Jacobs Journal of Nephrology and Urology

Impact of Renal Denervation and Vagal Nerve Stimulation in Ischemia-Reperfusion Injury in Rat Model

*Zeinab Mohammed Altaib
Department Of Medicine, Histology And Cell Biology, Helwan University, Egypt

*Corresponding Author:
Zeinab Mohammed Altaib
Department Of Medicine, Histology And Cell Biology, Helwan University, Egypt
Email:zaltaib@gmail.com

Published on: 2019-03-30

Abstract

Abstract Ischemia/reperfusion injury (IRI) is characterized by restriction of blood supply to an organ followed by restoration of blood flow and re-oxygenation. In the kidney, IRI contributes to acute kidney injury (AKI) with rapid kidney dysfunction and high mortality rates. A surgical or pharmacological blockade of renal sympathetic nerve prevents, partially, the progression of IR-induced AKI. Modulation of the cholinergic anti-inflammatory pathway (CAP) by vagus nerve stimulation (VNS) has also a delayed but effective role in renal IRI. Objectives This work aimed at investigating the combination effect of VNS and renal sympathetic denervation (RDN) in preventing deleterious effects of IRI in rats compared to the effects obtained by RDN alone and to elucidate the possible mechanisms. Methods: 32 adult male albino rats were equally allocated into 4 groups, sham group, IRI group, RDN group subjected to RDN before IRI and a group subjected to RDN and VNS before IRI. Results Compared to sham group, renal IRI led to elevation of BUN, serum creatinine and MDA levels, it also elevated TNFα and reduced GPX activity and nitrate levels in the renal tissue. In addition, IRI significantly increased BCL2 in immune-histochemical study and caused renal damage as observed by the histological light and electron microscopic examination. On the other hand, RDN demonstrated partial correction while, combination of RDN and VNS demonstrated nearly optimum recovery of renal functions, oxidant /antioxidant balance, inflammatory markers as well as marked amelioration of immunohistochemical , structural and ultra-structural studies. Conclusion VNS augmented and accelerated the reno-protective effects of RDN owing to its stimulating effect on CAP in addition to its antioxidant, anti-inflammatory as well as anti-apoptotic effects. Additionally, the delay of protective responses to VNS in the former literatures was abolished in our study when VNS was combined to RDN.

Keywords

Acute Kidney Injury; Ischemia Reperfusion Injury; Renal Denervation; Vagus Nerve Stimulation.

Introduction

Acute kidney injury (AKI) is associated with high mortality and morbidity. It has an expanding incidence, and is a strong risk factor for chronic kidney disease (CKD) and end stage renal disease (ESRD). One major cause of AKI is ischemia reperfusion injury due to decreased arterial and venous blood flow, (impaired tissue perfusion or ischemia) which results in cellular death. This is largely due to the depletion of energy stores and toxic metabolite accumulation. However, restoration of blood flow to the ischemic tissue may paradoxically exacerbate the injury.

Although there are many mechanisms underlying ischemic–reperfusion injury, the primary mechanism is the reintroduction of molecular oxygen to a previously hypoxic tissue, resulting in quickly formed reactive oxygen species [6]. As a result, multiple enzyme systems including proteases, nitric oxide synthases, phospholipases and endonuclease are activated causing cytoskeleton disruption, membrane damage, DNA degradation, and eventually cell death [7].

AKI is a clinical syndrome characterized by abrupt and sustained decline in glomerular filtration rate resulting in the accumulation of urea and other chemicals in the blood.The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation [8]. The inflammatory reflex referred to as the cholinergic anti-inflammatory pathway (CAP) regulates innate and adaptive immunity mainly with the spleen, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models. Therefore, vagus nerve– derived cholinergic signals provide tonic or continuous neurological modulation of cytokine synthesis, which limits the magnitude of the inflammatory immune response and mediates protective effect against AKI induced by IRI [9].

Nevertheless, protection against renal IRI by VNS was found to be time limited and at least 24 hours prior to IRI was needed for VNS to elucidate its protective effect (8). The limited and/or delayed response of VNS might be due to stimulation of renal sympathetic innervations [10] through a vagosympathetic reflex. Renal sympathetic hyperactivity is common in kidney injury and has been shown to contribute to glomerulonephritis and induce proteinuria both through and beyond its effect on blood pressure [11].

Renal denervation (RDN) at the time of injury improves histology and decreases proinflammatory, profibrotic, and apoptotic changes in the kidney (12). Therefore, we suppose that RDN may confer added and fast protective effect to VNS against renal IRI. Thereby, in this study, we hypothesize that RDN would protect the kidney and might potentiate or accelerate the Reno protective effects of VNS in a rodent model of AKI induced by IRI in a trial to elucidate such protective impacts and to investigate their possible mechanisms.

Material and Methods

This work was performed on adult male Wistar rats, initially weighting 150-220 g, Rats were obtained from the laboratory animal colony, Ministry of Health and Population, Helwan, Cairo, Egypt and maintained in the animal house of the Research Unit, Faculty of Medicine, Ain Shams university under standard conditions of boarding. Rats were kept at room temperature (25° C ± 5° C) under a 12 hour light/dark cycle. The rats were provided with regular Citation :ElSayed MH, Eltayeb Z, Abdellah AM, et al. Impact of Renal Denervation and Vagal Nerve Stimulation in Ischemia-Reperfusion Injury in Rat Model. JJ Nepho Urol 2019; 6 (2): 044. 3 diet consisting of bread, vegetables and milk. Tap water was provided ad libitum

 

Experimental Design

  1. After acclimatizing for 7 days, the animals were divided into 4 groups (with 8 rats each)
  2.  Group I, Sham Control Group: Underwent the same procedure of other groups without clamping of the arteries, renal denervation or vagal nerve stimulation
  3.  Group II, Ischemia Reperfusion (IR) Group: Subjected to bilateral clamping of renal pedicles for 40 min followed by reperfusion for24 hours.
  4.  Group III, Renal Denervation (RDN) Group: Subjected to bilateral renal denervation prior to bilateral renal IR by the same protocol of IR Group.
  5.  Group IV, Vagal Nerve Stimulation (RDN-VNS) Group: Underwent RDN followed by VNS 10 min before bilateral renal IR as in group II.

Experimental Procedure

Renal Denervation (RDN)

Rats of RDN group and RDN-VNS groups were anesthetized with an (i.p.) injection of ketamine (120 mg/ kg) and xylazine (12 mg/kg). An abdominal approach to the kidneys was used, with a midline incision then displacing abdominal content to gain access to each kidney one by time. Both kidneys underwent removal of the covering capsule to grant the removal of suprarenal glands and better denervation. Then nerves running along with the renal artery through renal pedicle were surgically removed [12]. 10 min later, bilateral renal artery clamping was performed for 40 min and ischemia was ensured visually by pale colored kidneys as well as by loss of arterial pulsation.

Vagal nerve stimulation (VNS)

Rats of RDN-VNS group were subjected to RDN followed by VNS by the following technique: The left cervical vagus nerve was isolated via a midline cervical incision and placed on a bipolar silver wire electrode for stimulation. Electrical stimulation (50 μA intensity; frequency, 5 Hz; duration, 1 ms) was applied for 1 minute using an isolated square wave stimulator [8,13]. We stimulated the left vagus nerve because this nerve is usually selected for stimulation in animal and human experiments [14-16]. 10 min later, bilateral renal artery clamping was performed for 40 min and ischemia was ensured visually by pale colored kidneys as well as by loss of arterial pulsation. In sham-operated animals, the vagus nerve was exposed but not stimulated and the kidneys were exposed also but not underwent IR technique.

Renal Ischemia/reperfusion injury

The skin of the anterior abdominal wall was sterilized by tincture iodine, a midline incision was performed, and the abdominal wall was retracted. Both renal pedicles were clamped off by atraumatic clamps for 45 min with a continuous intra-peritoneal instillation of saline to prevent dryness of the kidneys. The kidneys were inspected for immediate color change, indicating successful clamping, followed by 24 h of reperfusion [17]. After clamp removal, the kidneys were checked for a change in color within 3min to ensure reperfusion. The abdominal wall was closed with 3-0 silk after homeostasis and local application of broad spectrum antibiotic.

 

Anesthesia

After 24 hours of reperfusion, overnight fasted rats were weighed and injected intraperitoneally with 5000 IU/ Kg B.W heparin sodium (Nile Company, Egypt). Fifteen minutes later, the rats were anaesthetized with I.P. injection of thiopental sodium (Sandoz, Austria), in a dose of 40 mg/kg B.W.

Blood and Tissue Sampling

Blood samples were collected from retro-orbital plexus with the help of capillary tube into serum gel tubes which were centrifuged (6000 rpm for 15 min) to separate serum. The serum was then pipetted into clean storage tubes and stored at -20? C for later determination of serum urea, creatinine and MDA. Whereas, left kidneys were stored at -80? C for later determination of renal tissue GPX and renal tissue nitrate and renal tissue TNFα as well. On Citation :ElSayed MH, Eltayeb Z, Abdellah AM, et al. Impact of Renal Denervation and Vagal Nerve Stimulation in Ischemia-Reperfusion Injury in Rat Model. JJ Nepho Urol 2019; 6 (2): 044. 4 the other hand, right kidneys were divided into 2 halves, one of them was stored in 10% formalin for later light microscopic examination while, the other half was stored in gluteraldehyde for E/M examination.

 

Biochemical Analysis

Serum urea, creatinine and malondialdehyde (MDA) were estimated according to the methods described by Patton and Crouch (1977) [18] Bartels et al., (1972) [19 ] and Draper and Hadley (1991) [20] respectively, using kits supplied by Biodiagnostic, Egypt. The BUN is calculated as 28/60 or 0.446 of the blood urea

Biochemical Analysis in Renal Tissue

Left kidney homogenate (10%, w/v) was prepared with 0.1 M PBS and centrifuged at 12,000 g for 10 min. The supernatant was used to determine GPX levels by colorimetric method according to Paglia and Valentine [1967] [21], using kits supplied by Bio-diagnostic, Egypt. Tissue tumour necrosis factor-α (TNFα) was performed with ‘sandwich’ enzyme linked immunoadsorbant assay (ELISA) using a commercially available Mouse TNF-α DuoSet kit (Genezyme Diagnostics, Cambridge, MA) [22] while nitrate, the metabolic end product of nitric oxide (NO) was assayed according to Bories and Bories (1995) [23].

