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Saudi Journal of Kidney Diseases and Transplantation
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ORIGINAL ARTICLE  
Year : 2020  |  Volume : 31  |  Issue : 5  |  Page : 982-997
A Comparative Study of the Use of Dexamethasone, N-acetyl Cysteine, and Theophylline to Ameliorate Renal Ischemia–Reperfusion Injury in Experimental Rat Models: A Biochemical and Immuno-histochemical Approach


1 Department of Pediatrics, Pediatric Nephrology Unit, Faculty of Medicine, Cairo University, Cairo, Egypt
2 Department of Medical Histology and Cell Biology, Faculty of Medicine, Cairo University, Cairo, Egypt

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Date of Web Publication21-Nov-2020
 

   Abstract 


Renal ischemia–reperfusion injury (IRI) is commonly encountered in clinical practice during renal transplantation. In a trial to find the drug that best safeguards the kidney against IRI, dexamethasone (Dex), N-acetyl cysteine (NAC), and theophylline (Theo) were tested in experimental rat models. This study included 105 adult male albino rats, which were randomly assigned to the following five groups: Group I – sham-operated, n = 5, Group II – IRI n = 25, Group III – IRI + Dex n = 25, Group IV – IRI + NAC n = 25, and Group V –IRI + Theo n = 25. IRI was induced for 40 min followed by reperfusion. Rats were sacrificed 1, 2, 4, 6, and 24 h after reperfusion. This was preceded by blood and urine sampling for biochemical study of serum Cystatin C (Cys C), serum creatinine, and urinary Cys C. Kidneys were processed for histopathological evaluation and immune-histochemical staining for Cys C. The expression of Cys C in the proximal tubular cells was significantly lower in the IRI group compared to that of the sham group. There was a significant rise in the levels of serum and urinary Cys C after 1 h in the IRI group, while the rise in creatinine occurred later. Dex was superior to NAC and Theo 24 h after the IR insult, and the serum levels of creatinine and Cys C were significantly lower in this group than the other two drug groups (P <0.001 in both cases). Our study revealed a clear benefit for the use of Dex to ameliorate IRI over NAC and Theo if used immediately following the insult. The effect is evident 24-h after its use. The role of serum Cys C as an early marker of acute kidney injury compared to serum creatinine is confirmed.

How to cite this article:
Sayed Zeid AS, Sayed SS. A Comparative Study of the Use of Dexamethasone, N-acetyl Cysteine, and Theophylline to Ameliorate Renal Ischemia–Reperfusion Injury in Experimental Rat Models: A Biochemical and Immuno-histochemical Approach. Saudi J Kidney Dis Transpl 2020;31:982-97

How to cite this URL:
Sayed Zeid AS, Sayed SS. A Comparative Study of the Use of Dexamethasone, N-acetyl Cysteine, and Theophylline to Ameliorate Renal Ischemia–Reperfusion Injury in Experimental Rat Models: A Biochemical and Immuno-histochemical Approach. Saudi J Kidney Dis Transpl [serial online] 2020 [cited 2020 Dec 2];31:982-97. Available from: https://www.sjkdt.org/text.asp?2020/31/5/982/301203



   Introduction Top


In clinical practice, nephrologists are faced with renal ischemia/reperfusion injury (IRI) and its consequences in several situations, for example, during renal transplantation, patients recovering after cardiopulmonary resuscitation, and newborns experiencing hypoxia as a result of prolonged or difficult delivery.

In IRI, the damage caused by the ischemia itself can be explained by the lack of oxygen, which leads to a shift to anerobic glycolysis. Hence, mitochondrial oxidative phosphorylation is impaired. The resulting ATP depletion not only accelerates cell death but also leads to an increase in intracellular calcium. Increased cytosolic calcium can promote apoptosis by activating phospholipase-A2. The increase in calcium also leads to the formation of reactive oxygen species (ROS). However, upon reperfusion, the damage incurred is even more because the normalization of oxygenation and pH leads to more increase in calcium and ROS.[1],[2]

Inflammatory mediators and adhesion molecules such as P-selectin and intracellular adhesion molecule-1 released from the proximal tubular cells (PTCs) allow the infiltration of neutrophils and leukocytes in the renal tissue after the ischemia.[3] The ROS, hypochlorous acid, and peroxynitrite, produced by catalytic conversion from the damaged epithelial cells, cause peroxidation of membrane lipids, damage of cell proteins and DNA, and, subsequently, apoptosis.[4]

