linknary protein.
(b) Measurement of γ-glutamyl transferase (γ-GT) activity, a
proximal tubule epithelial cell injury marker. The white bar denotes
rats injected with saline. The blue bar denotes rats injected with
gentamicin. Data were presented in the mean±standard error of the mean
(SEM).
[Fig. 8] shows the assessment of
renal cortex structure. No modifications were observed among the experimental
groups when assessing the total glomerular area, glomerular tuft area, Bowman’s
space area, as well as glomerular and tubular cellularity ([Fig. 8a-f]). We observed increased
rates of cellular vacuolization and pyknotic nuclei (which are characteristics
of ATN) as well as an increased number of interstitial cells in the NT+AKI and
T+AKI groups ([Fig. 8g-i]).
However, these features were attenuated by previous moderate training (T+AKI
group).
Fig. 8 The beneficial effect of preconditioning by
moderate-intensity aerobic exercise on the kidney injury observed in
gentamicin-induced AKI. The rats were submitted to moderate-intensity
aerobic training protocols as depicted in [Table 1]. After the exercise
training the animals were treated with gentamicin to develop AKI
according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=4);
2) AKI+NT (n=5); 3) SAL+T (n=4) and 4) AKI+T (n=5). (a)
Representative images of 5-μm kidney slices stained with
hematoxylin-eosin. Subpanel A shows SAL+NT; subpanel B shows AKI+NT;
subpanel C shows SAL+T; subpanel D shows AKI+T. Cellular vacuolization
is denoted by (*) and pyknotic nuclei is denoted by (arrows).
Magnification 400X. Bar=50μm. Morphometric analysis has been performed
showing: (b) glomerular total area; (c) glomerular tuft
area; (d) Bowman space area; (e) Glomerular cellularity;
(f) Number of tubular epithelial cells; (g) Tubular
vacuolization; (h) Tubular pyknotic cells; (i)
Interstitial cells. Circles denote the number of animals (n) analyzed.
Data were presented in the mean±standard error of the mean (SEM).
Preconditioning by moderate-intensity aerobic exercise modulates components
from RAS and KKS altered during gentamicin-induced acute kidney injury
[Fig. 9] shows the measurement of
mRNA levels of renal RAS components. Our findings revealed that the NT+AKI,
T+SAL, and T+AKI groups exhibited higher levels of ACE mRNA. However, this
effect was less pronounced in the T+SAL and T+AKI groups ([Fig. 9a]). AT1R mRNA was
significantly increased only in the T+AKI group ([Fig. 9b]). Conversely, no
significant changes were observed in ACE2 and Mas receptor mRNA ([Fig. 9c, d]).
Fig. 9 The effect of preconditioning by moderate-intensity aerobic
exercise on the modulation of RAS components by gentamicin-induced AKI.
The rats were submitted to moderate-intensity aerobic training protocols
as depicted in [Table 1].
After the exercise training the animals were treated with gentamicin to
develop AKI according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=6);
2) AKI+NT (n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). NT, non-trained
rats; T, trained rats. Then, the measurement of mRNA expression on
kidney samples through real-time PCR was carried out. (a) ACE
mRNA. (b) ACE2 mRNA. (c) AT1R mRNA. (d) Mas mRNA.
The white bar denotes rats injected with saline. The blue bar denotes
rats injected with gentamicin. Data were presented in the mean±standard
error of the mean (SEM).
Our results showed that the NT+AKI group had a higher mRNA expression for
B1R than the NT+SAL group ([Fig. 10a]). Interestingly, the MIAE
was able to counteract this stimulatory effect induced by AKI. Additionally, we
observed no changes in the mRNA expression of B2R ([Fig. 10b]).
Fig. 10 The effect of preconditioning by moderate-intensity
aerobic exercise on the modulation of KKS components by
gentamicin-induced AKI. The rats were submitted to moderate-intensity
aerobic training protocols as depicted in [Table 1]. After the exercise
training the animals were treated with gentamicin to develop AKI
according to the experimental design shown in [Fig. 3a]: 1) SAL+NT (n=6);
2) AKI+NT (n=6); 3) SAL+T (n=6) and 4) AKI+T (n=7). NT, non-trained
rats; T, trained rats. Then, the measurement of mRNA expression on
kidney samples through real-time PCR was carried out. (a) B1R
mRNA. (b) B2R mRNA. The white bar denotes rats injected with
saline. The blue bar denotes rats injected with gentamicin. Data were
presented in the mean±standard error of the mean (SEM).
Discussion
In this present work, we found that the preconditioning by MIAE attenuated the
development of renal dysfunction in gentamicin-induced AKI, associated with
amelioration of tubular injury and proteinuria. This effect was correlated with a
reduction of renal ACE and B1R mRNA expression. These findings help
elucidate the possible beneficial interactions between systemic adaptations
following preconditioning by MIAE.