Histological and Immunohistochemical Studies

One half from right kidney was processed for histological examination by light microscope, the kidney specimens were fixed in 10% neutral buffered formalin and paraffin blocks were prepared. Serial 4-6 μm-thick sections were cut and stained by Heamatoxylin and Eosin (H&E), Masson trichrome (MT) stain and Bcl2 immune reaction. Another half from right kidney was divided into 2 parts, one (1/4 kidney) processed for transmission electron microscopic examination, small kidney specimens (1 mm3) were fixed in a 4% glutaraldehyde solution. Ultrathin sections were cut and examined using a 1200 EX Jeol, Japan, transmission electron microscope at the Electron Microscopic Unit – Faculty of Science, Ain Shams University. The second part (1/4 kidney) was placed on the ice quickly and homogenized with lysis buffer. Aliquots were stored in a -70° C refrigerator with sodium orthovanadate (2 mM), phenylmethylsulfonyl fluoride (0.2 mM), leupeptin (2 μg/mL), and aprotinin (2 μg/mL) on ice for 30 min, then were centrifuged at 13,000 g for 15 min at 4° C. Proteins from homogenization (50 μg protein) were electrophoretically separated by 8% or 12% SDS-PAGE and then transferred onto nitrocellulose membrane. After blockade of non-specific sites with 5% none fat milk for 1 h at room temperature, membranes were incubated with a rabbit polyclonal anti-CTGF, AKT/PKB.

Immunohistochemical BCL2 Study

BCl2 imunohistochemical (anti-apoptotic protein) evaluation was carried out as previously described by Bancroft et al. (2013) [24]. Five mm tissue sections were deparaffinized and rehydrated in gradient alcohols and processed using the streptavidin immunoperoxidase method. In brief, sections were submitted to antigen retrieval by microwave oven treatment for 10 min in 0.01mol/L citrate buffer (pH 6.0). Slides were then incubated in10% normal serum for 30 min, followed by an overnight incubation at 4? with the appropriately diluted primary Bcl2 antibody (Beyo time Inc., China) were used at 1:100 dilution. Next, samples were incubated with biotinylated anti-rabbit immunoglobulins for 15 min at 37?. Then, they were incubated with streptavidin peroxidase complexes for 15 min at 37?. After rinsing in PBS, the reaction products were visualized by immersing the section into the chromogen diaminobenzidine. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted. The negative control was processed in the same way, but omitting the step of 1ry Ab. The positive cells showed brown nuclei reaction.

Image Analysis

The percentage of BCL2 immunoreactivity was quantified in five images from five non-overlapping fields of each slide using Image-Pro Plus program version 6.0 (Media Cybernetics Inc., Bethesda, Maryland, USA).

Statistical Analysis

The data collected, tabulated and statistically analyzed, using SPSS 22.0 for windows (SPSS Inc., Chicago, Citation :ElSayed MH, Eltayeb Z, Abdellah AM, et al. Impact of Renal Denervation and Vagal Nerve Stimulation in Ischemia-Reperfusion Injury in Rat Model. JJ Nepho Urol 2019; 6 (2): 044. 5 IL, USA). All tests were two sided. A p-value<0.05was considered significant. Continuous variables were expressed as mean, standard deviation (SD) and median, checked for normality by using Shapiro-Wilk test. One way (ANOVA) F test used to compare between more than two groups, with Holm Sidiac methods for multiple comparisons, Kraskall Wallis test was used to compare between more than two groups of non-normally distributed data, with Turkey methods for multiple comparisons. Pearson’s correlation was calculated to assess the correlations between various study parameters. (+) sign indicate positive correlation and (-) sign indicate negative correlation

Results

In our study, there was a significant elevation in the renal function tests of the IR group compared to the sham control group (BUN p<0.001, serum creatinine p<0.05). A significant decrease of BUN level was observed in RDN group compared to IR group (p<0.001) while non-significant difference was found between both groups regarding serum creatinine level (p=0.09). Though, the BUN levels remained significantly higher in RDN group compared to sham controls (p<0.001) but serum creatinine was nearly normalized and showed no significant difference between both groups.

A significant reduction was found concerning the renal function tests of the RDN-VNS group compared to that of the IR group (BUN p<0.001, serum creatinine p<0.05) becoming comparable to the sham control group (BUN p=0.683, serum creatinine p<0.3).

Moreover, a significant reduction of renal tissue nitrate levels and significant incline of renal tissue TNFα were noticed in IR group as compared to the sham control group (p

Regarding oxidative/antioxidant biomarkers, a significant rise of serum MDA levels was found in the comparison of the IR group to the sham control group (p<0.001), while a significant decline of GPx enzyme activity was demonstrated in the renal tissue GPx levels of IR group compared to sham group (p<0.001). Serum MDA levels in the RDN group were significantly lower than that of the IR group (p=0.005) but remained significantly elevated compared to the normal MDA levels demonstrated by the sham control group (p<0.001). Moreover, renal tissue GPx levels of the RDN group exhibited no significance in comparison to the IR group (p=0.015) and became normalized when put into comparison with the sham control group (p=0.026).

A significant decrease of serum MDA levels of the RDN-VNS group compared to that of the IR group (p<0.001) and became comparable to the sham control level (p=0.402). Likewise, a significant increase was found regarding renal tissue GPx levels of the RDN-VNS group compared to that of the IR group (p<0.001) but none significant compared to the sham control group (p=0.264).

Table 1: Mean ± Standard deviation (SD) of BUN, Serum creatinine, Renal tissue nitrate, Renal tissue TNFα , BCL2 immune expression Serum MDA, and Renal tissue GPx in the different studied groups.

Figure 1: Correlation Study between Serum MDA and BUN, Serum Creatinine and Renal Tissue Nitrate.

Figure 2: Correlation Study between Renal Tissue GPx and BUN Serum Creatinine and Renal Tissue Nitrate.

Figure 3: Correlation Study between Serum TNFa and BUN, Serum Creatinine and Renal Tissue Nitrate.

Histological
results H&E-Stain 
 Control (sham) group: (Fig. 4-A, B, C, and D) 

Examination of H&E-stained sections obtained from the kidney of the control (sham) rats revealed average cortical thickness, average medullary thickness and intact renal capsule (Fig.4A). A normal histological structure of the renal cortex showed average Malpighian renal corpuscles which were composed of the glomerulus (a tuft of blood capillaries) surrounded by Bowman’s capsule with capsular space in between. The parietal layer of the capsule had a single layer of simple squamous cells, whereas the visceral layer was lined by podocytes with oval nuclei, surrounded by average proximal tubule were lined by three to five pyramidal cells characterized by acidophilic cytoplasm, apical brush border of microvilli, and rounded, vesicular, basally located, basophilic nuclei with free lumens and distal tubules were lined by six to eight cubical epithelial cells with a wider lumen. The cells of the DCT have pale acidophilic cytoplasm and rounded, centrally situated, vesicular, basophilic nuclei with free lumens and collecting tubule and interstitium contain average blood vessels and intact renal capsule. (Fig.4B, C). Also, average renal medulla showing average collecting tubules, lined by cuboidal cells had very pale acidophilic cytoplasm with free lumens, and average intervening stroma containing normal blood vessels were observed (Fig. 4D).

Figure 4: Photomicrograph of a section in the rat Kidney of control (sham) group Stained with H&E showing.

A.Renal tissue with average thickness of the cortex and medulla with intact renal capsule (blue arrow) (x40).

B.Renal cortex containing Malpighian renal corpuscles with average glomeruli (green arrow), average Bowman,s space (yellow arrow) and intact Bowman,s capsule (blue arrow). Also, proximal tubules (P), distal tubules (D), collecting tubules (CL) are average and interstitium contains blood vessels (BV) in between them. Part of intact renal capsule could be detected (blue arrow) (X 200).

C.Renal cortex with normal Malpighian renal corpuscle containing average of the glomerular capillary tufts (G), with average cellularity surrounded by Bowman’s space (BS) , and patent Bowman,s capsule with flat epithelium Lining (black arrow) surrounded by normal shaped proximal tubule (P) lined by pyramidal cells having apical brush border , rounded basal vesicular nuclei and free lumens and distal tubules (D) are lined by cubical cells having rounded central vesicular nuclei with free wide lumens and collecting tubule (CL). Part of patent renal capsule (blue arrow) can be observed. Note acidophilia of proximal and distal convoluted tubular cells. ( × 400).

D.Normal renal medulla showing average collecting tubules (CT) with free lumens, and average intervening stroma containing normal blood vessels (blue arrows) (X 400).

Renal Ischemia-Reperfusion (IR) group: (Fig. 5-A, B, C, D)

The H&E stained sections of the rat kidney in this group revealed that, thin cortical thickness in relation to medulla. Medullary rarefaction at focal areas and partially detached renal capsule (Fig. 5A). Cortical area by high power of magnification showed renal corpuscle with shrunken, markedly distorted and congested small glomeruli have a widened bowman’s space and the abnormal configuration of renal tubules were observed. In some proximal and distal tubules showed the cytoplasm of the some lining cells rarefied exhibiting cytoplasmic vacuoles, epithelial exfoliation at focal areas of some tubules.…Flattening of the epithelial lining of some tubules with apparent tubular dilatationand few were destructed and degenerated but few of them were average. Also, haemogenous materials, Cellular debris is dislodged within their lumina and congested in more than 60%. Dilated congested blood vessels were also observed (Fig. 5B).

In all stained sections showed necrotic changes in most of the cortical tubular epithelial cells in the form of nuclear changes (pyknotic and karyolitic nuclei), in addition to vacuolation. Interstitial Inflammatory mononuclear cellular infiltrations were noticed.(Fig. 5C)

Also, medullary area showed mononuclear cellular infiltration between disorganized degenerated tubules in addition to peritubular congestion (Fig. 5D)

Figure 5: Photomicrograph of a section in the rat Kidney of renal ischemia-reperfusion (IR) group stained with H&E showing.

A.Thin cortical thickness in relation to medulla. Focal areas of medullary rarefaction and partially detached renal capsule (blue arrow) and dilated congested blood vessels (BV) can be observed (x 40).

B.Cortical area showing two Malpighian corpuscles with shrunken markedly distorted congested glomeruli (G) having a widened bowman’s space (BS) and abnormal configuration of renal tubules. In some proximal (P) and distal (D) dilated tubules the cytoplasm of some lining cells was rarefied exhibiting vacuoles (yellow arrows) and pyknotic nuclei. Also, Cellular debris (green arrow) is dislodged in the tubular lumina and haemogenous materials (black arrow). Interstitial mononuclear infiltration (gray arrow) and red blood corpuscles (RBCs) are noticed. (x400).

C.Cortical area showing two Malpighian renal corpuscles with marked distorted congested glomeruli (G) and widened Bowman’s space (BS), congested dilated interstitial blood vessels (BV), exfoliation of some lining epithelial cells of some tubules (blue arrow), flattening of the epithelial lining of some tubules (red arrow). Some tubular cells showed vacuolated cells (black arrow) with pyknotic nuclei (yellow arrows). Inflammatory cells infiltration in interstitium (green arrow) are noticed and peritubular congestion could be seen (red arrows) (× 400).

D.Medullary area showing mononuclear cellular infiltrations (green arrow) and peritubular congestion could be seen (red arrows) (× 400)

RDN Group: (Fig. 6-A, B)

 The H&E stained renal sections showed renal cortex with some of the renal corpuscles, glomeruli and Bowman’s spaces looked average in size and shape but others showed slightly congested glomeruli and little widening of Bowman’s space. Also, some tubules seemed free from exfoliation in their luminae but others contained exfoliation in their luminae and intracellular cytoplasmic vacuoles. In addition, some of the blood vessels were dilated and moderately congested and few of the mononuclear cells infiltrated in the interstitium (Fig. 6A).

Medullary area showed mononuclear cellular infiltrations in interstitium of averege tubules but some of them showed exfoliated cell debris and haemogenous materials in their lumina and some pyknotic nuclei in their lining epithelium. Slightly dilated congested blood vessel was also observed. (Fig. 6B).

RDN-VNSGroup:(Fig.7-A,B,C,D) 

B,C,D) SGroup:(Fig.7-A,B,C,D)The H&E stained renal sections showed average diameter of cortex and medulla with average glomeruli and tubules covered by intact capsule (Fig. 7A). Higher magnification showed that most of the renal corpuscles were intact as the glomeruli were normal and Bowman’s capsules were patent. Proximal, distal and collecting tubules were also normal. The rest showed slightly wide Bowman’s space and some of the tubules contained few pyknotic nuclei and cytoplasmic vacuoles. Few mononuclear cellular infiltrations in the interstitium were also observed (Fig. 7B)

Renal medulla showed that most of the tubules were normal but some of them were dilated, with avA.Renal cortex with average Malpighian renal corpuscle having average glomeruli (G) Bowman's space(BS), proximal(P) and distal(D) tubules, some of them containing exfoliated materials in their luminae (blue arrow) in addition to the presence of few infiltrating inflammatory cells (black arrow) in interstitium could be seen. B.Renal medulla showed focal areas of mononuclear cellular infiltrations in interstitium (green arrow), average tubules but some of them show exfoliated cell debris and homogenous materials in their luminae (yellow arrow) and some pyknotic nuclei in their lining epithelium (red arrow). Slightly dilated congested blood vessel can be also observed.erage epithelial lining. Most of the nuclei were rounded and vesicular, but few of them were flat and pyknotic. Very little number of mononuclear cellular infiltrations in the interstitium was noticed (Fig. 4C).Renal medulla was with average tubules. Some of the dilated collecting tubules showed average epithelial lining. Most of their nuclei were rounded vesicular, but few of them were flat and pyknotic. A scanty mononuclear cellular infiltration in the interstitium could be observed. (Fig. 7D).

Figure 7: Photomicrograph of a section in the rat kidney in vagal nerve stimulation (RDN-VNS group) stained with H&E. capillary loops of glomeruli, mononuclear cellular infiltrations and the tubules (Fig. 8B, C)

A.Average diameter of cortex and medulla with average glomeruli (yellow arrows), and tubules (green arrow) covered with intact capsule (blue arrow) (x100). B.Renal cortex with renal corpuscle containing average glomeruli (G) slightly widened patent Bowman’s space (BS), average proximal (P), distal (D) and collecting tubules(CL). Some of them contain few pyknotic nuclei (green arrow) and few of cytoplasmic vacuoles (red arrows) in their lining cells. Few of the mononuclear cellular infiltration in the interstitium (black arrow) can be observed (x 400). C.Renal medulla with average tubules. Some of the dilated collecting tubules (Cl) showing average epithelial lining, most of the nuclei are rounded vesicular (green arrows), but few of them are flat and pyknotic (blue arrow). There were few mononuclear cellular infiltration in the interstitium could be observed. (x 400).

II-Masson’s Trichrome Stain

Sham Group: The renal cortex showed very thin blue collagen around Bowman’s capsules and renal tubules (Fig. 8A)

IR Group: The renal cortex showed marked increase of theRenal medulla showed marked increase of the collagen fibers around mononuclear cellular infiltrations, tubules, septa between tubules (Fig. 8D).

RDN Group:The renal cortex showed few collagen fibers around the renal capsules, the glomeruli and the tubules. Also, septa between tubules were intact in comparison to I/R group (Fig. 5E).

RDN-VNS Group: The renal cortex and medulla showed minimal collagen fibers around the Bowman’s capsules and the tubules. (Fig. 8E&F).

Figure 8: Photomicrograph of a section in the rat renal tissue from all groups stained by Masson’s Trichrome (× 400) showing.

A.Sham group: thin blue collagen around renal tubules (black arrow), Bowman’s capsules (green arrow).

B.In I/R group: The renal cortex with marked increase of the collagen fibers around mononuclear cellular infiltrations (black arrows), convoluted tubules (red arrow), Bowman’s capsules (green arrow) and capillary loops of glomerulus (G).

C.IR group: renal medulla with marked increase of the collagen fibers around mononuclear cellular infiltrations (green arrow), tubules (blue arrow) and septa between tubules (red arrow). D.RDN group showed renal cortex with few collagen fibers around glomeruli (G) and convoluted tubules (red arrow). It showed also interstitium (black arrow) and part of renal capsule (yellow arrow).

E.RDN-VNS group showed minimal collagen fibers around Bowman’s capsules (green arrow) and convoluted tubules (blue arrow) in comparison to IR group. F: RDN-VNS group showed minimal collagen fibers around collecting tubules (yellow arrows) in renal medulla.

BCL2:

Sham-Operated Group:

The renal cortex showed positive immunoreactions of the nuclei in renal corpuscle andnuclei among the tubular lining cells (Fig. 9A).

IR Group: The renal cortex showed weak immunoreactions of the nuclei in glomeruli, Bowman’s capsule and among the tubular lining cells (Fig. 9B).

RDN Group: The renal cortex showed moderately positive immunoreactions of the nuclei in glomeruli, Bowman’s membrane and among the lining tubular epithelial cells compared to I/R group (Fig. 9C).

RDN-VNS Group: The renal cortex showed markedly positive immunoreactions of the nuclei in glomeruli, Bowman’s membrane and among the lining tubular epithelial cells compared to IR group. (Fig. 9D)

Immunohistochemical Study of BCL2 Reaction

Figure 9: Photomicrograph of a section in the renal cortex of rats of all groups for immunohistochemical reaction of BCL2 (x400) showing.

A.Sham group, positive immunoreactive nuclei (red arrows) among the tubular lining cells,

B.IR group, a weak immunoreactive nuclei in glomeruli (red arrows), Bowman,s capsule (green arrow) and among the tubular lining cells (blue arrows).

C.RDN group, moderately positive immunoreactive nuclei in glomeruli, Bowman’s membrane (green arrow) and among the lining tubular epithelial cells (blue arrows) compared to IR group

D.RDN-VNS group, markedly positive immunoreactive nuclei in glomeruli (white arrow), Bowman,s membrane (red arrow) and among the lining tubular epithelial cells (blue arrows).

EM

Sham Group: (Fig. 10-A, B, C)

Examination of ultra-thin sections revealed podocytes with primary long processes which branched to give rise to secondary processes. The latter produced feet processes or pedicles that were separated by narrow slits bridged by a slit membrane. The podocytes and glomerular capillaries shared uniform thickness of a basement membrane which exhibited a trilaminae composed of a central electron dense layer (lamina densa) and two electron-lucent layers (lamina rarae) on either side. The podocytes contained large irregular euchromatic indented nucleus with peripheral dense chromatin. Fenestrated endothelial lining of the glomerular blood capillaries was noticed. (Fig. 10A).

The cells of the proximal tubules showed high cubical cells exhibited apical closely packed microvilli and basal numerous enfolding enclosing elongated mitochondria with normal cristae. The cytoplasm contained some small pinocytic vesicles and few lysosomes. The large euchromatic nuclei with peripheral chromatin condensations were noticed (Fig. 10B)

The distal convoluted tubules which were lined with cubical cells appeared smaller than those of the proximal ones, with few short microvilli and large rounded euchromatic nuclei near to their luminal surface with peripheral chromatin condensations. The basement membrane was separating the cells of the tubule from the delicate capillary. The cytoplasm contained a few and small pinocytic vesicles and few lysosomes. The large numbers of elongated mitochondria with normal cristae were situated in the basal parts of the cells between basal enfolding (Fig. 10C).

Electron Microscopic Examination:

Figure 10: An electron micrograph of a section of the renal cortex of a rat in sham group showing.

A.Podocytes with large irregular euchromatic indented nucleus (N) with peripheral dense chromatin. The podocyte appears with primary process (P) and secondary interdigitating feet processes or pedicles (red arrow). The space between the feet or pedicles called filtration slits. Fenestrated endothelial lining of blood capillary (Yellow arrow) could be also noticed. Uniform thickness of glomerular basement membrane (GBM) is observed (blue arrows) and a central electron dense lamina densa and lamina rarae on either side are also detected. Mesangial cells (green arrows) are also noticed. (x 12000)

B.A cell in a proximal convoluted tubule with elongated mitochondria (M) having normal crestae. The brush border of the cells has normal microvilli (MV) and contains some pinocytic vesicles (green arrow) and lysosomes (L). Note the large nucleus (N) with peripheral chromatin condensations (red arrows) (x 12000).

C: A part of a distal convoluted tubule which is lined by cubical cells with short few micro¬villi (MV) and large rounded nucleus (N) near to their luminal surface(LU) with peripheral chromatin condensations ( red arrows). The basement membrane is separating the cells of the tubule from the delicate capillary (C). The cytoplasm contains some pinocytic vesicles (yellow arrow) and few lysosomes (L). Note the large number of elongated mitochondria (M) with normal crestae situ¬ated in the basal parts of the cells between basal enfolding (green arrows) (x 10000).

IR Group: (Fig. 11-A, B, C&D)

The podocytes showed 1ry process with elongated irregular nuclei. The other cells had shrunken, heterochromatic and pyknotic nuclei. Also, they showed disorganization, distortion and fusion in the feet processes. Irregular thickening of glomerular basement membranes was clearly seen. Dilated glomerular capillaries with lack of fenestrations and mesangial cells and their surrounding thick amorphous mesangial matrix were observed. Marked irregular thickening of Bowman’s membrane surrounded by thick layer of collagen fibers was also an observation (Fig. 11A)

The cells of the proximal tubules showed variable ultra-structural changes. Some cells had a disoriented basal mitochondrial, with few and dilated basal enfolding; in addition to the presence of multiple vacuoles. Nuclei of some cells showed a dilated perinuclear space. Multiple secondary lysosomes, abnormal-shaped mitochondria, and electron-dense bodies could be detected in the cytoplasm of other cells (Fig.11B). The cells of the proximal tubules contained areas of rarified cytoplasm, swollen mitochondria with ill-defined cristae. Many dense irregular bodies mostly lysosomes were found. Heterogenous, Pleomorphic, autophagic vacuoles containing whorls of membranous material were also observed. There were shrunken, condensed pyknotic nuclei, while the microvilli were mostly dilated and irregular together with irregular thickened basal laminae were detected (Fig. 11C).