The exercise training protocol used in this study was effective. The improvement in
physical performance observed in trained animals confirms that the velocity reached
in the MPET is adequate as described by Priviero et al. [34]. CS enzyme is a marker of oxidative
adaptation of skeletal muscle to aerobic training due to its role as a regulator of
carbon input in the Krebs cycle [39].
Our data are in agreement with the study that demonstrates an improvement in aerobic
capacity [40]. Herein, no changes were
observed in arterial pressures. This observation corroborates the effect of aerobic
physical training on blood pressure in normotensive animals and humans, which seems
to be minimal or even non-existent [41].
On the other hand, we did observe a reduction in resting heart rate.
Training-induced bradycardia may indicate some adaptation of the autonomic nervous
system [42] or even intrinsic
electrophysiological changes in the sinus node [43]. The reduction of heart rate has been
considered a physiological marker for aerobic adaptation to training [44].
In our study, we employed the preconditioning by MIAE protocol to investigate the
potential protective effects of aerobic exercise on kidneys. Our choice was based
on
several reports indicating the protective effects of MIAE on different diseases,
including kidney disease [22]
[23]
[24]
[25]. Additionally, it was
observed that MIAE did not change renal O2 supply, but it did increase
kidney mitochondria density and function [45]. On the other hand, it has been shown that high-intensity aerobic
exercise transiently reduces renal blood flow [46]. This could be dangerous during the development of AKI, usually
associated with PTECs hypoxia, leading to a worsening outcome. Moreover, it has been
shown that high-intensity physical activity, such as ultramarathons or strenuous
physical exercise, induces muscle damage and AKI [47]
[48]
[49]
[50]
[51]
[52].
Hungaro et al. [53] demonstrated that
preconditioning by 4-week MIAE did not alter the glomerular dysfunction in
LPS-induced AKI. On the other hand, a similar training protocol attenuated
glomerular dysfunction observed in cisplatin- and I/R-induced AKI [27]
[28]. Herein, the preconditioning by MIAE ameliorated the development of
glomerular dysfunction observed in gentamicin-induced AKI. These apparent contrast
effects may be explained by the origin site of injuries.
GFR decline could involve a tubuloglomerular feedback (TGF) mechanism [54]. Herein, the preconditioning by MIAE
attenuated the impairment of electrolytes and water reabsorption in
gentamicin-induced AKI. In this way, the recovery of PTEC tubular reabsorption of
electrolytes reduces the electrolytes delivery into the distal nephron, inhibiting
the TGF mechanism, and consequently, increasing the CCr levels.
Accordingly, preconditioning by MIAE only recovers GFR levels in AKI animal models
where amelioration of tubules is observed [27]
[28]
[53]. Thus, it is plausible to imagine
that: 1) glomerular dysfunction observed in gentamicin-induced AKI is secondary to
tubular dysfunction; 2) this renoprotective effect occurs mainly due to improvement
in tubular function.
Tubular dysfunction correlates with tubular injury and proteinuria in AKI and CKD
[12]
[13]
[17]
[55]. Proteinuria is a
good marker of kidney disease and an active factor in disease progression [56]
[57]. Herein, our findings showed that preconditioning by MIAE attenuated
the development of ATN and proteinuria in gentamicin-induced AKI, indicating that
the protective effect on tubular injury occurs due to amelioration of proteinuria.
Accordingly, it has been shown: 1) an association between proteinuria and tubular
injury in subclinical AKI and established AKI [17]
[28]
[58]
[59]
[60]; 2) compounds with
anti-proteinuric effect ameliorate the tubular injuries [60]
[61].
What is the possible correlation between the development of proteinuria and ATN in
gentamicin-induced AKI? It is known that gentamicin accumulates in PTECs through its
internalization into lysosomes in a process dependent on megalin-mediated
endocytosis [62]
[63]. Balaha et al. [64], using a gentamicin-induced AKI in
rats, showed that gentamicin increased megalin expression at mRNA and protein levels
in renal tissue. This could per se form a dangerous loop in
gentamicin-induced nephrotoxicity. In addition, it is known that gentamicin binding
to megalin inhibits megalin-mediated albumin uptake in PTECs and placenta [65]
[66]. This inhibitory effect leads to the development of proteinuria
during AKI [64]. The importance of this
process appears when megalin blockade reduced the internalization of gentamicin as
well as its nephrotoxicity [67].
Interestingly, in different renal diseases where the cause is megalin deficiency,
the main phenotype is proteinuria as well as the development of tubule-interstitial
injury [68]
[69]. In this way, it is plausible to
imagine that proteinuria associated with impairment of renal protein transport could
trigger the development of tubule-interstitial injury in gentamicin-induced AKI. In
agreement with this hypothesis, we observed the development of proteinuria and ATN
in our experimental model.