Figure 11: An electron micrograph of a section of the renal cortex of a ratof IR group showing.

A.Podocytes with 1ry process (P) are shown with an elongated irregular nucleus (yellow N). The part of other cell has shrunken, heterochromatic and pyknotic nucleus (blue N). Also they show disorganization, distortion and fusion of some of the feet processes (green arrow). Irregular thickening of glomerular basement membranes (yellow arrow) is clearly seen. Dilated glomerular capillary (C) with lack of fenestrations (blue arrow) is also found.Mesangial cell (MC) and its surrounding amorphous mesangial matrix (MM) and Marked thickening of Bowman’s membrane surrounded by thick layer of collagen fibers (CL) are observed (x 8000).

B.A proximal tubular cell contains endoplasmic reticulum, heterogenous pleomorphic autophagic vacuoles containing whorls of membranous material (blue arrows), many lysosomes (L) and shrunken apoptotic neuclus (N). Thickening in basal lamina (green arrow) , many of cytoplasmic vacuoles (red arrow) in addition to an area of partial loss of the apical microvilli (MV) and swollen mitochondria (M) with variable sizes were apparent (x 10000).

C.The epithelial lining cells of the proximal convoluted tubule show areas of rarified cytoplasm (yellow arrow), swollen mitochondria (M ) with ill-defined crestae. Numerous dense irregular bodies, mostly lysosomes (green arrow) are observed. Pleomorphic, autophagic vacuoles (V) containing whorls of membranous material and the shrunken condensed pyknotic nucleus (N) were found, while the microvilli (MV) were mostly dilated and irregular could be observed. (x 8000).

D.Some of the lining cells of the distal convoluted tubule have small pyknotic nucleus (N), but others have atrophied nucleus, disorganized basal enfolding with the appearance of swollen mitochondria (red arrows) and obliteration of tubular lumen (LU). Other cell lost most of their organelles and appeared to contain variable-sized large vacuoles (blue arrows). Large areas of rarified cytoplasm (green arrows), thick irregular basement membrane (BM) surrounded by capillary (C) and hemorrhagic area (H) were noticed. Apical microvilli could not be detected. (x 4000).

The cells of the distal convoluted tubule demonstrated that the majority of the cells with few basal enfolding and a palisade form of the mitochondria but their cytoplasm showed some rarefaction. On the other hand, some atrophied tubules with irregular thickening of the basement membranes surrounded by hemorrhagic areas and thick collagen fibers in between the tubules. The cells revealed shrunken condensed and/or pyknotic nuclei, but others had completely atrophied nuclei. Large areas of rarified cytoplasm with disorganized and dilated basal enfolding were shown. The mitochondria migrate towards the lumen with loss of their parallel arrangement, swollen with indistinct cristae and obliteration of tubular lumen (Fig.11D).

RDN Group: (Fig. 12-A, B, C)

The podocytes showed disorganized pedicles and electron-dense substances in the glomerular capillaries. The mesangial cells were surrounded by mesangial matrix. Thick regular Bowman’s capsule with thin layer of collagen was visible on the outer aspect of the basal lamina. The blood capillaries and red blood corpuscles were noticed (Fig. 12A).

Lumens of the proximal tubules were filled with secretions. The lining cells of the tubules showed microvilli, small cytoplasmic vacuoles and small dense lysosomes but some of their mitochondria were swollen (Fig. 12B).

The cells of the distal tubules revealed small lysosomes and some elongated mitochondria with mostly intact cristae. Areas of cytoplasmic rarefaction could be observed. Some nuclei were irregular in shape and shrunken, but others were nearly average in appearance. The brush borders couldn’t be detected but basal enfolding and thickening could be observed at some of the basal laminae (Fig. 12C).

A.A renal corpuscle with nuclei of podocytes (N), disorganized pedicles (green arrow). Note electron-dense substances (E) in the glomerular capillaries (GC). The mesangial cells (MC) surrounded by mesangial matrix (MM), thick regular Bowman,s capsule (yellow arrow), thin layer of collagen (red arrows) are visible on the outer aspect of the basal lamina of the Bowman capsule. Clood capillary(C) and red blood corpuscles (RBCs) are also noticed. (x 8000).

B.A part of proximal tubule and its lumen (LU) filled with secretion, microvilli (MV), small cytoplasmic vacuoles (red arrows), small dense lysosomes (L) averge sized mitochondrea (M) but some of them are swollen (black arrow). ( x 8000 ).

C.An epithelial cell of distal tubule revealed small lysosomes (yellow arrow) and some elongated mitochondria (M) with mostly intact crestae. Areas of cytoplasmic rarefaction (black arrow) could be observed.

Figure 12: An electron micrograph of a renal cortex of a rat in RDN group showing

Nuclei (N) are irregular in shape and shrunken, but others (red arrow) are nearly normal appearance. The brush borders couldn’t be detected and basal enfolding (green arrow) could be observed at some areas. Thickening of basal lamina (white arrow) is also found. ( x 4000)

RDN- VNS Group: (Fig. 13-A, B, C

The podocytes showed euchromatic indented nuclei and mitochondria and few small vacuoles were seen in their cytoplasm. They appeared with normal arranged secondary feet processes. The space between the pedicles (filtration slits) could be seen and laid on a regular glomerular basement membrane. Glomerular capillaries lacking fenestrae were seen. Though, some of the podocytes showed distorted and fused feet processes with the appearance of few mesangial matrixes around mesangial cells (Fig. 13A).

The cells of the proximal tubules revealed long, dense apical microvilli and basal enfolding in addition to presence of small pinocytic vesicles near to the lysosomes. Many elongated mitochondria with distinct cristae and large nuclei with peripheral condensed chromatin could be observed (Fig. 13B).

The cells of the distal tubules revealed elongated mitochondria with mostly intact cristae. Small areas of cytoplasmic rarefaction could be observed. Most of the nuclei were normal in appearance, but scanty of them revealed pyknotic. The brush borders couldn’t be detected but basal enfolding could be observed at some areas. Little thickening of the basement membrane and very small vacuoles were noticed (Fig. 13c)

Figure 13: An electron micrograph of a renal cortex of albino rat from RDN-VNS group showing .

A.Podocyte (P) with euchromatic indented nuclei (N) mitochondria (m) and few small vacuoles are seen in the podocyte cytoplasm. The podocyte appears with primary and normally arranged secondary feet processes (green arrow). The space between the pedicles (filtration slits) is clearly seen and laid on a regular glomerular basement membrane (yellow arrows). Glomerular capillaries (c) with lack of fenestrations (yellow arrow) are seen. A podocyte (P1) is shown with distorted, fused feet processes. Mesangial matrix (MM) around mesangial cells (MC) is also present (x 12000 ).

B.Cells in the proximal tubule reveal long dense apical microvilli (MV), and basal enfolding (red arrow) as well as small pinocytotic vesicles (blue arrow) near to the lysosomes (L). Many elongated mitochondria (M) with distinct creastae are situated. Note the large nucleus (N) with peripheral chromatin condensations. (x 10000).

C.Epithelial cells of the distal tubule revealed elongated mitochondria (M) with mostly intact crestae. Small areas of cytoplasmic rarefaction (green arrow) could be observed. Some nuclei (red N) are nearly normal in appearance, but few of them (blue N) appear pyknotic. The brush borders couldn’t be detected and basal enfolding (blue arrow) could be observed at some areas. Some thickening of basement membrane (BM) is detected and very small vacuoles (yellow arrow) are noticed (x 6000).

Discussion

Ischemia/reperfusion injury (IRI) is characterized by restriction of blood supply to an organ followed by restoration of blood flow and re-oxygenation. The inevitable injuries may occur after infarction, sepsis and organ transplantation and this phenomena exacerbate tissue damage by initiating an inflammatory cascade including reactive oxygen species (ROS), cytokines, chemokines, and leukocytes activation [25,26].

In the kidney, IRI remains popular in the development of kidney-related surgery and transplantation and contributes to pathological conditions called acute kidney injury (AKI) [25]. AKI is a clinical syndrome that occurs within a few hours or up to a few days after an injury and is associated by inflammation and tissue damage. AKI gives rise to dangerous complications such as excess fluid, hyperkalemia, uremia, and metabolic acidosis and can also have systemic effects on other organs including heart, brain, and lung. In hospitalized patients, AKI is a common and major concern because of its high morbidity and mortality [26, 27]. In addition, acute kidney damage is one of the most important reasons for mortality in intensive care units [28, 29, 30]

. The early diagnosis and treatment of acute kidney damage is one of the most important factors in the correction of the progress of this disease. The most well-known diagnostic methods for determining kidney damage are the BUN and serum creatinine levels [31]. When taking this into account, in agreement with other reports [32] we found that animals that underwent renal IRI exhibited significant increase in the BUN and creatinine, compared to sham-operated group.

Interestingly enough, this increase was partially corrected by RDN and their values were normalized in RDNVNS group. We had supported these results by histological study (light and electron microscopic examinations).

Our study also showed that the amount of serum MDA was significantly increased, and the anti-oxidant enzyme GPX activity was significantly reduced in I/R group when compared to sham control group. The MDA is the most important indicator showing lipid peroxidation in the ischemia–reperfusion induced tissue damage [33].

In addition, the return to normoxia after reperfusion causes a large production of ROS and lipid peroxidation as main pathway of free radical tissue injuries [39] in addition to reduction in antioxidant capacity level [40, 41]. Formation of free radicals de¬velops renal tissue injury via peroxidation of membrane lipids and oxidative damage of proteins and DNA which contrib¬utes to apoptosis and cell death [42]. Also the down regu¬lation of the antioxidant enzyme system such as catalase, superoxide dismutase, and glutathione peroxidase were found to play a crucial role in the pathophysiology of ischemia-re¬perfusion injury [43].

Additionally, it was reported that NO in low concentrations considered as renoprotective against renal ischemia due to its vasodi¬latory, antioxidant and anti-inflammatory properties, as well as its beneficial effects on cell signaling and inhibition of nuclear proteins [44-46]. Interestingly, the half-life of NO is very short which limited its direct measure¬ment, therefore its metabolites; nitrite and nitrate usually are measured [44-46]. Several reports [47- 50] have demonstrated nitrate-medi¬ated cytoprotection in IRImodels. In our work, renal tissue nitrate was inversely related to BUN and serum creatinine levels. In addition, it was seen herein, that renal tissue nitrate level was significantly decreased in IRI group which may provide another explanation for the reduced oxidant defense mechanisms in this group, as nitrate was proved to have anti-oxidant effects [51].

Histological findings in IR rats revealed thin cortical thickness in relation to medulla in addition to the appearance of medullary rarefaction at focal areas and partially detached renal capsule. Some cortical areas showed renal corpuscle with shrunken, markedly distorted and congested small glomeruli having a widened bowman's space and abnormal configuration of renal tubules were observed. Moreover, some proximal and distal tubules showed rarefied cytoplasm of lining cells exhibiting cytoplasmic vacuoles and epithelial exfoliation at focal areas. Flattening of the epithelial lining of some tubules and apparent tubular dilatation were found. Also, homogenous materials and cellular debris were dislodged within their luminae and dilated congested blood vessels were detected.

Additionally, necrotic changes in most of the cortical tubular epithelial cells in the form of nuclear changes (pyknotic, karyorrhectic, and karyolitic nuclei), in addition to vacuolation and interstitial inflammatory mononuclear cellular infiltrations were noticed. Moreover, the renal cortex showed also marked increase of the collagen fibers around the Bowman’s capsules and the capillary loops of glomeruli with the appearance of mitochondrial transition pore (mPTP) after reperfusion, leading to apoptosis, necrosis and autophagy [37, 38].

In addition, the return to normoxia after reperfusion causes a large production of ROS and lipid peroxidation as main pathway of free radical tissue injuries [39] in addition to reduction in antioxidant capacity level [40, 41]. Formation of free radicals de¬velops renal tissue injury via peroxidation of membrane lipids and oxidative damage of proteins and DNA which contrib¬utes to apoptosis and cell death [42]. Also the down regu¬lation of the antioxidant enzyme system such as catalase, superoxide dismutase, and glutathione peroxidase were found to play a crucial role in the pathophysiology of ischemia-re¬perfusion injury [43].

Additionally, it was reported that NO in low concentrations considered as renoprotective against renal ischemia due to its vasodi¬latory, antioxidant and anti-inflammatory properties, as well as its beneficial effects on cell signaling and inhibition of nuclear proteins [44-46]. Interestingly, the half-life of NO is very short which limited its direct measure¬ment, therefore its metabolites; nitrite and nitrate usually are measured [44-46]. Several reports [47- 50] have demonstrated nitrate-medi¬ated cytoprotection in IRImodels. In our work, renal tissue nitrate was inversely related to BUN and serum creatinine levels. In addition, it was seen herein, that renal tissue nitrate level was significantly decreased in IRI group which may provide another explanation for the reduced oxidant defense mechanisms in this group, as nitrate was proved to have anti-oxidant effects [51].

Histological findings in IR rats revealed thin cortical thickness in relation to medulla in addition to the appearance of medullary rarefaction at focal areas and partially detached renal capsule. Some cortical areas showed renal corpuscle with shrunken, markedly distorted and congested small glomeruli having a widened bowman’s space and abnormal configuration of renal tubules were observed. Moreover, some proximal and distal tubules showed rarefied cytoplasm of lining cells exhibiting cytoplasmic vacuoles and epithelial exfoliation at focal areas. Flattening of the epithelial lining of some tubules and apparent tubular dilatation were found. Also, homogenous materials and cellular debris were dislodged within their luminae and dilated congested blood vessels were detected.

Additionally, necrotic changes in most of the cortical tubular epithelial cells in the form of nuclear changes (pyknotic, karyorrhectic, and karyolitic nuclei), in addition to vacuolation and interstitial inflammatory mononuclear cellular infiltrations were noticed. Moreover, the renal cortex showed also marked increase of the collagen fibers around the Bowman’s capsules and the capillary loops of glomeruli with the appearance of mononuclear cellular infiltrations. Renal medulla showed also marked increase of the collagen fibers around tubules and septa between tubules. Some medullary areas showed mononuclear cells infiltrations between disorganized, degenerated tubules and peritubular congestion. These results were in accordance with previous histological studies which revealed severe acute tubular damage in renal sections of the rats subjected to IR [52, 53].

The peritubular cellular infiltration observed in IRI rats could be explained by the fact that the cellular infiltration could be considered as a defense mechanism of the kidney to aid the rapid removal of necrotic tissue. In addition, congestion leads to slowing of the circulation and an increase in the permeability of the blood capillaries, leading to extravasation of red blood corpuscles, protein rich inflammatory fluid, and leukocytes migration that initiate the inflammatory reaction and attraction of infiltrating cells [54]. Mononuclear cellular infiltration was also observed in between the tubules, which may explain the presence of collagen fibrils in relation to the basement membranes [55].

Adaptation of the inflammatory cells and leukocyte infiltration into post-ischemic regions in the kidney occurs within several hours of tissue injury, and these cells contribute to initiation of the inflammatory series. Leukocyte infiltration into postischemic regions in the kidney is promoted by activation of the complement system in addition to enhanced proinflammatory cytokines and chemokines production [56]. In our study, TNFα level is increased in renal tissue of IR group. Consistently, it was found that inflammation has very important role in IR damage, and studies have shown that neutrophils, T and B lymphocytes and macrophages controlled the dominant damage in I/R [57- 59] and several chemokines are released in these inflammatory cells; in particular, IL-1b, IL-6 and TNFα that are capable of damaging endothelial cells and increasing vascular permeability, with a subsequent reduction in renal blood flow [60, 61].

In addition, renal dendritic cells were found to increase in number in the injury site and mediate inflammation. Likewise, inflammatory monocytes infiltrate the injury site, and then they differentiate into macrophages. In the injury phase, macrophages are polarized into proinflammatory macrophages (M1) and cause tissue damage [62, 63]. Tubular cells also contribute to inflammation in response to kidney IRI by generating proinflammatory and chemotactic cytokines such as TNF-α, MCP-1, IL-8, IL-6, IL-1β, and TGF-β [64].

Moreover, in the present study, E/M examination of kidneys of I/R group revealed that the podocytes had 1ry process with an elongated irregular nuclei together with disorganization, distortion and fusion in the some feet processes. Other cells showed shrunken, heterochromatic and pyknotic nuclei. Irregular thickening of glomerular basement membranes and dilated glomerular capillaries with lack of fenestrations were clearly seen. Marked irregular thickening of Bowman’s membrane surrounded by thick layer of collagen fibers was also observed. Shrinkage of the glomeruli and shrinkage of the capillary network resulting in widening the space between the wall and the network were found in the current study as proved by other study [65].

In accordance to our findings, Transmission and scanning electron microscopic examinations of other studies revealed degeneration of the capillaries endothelium, destruction of the podocytes, and extravasations of many RBCs in some of the glomeruli [66] and irregular thickening in the basal lamina of the glomeruli and fusion of the foot processes of the podocytes were also reported [67]. Glomerular injury in IRI may be attributed to adherence of neutrophils to the endothelium, thus impairing or obstructing blood flow in small blood vessels, leading to local tissue hypoxia [68] or may be a result of unusual production of type-III collagen by mesangial cells [67]. Meanwhile, postischemia or reperfusion exacerbated tissue injury due to migration of the adlevel is increased in renal tissue of IR group. Consistently, it was found that inflammation has very important role in IR damage, and studies have shown that neutrophils, T and B lymphocytes and macrophages controlled the dominant damage in I/R [57-59] and several chemokines are released in these inflammatory cells; in particular, IL-1b, IL-6 and TNFα that are capable of damaging endothelial cells and increasing vascular permeability, with a subsequent reduction in renal blood flow [60, 61]. 

In addition, renal dendritic cells were found to increase in number in the injury site and mediate inflammation. Likewise, inflammatory monocytes infiltrate the injury site, and then they differentiate into macrophages. In the injury phase, macrophages are polarized into proinflammatory macrophages (M1) and cause tissue damage [62, 63]. Tubular cells also contribute to inflammation in response to kidney IRI by generating proinflammatory and chemotactic cytokines such as TNF-α, MCP-1, IL-8, IL-6, IL-1β, and TGF-β [64].

Moreover, in the present study, E/M examination of kidneys of I/R group revealed that the podocytes had 1ry process with an elongated irregular nuclei together with disorganization, distortion and fusion in the some feet processes. Other cells showed shrunken, heterochromatic and pyknotic nuclei. Irregular thickening of glomerular basement membranes and dilated glomerular capillaries with lack of fenestrations were clearly seen. Marked irregular thickening of Bowman’s membrane surrounded by thick layer of collagen fibers was also observed. Shrinkage of the glomeruli and shrinkage of the capillary network resulting in widening the space between the wall and the network were found in the current study as proved by other study [65].

In accordance to our findings, Transmission and scanning electron microscopic examinations of other studies revealed degeneration of the capillaries endothelium, destruction of the podocytes, and extravasations of many RBCs in some of the glomeruli [66] and irregular thickening in the basal lamina of the glomeruli and fusion of the foot processes of the podocytes were also reported [67]. Glomerular injury in IRI may be attributed to adherence of neutrophils to the endothelium, thus impairing or obstructing blood flow in small blood vessels, leading to local tissue hypoxia [68] or may be a result of unusual production of type-III collagen by mesangial cells [67]. Meanwhile, postischemia or reperfusion exacerbated tissue injury due to migration of the adherent neutrophil and the release of cytotoxic substances, destroying neighboring cells and tissue matrix [68].

Additionally, the current study revealed that the cells of the proximal tubules showed also variable ultrastructural changes. Some cells had a disoriented basal mitochondrial, with few and dilated basal enfolding; in addition to the presence of multiple vacuoles, nuclei of some cells showed a dilated perinuclear space. Multiple secondary lysosomes, abnormal-shaped mitochondria, and electron-dense bodies could be detected in the cytoplasm of other cells. Other cells of the proximal tubules contained areas of rarified cytoplasm, swollen mitochondria with ill-defined crestae and many dense irregular bodies mostly lysosomes. Heterogeneous pleomorphic autophagic vacuoles containing whorls of membranous material were present. In addition to the appearance of shrunken, condensed pyknotic nuclei, while the microvilli were mostly dilated and irregularly thickened basal lamina were detected

The cells of the distal convoluted tubule showed, in the majority of cells, few basal enfolding and a palisade form of the mitochondria but the cytoplasm showed some rarefaction. Additionally, some atrophied tubules with irregular thickening of the basement membranes surrounded by hemorrhagic areas and thick collagen fibers in between the tubules were observed. The cells revealed shrunken condensed and pyknotic nuclei, but others had completely atrophied nuclei. The mitochondria migrated towards the lumen with loss of their parallel arrangement. They were swollen in addition to the appearance of indistinct crestae and obliteration of their tubular lumen. Our study additionally revealed that the DCTs showed loss of their microvilli, with shedding of their apical cytoplasm into the lumen. This was in agreement with the result obtained by other authors [69, 70].

These findings might indicate that IRI has induced cellular necrosis and/or apoptosis of the tubular cells.