RAS and KKS components are involved in the development and progression of different
forms of AKI [14]
[15]
[16]
[17]. Different works
showed that blockade of the classical arm of RAS [18]
[19]
[70]
[71] or activation of the alternative arm
of RAS [4]
[72] attenuates the gentamicin-induced
AKI. Regarding KKS, Bledsoe et al. [20]
showed the activation of B2R ameliorated tubular injury induced by
gentamicin. On the other hand, to our knowledge, no report has described the role
of
B1R on gentamicin-induced AKI yet. Moreover, higher levels of
angiotensin II and bradykinin impair the tubular protein uptake by PTECs [60]
[73], which contributes to proteinuria and tubular injury. So, all these
findings suggest that gentamicin enhances intrarenal RAS and KKS, and then promotes
the development of ATN and proteinuria. Interestingly, our work is the first to
describe the modulatory effect of MIAE on intrarenal RAS and KKS components. Our
results showed that preconditioning by MIAE attenuated the increase of ACE and B1R
mRNA expression triggered by gentamicin. In agreement with these findings, it has
been proposed that MIAE modulates systemic and local RAS components in skeletal
muscle, lungs, and liver [30]
[31]
[32]
[33]. Altogether, these
results allow us to suggest that the protective effects of MIAE-induced renal
adaptations avoid the upregulation of intrarenal RAS and KKS triggered by
gentamicin.
One question arises: what is the proposed mechanism for the protective direct effects
of preconditioning by MIAE on kidneys during gentamicin-induced AKI? It is known
that the common adaptations to MIAE lead to the maintenance of renal blood flow as
well as renal O2 supply [74].
In addition, other MIAE-induced renal adaptations are the increase of mitochondria
biogenesis and activity as well as an increased of NO in the renal cortex of humans
and rodents [45]. These effects are
correlated with an increased life span and the tightrope test. Herein, higher CS
activity was observed in skeletal muscle indicating the maintenance of MIAE-induced
systemic adaptation even after the exercise period. Since it has been reported that
gentamicin-induced PTEC injury involves mitochondrial dysfunction and PTEC death
[7]
[10]
[11] and AKI onset depends on the balance between the intensity of injury
and effective recovery phase [12]
[13], the MIAE-induced renal adaptations
may reduce the impact of gentamicin-induced mitochondrial dysfunction favoring the
acceleration of recovering phase during the AKI episode. Moreover, it is worth
mentioning that MIAE also stimulates cellular turnover as well as an
anti-inflammatory microenvironment in different systems [21].
Gentamicin-induced AKI is observed in non-critical children and neonates as well as
adults [3]. Zappitelli et al. [9], in a retrospective cohort study
comprising ~8-years old 557 children, showed that 20–30% presented AKI after
gentamicin treatment through the pediatric Risk, Injury, Failure, Loss, End Stage
Kidney Disease (pRIFLE) and Acute Kidney Injury Network (AKIN) definitions. Huang
et
al. [8], in another retrospective study
with 8,049 patients, showed that 6.14% presented AKI. The authors identified several
risk factors involved in the development of AKI where the precondition of patients
can determine AKI severity. Interestingly, Costanti-Nascimento et al. [75] proposed the prescription of moderate
and regular aerobic exercise for patients who are at risk of developing AKI and for
those diagnosed with AKI. In this present study, we observed that preconditioning
by
MIAE attenuated the development of gentamicin-induced AKI and reduced kidney injury
biomarkers. Altogether, these findings allow us to suggest that regular aerobic
exercise (moderated intensity) may contribute to reducing AKI severity in patients
under antibiotic therapies, and consequently, avoiding AKI progression to CKD.
Possible limitations of our study might lie in the fact that the lack of a complete
adaptation of rats to treadmill could reduce the final number of samples available
for further analysis. In addition, despite CS activity in the skeletal muscle being
a marker of systemic adaptation to MIAE, our data do not allow us to conclude that
the same extent of modifications occur in the renal parenchyma. Limitations such as
the assessment of renal functional parameters only on the 5th day after
the initial injection of gentamicin made it impossible to precisely determine what
phase of AKI the MIAE-induced systemic adaptation could be playing a role. After
all, the RAS and KKS components in this study were assessed only by qRT-PCR, which
makes it hard to state that the functionality of the components is directly
correlated to protein expression.
In conclusion, our results show that the preconditioning by MIAE led to renal
adaptations that reduce the susceptibility to the development of ATN and proteinuria
triggered by gentamicin. Our study is the first to show that this protective
MIAE-induced renal adaptation involves the modulation of intrarenal RAS and KKS by
inhibiting ACE and B1R expression. Our findings highlight the importance
of MIAE bringing the practice of physical exercise as a complementary strategy to
prevent the development of AKI in individuals under antibiotic therapy. By reducing
the number of AKI episodes in the population, the precondition by MIAE will
contribute to preventing AKI progression to CKD and the development of other chronic
degenerative diseases.