The necrosis, characterized by the cell swelling with subsequent rupture of surface membranes [71] is a frequent consequence of the IR. The necrotic cells stimulate the immune system and lead to tissue infiltration of inflammatory-cells with consequent cytokine release. Generally the apoptosis process was considered as less immunostimulating than the necrosis process [72] however; the extracellular release of ATP from the apoptotic cells may attract phagocytes [73].

In our point of view, hypoxia and/or reperfusion with their subsequent events of oxidative stress and inflammation in addition to deficient antiapoptotic responses might explain the severe deleterious functional and structural effects appeared on kidneys of IR group of rats. In our study, week positive Bcl2 anti-apoptotic reaction in immune-histochemical study of IR group was observed. In parallel, apoptosis was previously reported by Kunduzova et al (2003) [74] in renal tubular cells after IR in an in-vivo and in-vitro model of renal injury and attributed these findings to caspase-3 activation, which represents a direct mechanism for tubular damage

Also, In renal cells, ischemia activates Bax [75] and reduces Bcl2 markedly altering the Bax/Bcl2 ratio in a pro-apoptotic direction [76, 77]. In addition, renal tubular cells also express cell surface ‘death receptors’ of the tumor necrosis factor (TNF) superfamily including Fas, TNF receptor 1, and Fn14 receptor that also induce apoptosis [78] and activate caspases by binding to distinct receptors during renal ischemia, presumably contributing to AKI [79].

The kidney is extremely sensitive to anoxia because of its special features, which makes it vulnerable to hypoxic injury and oxidative stress is considered the key step in the initiation and development of renal IRI [67, 68]. AS shown in our study, there was oxidant/anti-oxidant imbalance in the IR rats which was demonstrated by the high lipid peroxidation marker (MDA) versus low GPX anti-oxidant activity. Oxidative stress plays an important role in renal apoptosis, especially during reperfusion either by acting as signal transduction molecules or by directly causing cellular damage. Reactive oxygen species (ROS) activate apoptosis at multiple steps in the cell death pathway [76, 80].

Moreover, it has been reported that the inflammatory response generated by activated neutrophils could also generate large amounts of reactive oxygen species (ROS), leading to lipid peroxidation within the cell membranes, followed by disintegration of the cells and cell death [81]. B cells were also found to be activated in the injury phase of IR and limit tubular regeneration in the recovery phase, leading to tubular atrophy and apoptosis [82].

On the other hand, in our work, serum MDA was significantly lowered while, GPX activity was significantly elevated in renal tissues of RDN group compared to IRI group but not normalized and remained significantly different compared to sham control group. Whereas, normalization of such values in the group of rats subjected to RDN and VNS (RDN-VNS group) was observed, this may explain the return of renal functions (BUN and serum creatinine) in this group towards normal. In addition, renal tissue nitrate was substantially increased in response to RDN alone or combined to VNS compared to IRI group. Tissue nitrate level was significantly increased in the RDN group and further elevated in the RDN-VNS group to the normal level. Therefore, VNS conferred more protective effect when combined to RDN

The significant positive correlation between serum MDA and renal functions (BUN & serum creatinine) in addition to the significant negative relationship between both renal tissue GPX and renal tissue nitrate on one hand and renal function biomarkers on the other hand essentially support the partial Reno-protective effect provided by RDN especially when combined to VNS. In parallel, renal denervation showed protective effects against renal failure in both animals and humans. Although the mechanisms remain to be fully elucidated, it may include decrease in blood pressure and downregulation of the renin-angiotensin system [83, 84] against renal failure in both animals and humans.

Herein also, the inflammatory marker TNFα level in renal tissue was decreased in the RDN and further decreased in the RDN-VNS groups compared to the IR group. Additionally, renal tissue nitrate was significantly increased which has an anti-inflammatory in the group of rats subjected to RDN and followed by VNS.

The aforementioned changes observed in RDN-VNS group led to prevention of renal damage following IRI as the renal function tests (BUN& Serum creatinine) were positively correlated with TNFα and inversely related to tissue nitrate level

The H&E study of sections of the RDN kidneys revealed in the renal cortex that some of the renal corpuscles, glomeruli and Bowman’s spaces were average in size and shape but others showed slightly congested glomeruli and little widening of Bowman’s space. Also, some tubules seemed free from exfoliation in their luminae but others contained exfoliation in their luminae and intracellular cytoplasmic vacuoles. In addition, some of the blood vessels were dilated and moderately congested and few of the mononuclear cells infiltrated in the interstitium. The renal cortex showed few collagen fibers around the renal capsules, the glomeruli and the tubules. Also, septa between tubules were intact in comparison to IR group. Medullary area showed mononuclear cellular infiltrations in interstitium of avarege tubules but some of them showed exfoliated cell debris and homogenous materials in their luminae and some pyknotic nuclei in their lining epithelium. Slightly dilated congested blood vessels were also observed.

EM study showed that the podocytes had disorganized pedicles and electron-dense substances in the glomerular capillaries. The mesangial cells were surrounded by mesangial matrix. Thick regular Bowman’s capsules with thin layers of collagen were visible on the outer aspect of the basal lamina. Lumens of the proximal tubules were filled with secretions. The lining cells of the tubules showed microvilli, small cytoplasmic vacuoles and small dense lysosomes but some of their mitochondria were swollen.

The cells of the distal tubules revealed small lysosomes and some elongated mitochondria with mostly intact crestae. Areas of cytoplasmic rarefaction could be observed. Some nuclei were irregular in shape and shrunken, but others were nearly average in appearance. The brush borders couldn’t be detected but basal enfolding and thickening could be observed at some of the basal laminae

Regarding Bcl2 immune-histochemical study, the renal cortex showed moderately positive immunoreactions of the nuclei in glomeruli, Bowman’s membrane and among the lining tubular epithelial cells compared to IR group

In agreement to our findings, in denervated kidneys, a significant reduction of inflammatory parameters such as accumulation of interstitial macrophages and renal expression of TNF-α as well as reduced accumulation of macrophages were observed [85]. Fibrosis was also reduced as measured by TGF-β expression and glomerular collagen IV deposition in denervated rats [85]. Consistently, the associated ischaemia-reperfusion induced renal dysfunction and tissue damage were reported to be attenuated by both surgical and pharmacological blockade of renal sympathetic nerve activity [86]. Interestingly, it has been demonstrated that renal sympathetic nerve stimulation is a primary mechanism instigating fibrogenesis in the ischaemia-reperfusion induced AKI [87].

Additionally, it was also found that RDN prevented unilateral ureteral obstruction-induced inflammation, interstitial fibrosis and renal nerve derived signaling molecules (noradrenaline and CGRP) mediating the inflammatory and fibrotic response [88]. Moreover, RDN prior to or up to 1 day post-ischaemic renal injury attenuated the associated tubular injury, apoptosis, tubulointerstitial inflammation and fibrosis and preserved kidney function during the early post-ischaemic period [87]. Furthermore, centrally acting sympatholytics (i.e. clonidine and moxonidine) have been shown to prevent IR induced AKI [89].

Certain sympathetic nerve fibers in the kidney were found to lie in very close proximity to inflammatory and antigen-presenting cells, such as ED1-positive macrophages and denderitic cells [90]. As the immunocytes co-localize with both sympathetic and primary sensory nerve fibers, it is likely that both neuronal systems are involved in neuroimmune interactions in the kidney [90, 91].In the kidney, neuroimmune interactions might result from local production of the proinflammatory neuropeptide SP or indirectly via increased cytokine-stimulated sympathetic nerve activity [92]. . In addition, activation of β1 receptors on juxtaglomerular cells by catecholamines leads to production of AngII which is widely known as a hypertensive and proinflammatory mediator [93]. Therefore, it is demonstrated that renal innervation is critical for the development of experimental nephritis [93].

Taken together, our results argue for a significant role of the RDN for preventing kidney deterioration after IRI. This study provides evidence that inflammation, oxidation and apoptosis and their effects were partially lowered in the kidney of rats exposed to RDN. However, in this work, the functional and histological study of rats subjected to RDN and followed by vagal nerve stimulation (RDN-VNS group) showed marked preservation of various changes produced by IR injury. The oxidant/anti-oxidant imbalance observed in the IR group was improved in the group which was underwent RDN. Meanwhile, the combination of RDN and VNS reduced the oxidative stress dependent tissue damage, and restored the antioxidant defense mechanism as serum MDA was significantly decreased and the renal tissue GPX anti-oxidant enzyme activity was significantly increased to the normal levels in RDN-VNS group becoming comparable to the sham control group.

Structurally, H&E study by light microscopy showed that most of the glomeruli of RDN-VNS group were to some extent as those of the sham group. The mononuclear cellular infiltration in the interstitial tissues around the injured areas in IR group was also declined in RDN group and markedly decreased in RDN-VNS group. In addition, renal sections demonstrated average diameter of cortex and medulla with average glomeruli and tubules covered by intact capsule. Higher magnification showed that most of the renal corpuscles of the RDN-VNS rats were intact as the glomeruli were normal and Bowman’s capsules were patent. Proximal, distal and collecting tubules were also normal. The rest of renal corpuscles showed slightly wide Bowman’s space and some of the tubules contained few pyknotic nuclei and cytoplasmic vacuoles. Moreover, the renal cortex and medulla showed minimal collagen fibers around the Bowman’s capsules and the tubules.

Upon EM examination of RDN-VNS group, the podocytes showed euchromatic indented nuclei and mitochondria and few small vacuoles were seen in their cytoplasm. They appeared with normal arranged secondary feet processes. The space between the pedicles (filtration slits) could be seen and laid on a regular glomerular basement membrane. Glomerular capillaries lacking fenestrae were seen. Though, some of the podocytes showed distorted and fused feet processes with the appearance of few matrixes around mesangial cells.

The cells of the proximal tubules revealed long, dense apical microvilli and basal enfolding in addition to presence of small pinocytotic vesicles near to the lysosomes. Many elongated mitochondria with distinct crestae and large nuclei with peripheral condensed chromatin could be observed. On the other hand, the cells of the distal tubules revealed elongated mitochondria with mostly intact crestae. Small areas of cytoplasmic rarefaction could be observed. Most of the nuclei were normal in appearance, but scanty of them were pyknotic

Regarding immune-histochemical study, the renal cortex of the RDN-VNS rat group showed markedly positive anti-apoptotic Bcl2 immune reactions of the nuclei in glomeruli, Bowman’s membrane and among the lining tubular epithelial cells compared to other groups

Additionally, our results revealed in the RDN-VNS group that TNFα was significantly reduced and renal tissue nitrate was increased markedly compared to IR group, indicating reduced inflammatory responses to renal IRI. Such results were supported by the histological findings of reduced cellular infiltration, fibrosis and collagen deposition in renal corpuscles and tubules. Parallel to our findings, other investigators deduced that electrical stimulation of the efferent VN significantly decreased the amounts of TNFα in the serum [94

Moreover, VNS when performed 24 h prior to IRI, renal function and tissue morphology were preserved. Plasma TNF-α induction by IRI was also suppressed by prior VNS. This renal protection was observed after either afferent or efferent VNS. [8]. Additionally, VNS was reported to activate a neuroimmune pathway called cholinergic anti-inflammatory pathway (CAP) which inhibits inflammation through the suppression of cytokine production and prevent damage [95, 96, 97]. Interestingly, the VN possesses a double role within the framework of inflammation: informing the CNS via its afferents of the presence of inflammation and modulating inflammation via its efferents [95]. stimulation of the efferent VN significantly decreased the amounts of TNFα in the serum [94].

Moreover, VNS when performed 24 h prior to IRI, renal function and tissue morphology were preserved. Plasma TNF-α induction by IRI was also suppressed by prior VNS. This renal protection was observed after either afferent or efferent VNS. [8]. Additionally, VNS was reported to activate a neuroimmune pathway called cholinergic anti-inflammatory pathway (CAP) which inhibits inflammation through the suppression of cytokine production and prevent damage [95, 96, 97]. In

Conclusion

RDN given alone or in combination with VNS might be future options for treatment of inflammatory disease in the kidney. Synergism between RDN and VNS enhanced and accelerated the Reno protection via restoration of oxidant/ antioxidant balance, anti-inflammatory and anti-apoptotic effects. Taken together, RDN and VNS in combination conferred an optimized functional, structural and ultra-structural renal responses in IRI induced AKI.

Competing Interests
The authors declare no competing or financial interests

Author Contributions
Conceptualization:M.; Methodology: M.Z.W.A.K.G.F.; Formal analysis: M.Z.W.A.K.G.F.; Investigation: M.Z.W.A.K.G.F.; Writing - original draft: M.Z.W.K.G.F.; Writing - review & editing: M.Z.W.A.K.G.F.; Supervision: M.A.; Project administration: M.

Acknowledgment

We would like to thank and acknowledge Dr Shadwa Khaled, Dr Nada Abdelhameed , DrAbdalla Magdy, Dr Mohamed Mahmoud Ismaiel and Dr Mohamed Fahmy for their hard and kind support during the practical work of the study.

References

1.Coca SG, Singanamala S, Parikh CR. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis. Kidney Int 2012; 81(5): 442–448.

2.Schiffl H, Lang SM, Fischer R. Long-term outcomes of survivors of ICU acute kidney injury requiring renal replacement therapy: a 10-year prospective cohort study. Clin Kidney J 2012; 5(4): 297–302.

 3.Rifkin DE, Coca SG, Kalantar-Zadeh K. Does AKI truly lead to CKD? J Am Soc Nephrol 2012; 23(6): 979– 984.

4.Kellum JA, Unruh ML, Murugan R. Acute kidney injury. BMJ Clin Evid 2011. 2001.

5.Hoste EA, Clermont G, Kersten A, et al. RIFLE criteria for acute kidney injury are associated with hospital mortality in critically ill patients: a cohort analysis. Crit Care 2006; 10(3): R73

6.Zimmerman BJ, Granger DN. Reperfusion injury. Surg Clin North America 1992; 72(1): 65–83.

7. Bonventre JV, Zuk A. Ischemic acute renal failure: an inflammatory disease? Kidney Int 2004; 66(2): 480- 485.

8. Inoue T, Abe C, Sung S.S, et al. Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes. J. Clin. Invest 2016; 126(5): 1939–1952.

9. Gallowitsch-Puerta M, Pavlov VA. Neuro-immune interactions via the cholinergic anti-inflammatory pathway. Life Sci 2007; 80(24-25): 2325–2329.

10. Iaina A, Eliahou HE. The Sympathetic Nervous System in the Pathogenesis of Acute Renal Failure. Renal Failure 1983; 7(1-2): 115–125.

11. Schlaich MP, Socratous F, Hennebry S, et al. Sympathetic activation in chronic renal failure. J Am Soc Nephrol 2009; 20(5): 933–939.

12. Kim J, Padanilam BJ. Renal denervation prevents long-term sequelae of ischemic renal injury. Kidney Int 2015; 87(2): 350–358.

13. Borovikova LV. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature 2000; 405: 458–462.

14. Stacey WC, Litt B. Technology insight: neuroengineering and epilepsy-designing devices for seizure control. Nat Clin Pract Neurol 2008; 4(4): 190–201.

15. Rosas-Ballina M. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science 2011; 334(6052): 98–101.

16. Levine YA. Neurostimulation of the cholinergic anti-inflammatory pathway ameliorates disease in rat collagen-induced Arthritis. PLoS One 2014; 9(8).

17. Zhang YL, Zhang J, Cui LY, Yang S. Autophagy activation attenuates renal ischemia-reperfusion injury in rats. Exp Biol Med (Maywood) 2015; 240(12): 1590–1598.

18. Patton C.J, and Crouch S.R: Spectrophotometric and kinetics investigation of the Berthelot reaction for the determination of ammonia. Anal Chem 1977; 49(3): 464-469.

19. Bartels H, Bohmer M, and Heierli C. Serum creatinine determination without protein precipitation. Clin Chem Acta 37:193-197.

20. Draper H. and Hadley M. Malondialdehyde determination as index of lipid peroxidation. In Methods in enzymology. New York: Academic Press 1991; 421- 443.

21. Paglia DE and Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70(1): 158-69.

22. Marín-García J, Goldenthal MJ, Moe GW. Abnormal cardiac and skeletal muscle mitochondrial function in pacing-induced cardiac failure. Cardiovasc Res 2001; 52(1): 103-10.

23. Bories H and Bories C. Nitrate determination in biological fluids by an enzymatic one step assay with nitrate reductase. Clin Chem 1995; 41(6): 904-907.

24. Bancroft JD, Layton C. The hematoxylin and eosin, connective and mesenchymal tissues with their stains. In: Suvarna SK, Layton C and Bancroft JD, editors. Bancroft’s theory and practice of histological techniques.7th edition. Churchill Livingstone: Philadelphia 2013; 173- 212.

25.Y, et al. The role of SDF-1-CXCR4/ CXCR7 axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury. PLoS One 2012; 7(4): e34608 Liu H, Liu S, Li .

26. Chatterjee PK, Brown PA, Cuzzocrea S, et al. Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat. Kidney Int 2001; 59(6): 2073-2083.

27. Sadek ME, Afifi MN, Abd Elfattah I L, et al. Histological Study on Effect of Mesenchymal Stem Cell Therapy on Experimental Renal Injury Induced by Ischemia/Reperfusion in Male Albino Rat. Int J Stem Cells 2013; 6(1): 55-66.

28. Shimizu S, Saito M, Kinoshita Y, et al. Nicorandil ameliorates ischaemia-reperfusion injury in the rat kidney. Br J Pharmacol 2011; 163(2): 272-282.

29. Schrier RW, Wang W. Acute renal failure and sepsis. N Engl J Med 2004; 351(2): 159–169.

30. Hong X, Zhao X, Wang G, et al. Luteolin Treatment Protects against Renal Ischemia-Reperfusion Injury in Rats. Mediators of Inflammation 2017.

31. Waikar SS, Bonventre JV. Biomarkers for the diagnosis of acute kidney injury. Curr Opin Nephrol Hypertens 2007; 16(6): 557–564.

32. Ziypak T, Halici Z, Alkan E, et al. Renoprotective effect of aliskiren on renal ischemia/reperfusion injury in rats: electron microscopy and molecular study. Ren Fail 2015; 37(2): 343-354.

33. Kacmaz A, Polat A, User Y, et al. Octreotide improves reperfusion-induced oxidative injury in acute abdominal hypertension in rats. J Gastrointest Surg 2004; 8(1):113–119.

34. Johnson KJ, Weinberg JM. Postischemic renal injury due to oxygen radicals. Curr Opin Nephrol Hypertens 1993; 2(4): 625-635.

35. Paller MS. The cell biology of reperfusion injury in the kidney. J Investig Med 1994; 42(4): 632-639.

36. Bonventre JV. Mechanisms of ischemic acute renal failure. Kidney Int 1993; 43(5): 1160-1178.

37https://nyaspubs.onlinelibrary.wiley.com/doi/abs/10.1111/j.1749-6632.2010.05634.x.

38. Kalogeris T, Baines CP, Krenz M, et al. Cell biology of ischemia/reperfusion injury. Int Rev Cell Mol Biol 2012; 298: 229–317.

39. Paller MS, Hoidal J, Ferris TF. Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 1984; 74(4): 1156-1164.

40. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002; 282(2): C227–C241.

41. Bayrak O, Bavbek N, Karatas OF, et al. Nigella sativa protects against ischaemia/reperfusion injury in rat kidneys. Nephrol Dial Transplant 2008; 23(7): 2206–2212.

42. Kehrer JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 1993; 23(1):21-48.

43. Singh I, Gulati S, Orak JK, et al. Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury Mol Cell Biochem 1993; 125(2): 97- 104.

44. Kanner J, Harel S, Rina G. Nitric oxide as an antioxidant. Arch Biochem Biophys 1991; 289(1):130-136.

45. Granger DN, Kubes P. Nitric oxide as anti-inflammatory agent. Methods Enzymol 1996; 269: 434-442.

46. Phillips L, Toledo AH, Lopez-Neblina F, et al. Nitric oxide mechanism of protection in ischemia and reperfusion injury. Investigative Surgery 2009; 22(1): 46-55.

47. Duranski MR, Greer JJ, Dejam A, et al. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest 2005; 115(5): 1232-1240.

48. Jung KH, Chu K, Ko SY et al. Early intravenous infusion of sodium nitrite protects brain against in vivo ischemia-reperfusion injury. Stroke 2006; 37(11): 2744-2750.

49. Lii P, Liu F, Yao Z, et al. Nitrite-derived nitric oxide by xanthine oxidoreductase protects the liver against ischemia-reperfusion injury. Hepatobiliary Pancreat Dis Int 2005; 4(3): 350-355.

50. Tripatara P, Patel NS, Webb A et al. Nitrite-derived nitric oxide protects the rat kidney against ischemia/ reperfusion injury in vivo: role for xanthine oxidoreductase. J Am Soc Nephrol 2007; 18(2): 570-580.

51. Hammad FT, Al-Salam S, Lubbad L. Does aliskiren protect the kidney following ischemia reperfusion injury? Physiol Res 2013; 62(6): 681–690.

52. Mehta RL, Burdmann EA, Cerda J, et al. Recognition and management of acute kidney injury in the International Society of Nephrology 0by25 Global Snapshot: a multinational cross-sectional study. Lancet 2016; 387(10032): 2017–25.

53. Gao Y, Chen L, Ning Y, et al. Hydro-Jet-assisted laparoscopic partial nephrectomy with no renal arterial clamping: A preliminary study in a single center. Int Urol Nephrol 2014; 46(7): 1289–1293.

54. Huen SC, Cantley LG. Macrophage-mediated injury and repair after ischemic kidney injury. Pediatr Nephrol 2015; 30(2): 199–209.

55. Okasha FE .Effect of high-fructose diet on the renal cortex of adult male albino rats: histological and immunohistochemical study. Egypt J Histol 2011; 34: 639-649.

56. Jang HR, Rabb H. Immune cells in experimental acute kidney injury. Nat Rev Nephrol 2015; 11(2):88–101.

57. Friedewald JJ, Rabb H. Inflammatory cells in ischemic acute renal failure. Kidney Int 2004; 66(2): 486–491.

58. Farrar CA, Wang Y, Sacks SH, et al. Independent pathways of P-selectin and complement-mediated renal ischemia/reperfusion injury. Am J Pathol 2004; 164(1): 133–141.

59. Singbartl K, Ley K. Leukocyte recruitment and acute renal failure. J Mol Med 2004; 82(2): 91–101.

60. Singh V, Jain S, Gowthaman U, et al. Co-administration of IL-1 + IL-6 + TNF-alpha with Mycobacterium tuberculosis infected macrophages vaccine induces better protective T cell memory than BCG. PLoS One 2011; 6(1): e16097.

61. Schreiber S, Nikolaus S, Hampe J, et al. Tumour necrosis factor alpha and interleukin 1beta in relapse of Crohn’s disease. Lancet 1999; 353(9151): 459– 461.

62. Cao Q, Harris DC, Wang Y. Macrophages in kidney injury, inflammation, and fibrosis. Physiology (Bethesda) 2015; 30(3): 183–94.

63. Huen SC, Cantley LG. Macrophage-mediated injury and repair after ischemic kidney injury. Pediatr Nephrol 2015; 30(2): 199–209.

64. Van Kooten C, Woltman AM, Daha MR. Immunological function of tubular epithelial cells: the functional implications of CD40 expression. Exp Nephrol 2000; 8(4-5): 203-7.

65. Aziz JNS. The structural and functional changes indcuced by lithium on the renal cortex of Growing albino Rats: Ultrastructure and laboratory study. Acta Medica International 2015; 2(1):70-78.

66. Szeto HH, Liu S, Soong Y, et al. Mitochondria protec-tion after acute ischemia prevents prolonged upregulation of IL-1beta and IL-18 and arrests CKD. J Am Soc Nephrol 2017; 28: 1437–1449.

67. Bonventre JV, Yang L. Cellular pathophysiology of ischemic acute kidney injury. J Clin Invest 2011; 121: 4210-4221.

68. Bonventre JV, Zuk A. Ischemic acute renal failure: an inflammatory disease?. Kidney Int 2004; 66(2): 480–485.

69. Forbes JM, Hewitson TD, Becker GJ, et al. Ischemic acute renal failure: Long-term histology of cell and matrix changes in the rat. Kidney Int 2000; 57: 2375-2385.

70. Ahmed S.M, Mahmoud A.A, Hassen E.Z , et al. Light and Electron Microscope Study on the Effect of Platelet-Rich Plasma in Induced Renal Ischaemia-Reperfusion Injury in the Renal Cortex of Adult Male Albino Rats. J Biochem Cell Biol 2018; 1(2).

71. Hotchkiss R.S, Strasser A, McDunn J.E, et al. Cell death. N. Engl. J. Med 2009; 361:1570–1583.

72. Chen X, Sun C, Chen Q, et al. Apoptotic engulfment pathway and schizophrenia. PLoS One 2009; 4: e6875.

73. Elliott M.R, Chekeni F.B, Trampont P.C, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 2009; 461: 282–286.

74. Kunduzova O.R, Escourrou G, Seguelas M.-H, et al. Prevention of apoptotic and necrotic cell death, caspase-3 activation, and renal dysfunction by melatonin after ischemia/reperfusion. FASEB J 2003; 17: 872–874.

75. Havasi A, Li Z, Wang Z, et al. Hsp27 inhibits Bax activation and apoptosis via a phosphatidylinositol 3-kinase-dependent mechanism. J Biol Chem 2008; 283: 12305–12313.

76. Chien CT, Chang TC, Tsai CY, et al. Adenovirus-mediated bcl-2 gene transfer inhibits renal ischemia/ reperfusion induced tubular oxidative stress and apoptosis. Am J Transplant 2005; 5: 1194–1203.

77. Wolfs TG, de Vries B, Walter SJ, et al. Apoptotic cell death is initiated during normothermic ischemia in human kidneys. Am J Transplant 2005; 5: 68–75.

78. Feldenberg LR, Thevananther S, del Rio M, et al. Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells. Am J Physiol 1999; 276: F837–F846.

79. Justo P, Sanz AB, Sanchez-Nino MD, et al. Cytokine cooperation in renal tubular cell injury: the role of TWEAK. Kidney Int 2006; 70: 1750–1758.

80. Le Bras M, Clement MV, Pervaiz S, et al. Reactive oxygen species and the mitochondrial signaling pathway of cell death. Histol Histopathol 2005; 20(1): 205–219.

81. Birben E, Sahiner U.M, Sackesen C, et al. Oxidative stress and antioxidant defense. World Allergy Organ. J 2012; 5:9-19.

82. Jang HR, Gandolfo MT, Ko GJ, et al. B cells limit repair after ischemic acute kidney injury. J Am Soc Nephrol 2010; 21(4): 654–65.

83. DiBona GF. Physiology in perspective: The Wisdom of the Body. Neural control of the kidney. Am J Physiol Regul Integr Comp Physiol 2005; 289: R633–641.

84. Clayton SC, Haack KKV, Zucker IH. Renal denervation modulates angiotensin receptor expression in the renal cortex of rabbits with chronic heart failure. American Journal of Physiology - Renal Physiology 2011; 300: F31–F39.

85. Veelken R, Vogel EM, Hilgers K, et al. Autonomic renal denervation ameliorates experimental glomerulonephritis. J Am Soc Nephrol 2008; 19(7): 1371- 1378

86. Fujii T, Kurata H, Takaoka M, et al.The role of renal sympathetic nervous system in the pathogenesis of ischemic acute renal failure. Eur J Pharmacol 2003; 481: 241-248.

87. Kim J, Padanilam BJ. Renal denervation prevents long-term sequelae of ischemic renal injury. Kidney Int 2014; 87(2): 350-358.

88. Kim J, Padanilam BJ. Renal nerves drive interstitial fibrogenesis in obstructive nephropathy. J Am Soc Nephrol. 2013; 24: 229–242.

89. Tsutsui H, Sugiura T, Hayashi K, et al. Moxonidine prevents ischemia/reperfusion-induced renal injury in rats. European journal of pharmacology 2009; 603(1-3): 73-78.

90. Kaissling B, Le Hir M. Characterization and distribution of interstitial cell types in the renal cortex of rats. Kidney Int 1994; 54: 709–720.

91. Soos TJ, Sims TN, Barisoni L, et al.CX3CR+ interstitial dendritic cells form a contiguous network throughout the entire kidney. Kidney Int 2006; 70: 591–596.

92. Zhang ZH, Wei SG, Francis J, et al. Cardiovascular and renal sympathetic activation by blood-borne TNFα in rat: The role of central prostaglandins. Am J Physiol Regul Integr Comp Physiol 2003; 284: 916–927.

93. Phillips MI, Kagiyama S. Ang II as a pro-inflammatory mediator. Curr Opin Investig Drugs 2002; 3: 569–577.

94. Bonaz B, Picq C, Sinniger V, Mayol JF, Clarencon D. Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway. Neurogastroenterol Motil 2013; 25(3): 208–221.

95. Tracey KJ. Physiology and immunology of the cholinergic antiinflammatory pathway. J Clin Invest 2007; 117 (2): 289–296.

96. Olofsson PS, Rosas-Ballina M, Levine YA, et al. Rethinking inflammation: neural circuits in the regulation of immunity. Immunol Rev 2012; 248(1): 188–204

97. Pavlov VA, Tracey KJ. The vagus nerve and the inflammatory reflex--linking immunity and metabolism. Nat Rev Endocrinol 2012; 8(12): 743-754.

98. Pavlov VA, Wang H, Czura CJ, et al. The cholinergic anti-inflammatory pathway: a missing link in neuroimmunomodulation. Mol. Med 2003; 9: 125–134.

99. Nizri E, Hamra-Amitay Y, Sicsic C et al. Anti-inflammatory properties of cholinergic up-regulation: a new role for acetylcholinesterase inhibitors. Neuropharmacology 2006; 50: 540–547.

100. Buijs RM, van der Vliet J, Garidou ML et al. Spleen vagal denervation inhibits the production of antibodies to circulating antigens. PLoS One 2008; 3(9): 3152.

101. Bratton BO, Martelli D, McKinley MJ et al. Neural regulation of inflammation: no neural connection from the vagus to splenic sympathetic neurons. Exp Physiol 2012; 97: 1180–1185.

102. Bellinger DL, Lorton D, Hamill RW et al. Acetylcholinesterase staining and choline acetyltransferase activity in the young adult rat spleen: lack of evidence for cholinergic innervation. Brain Behav Immun 1993; 7: 191–204

103. Huston JM, et al. Splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis. J Exp Med 2006; 203: 1623–1628.

104. Gigliotti JC, et al. Ultrasound prevents renal ischemia-reperfusion injury by stimulating the spleniccholinergic anti-inflammatory pathway. J Am Soc Nephrol 2013; 24(9): 1451–1460.

105. Gigliotti JC. Ultrasound modulates the splenic neuroimmune axis in attenuating AKI. J Am Soc Nephrol 2015; 26(10): 2470–2481.

106. Rosas?Ballina M, Ochani M, Parrish WR, et al. Splenic nerve is required for cholinergic antiinflammatory pathway control of TNF in endotoxemia. Proc Natl Acad Sci USA 2008; 105: 11008–11013.

107. Rosas-Ballina M, Olofsson PS, Ochani M, et al. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science 2011; 334: 98–101.

108. Lee HT, Kim JY, Kim M, et al. Renalase protects against ischemic AKI [J] J Am Soc Nephrol 2013; 24(3): 445–455.

109. Shimokawa T, Tsutsui H, Miura T, et al. Renoprotective effect of yohimbine on ischaemia/reperfusion-induced acute kidney injury through alpha2C-adrenoceptors in rats. Eur J Pharmacol 2016; 781: 36-44.

110. Basile DP, Liapis H, Hammerman MR. Expression of bcl-2 and bax in regenerating rat renal tubules following ischemic injury. Am J Physiol 1997; 272 (5): 640–647.

111. Ajami M, Davoodi SH, Habibey R, et al. Effect of DHA+EPA on oxidative stress and apoptosis induced by ischemia-reperfusion in rat kidneys. Fundam Clin Pharmacol 2013; 27: 593–602