The endothelial cells produce vasoconstrictor agents namely platelet-derived growth factor-B and endothelin-1.[2] Renin–angiotensin–aldosterone system is activated as a consequence of IRI with resultant constriction of renal vessels, thus enhancing oxidative stress and promoting apoptosis.[5]

The pathophysiology in IRI involves epithelial cell injury. The cells most commonly affected are those lining the PTCs in the juxtamedullary part of the cortex. The affected PTCs lose the microvilli present at the apical brush border. If the injury is more severe, the cells are detached. Sloughed cells and Tamm-Horsfall glycoprotein form casts that can block the tubular lumen. A more severe injury can result in necrosis and apoptosis of cells.[6]

Cystatin C (Cys C) is a low-molecular-weight protein that is produced by all nucleated cells. It is freely filtered by the kidney and is not secreted. It is actively reabsorbed and catabolized by the proximal tubules and hence, it is normally present in very little amounts in urine. Although plasma Cys C level is used to estimate GFR, urinary cystatin is mainly regarded as a marker of proximal tubular injury.[7]

The method applied in this study to simulate the IRI resembles the warm – but not the cold – ischemia seen in renal transplantation.

In this study, we aim to compare inexpensive readily available drugs known to target the mechanisms involving renal IRI namely inflammation, ROS, and vasoconstriction. Dexamethasone (Dex) was chosen for its known anti-inflammatory properties.[8] N-acetyl cysteine (NAC) was used being a potent anti-oxidant.[9] Theophylline (Theo) is a phosphodiesterase inhibitor and hence, a vasodilator.[10]


   Materials and Methods Top


Animals

This study included 105 adult male albino rats, 180–200 g body weight. They were housed in hygienic stainless steel cages and kept in clean well-ventilated room. They were fed standard chow diet and had free access to water. This experiment was performed in the animal house of Faculty of Medicine, Cairo University, according to the ethical guidelines for the care and use of laboratory animals.

Drugs

  1. Dex was purchased from Amriya Pharmaceuticals (Alexandria, Egypt)
  2. NACwas was purchased from Sedico Pharmaceutical Co.(6 October city, Egypt)
  3. Theo provided as Minophylline-N was purchased from Alexandria Co for Pharmaceuticals (Alexandria, Egypt).


Induction of ischemia–reperfusion acute kidney injury

IRI was induced in ketamine xylasine anesthetized animals. Mid-abdominal laparotomy was performed after the disinfection of abdominal wall by povidone iodine (Betadine). Kidneys were exposed, and renal pedicles were clamped for 40 min by smooth-edged surgical clamp. Reflow was visually confirmed.

Four milliliters of warm normal saline was given intra-peritoneally (IP) before abdominal closure to avoid ileus.[11]

Sham operation

The procedure was performed similar to the operation of IRI, but the renal pedicle ligation was omitted.[12]

Rats were randomly assigned to the following five groups: Group I –sham-operated (n = 5): they were subjected to sham operation. They received saline IP. Rats were sacrificed after 24-h.

Group II – nontreated group, IRI (n = 25): They received single IP injection of 1 mL saline. Group III – mDex, IRI + Dex (n = 25): they received single IP injection of Dex in a dose of 3 mg/kg.[13]

Group IV – NAC-treated group, IRI + NAC (n = 25): they received single IP injection of NAC in a dose of 500 mg/kg.[14] Group V Theo– IRI + Theo (n = 25): they received single IP injection of Theo in a dose of 15 mg/kg.[15]

Drugs were injected immediately after reperfusion. Five rats from groups II, III, IV, and V were sacrificed 1, 2, 4, 6, and 24 h after reperfusion. This was preceded by blood and urine sampling for biochemical study.

Histopathological study

At the time of sacrifice, abdominal incision was performed; the left kidney was dissected and fixed in formol saline 10% and then processed to obtain paraffin blocks. Paraffin sections of 5–7 μm thick were stained by hematoxylin and eosin (H and E), and immune-histochemical staining for Cys C.

Immuno-histochemical staining

Rabbit antiCys C primary antibody was purchased from USBiological Life Sciences (catalogue, 139994, Salem, MA, USA). Sections were deparaffinized and rehydrated, and then incubated with hydrogen peroxide block for 10–15 min to reduce the non-specific staining due to endogenous perioxidase to be followed by washing in phosphate buffer. The sections were incubated with the primary antibody for 1 h at a dilution of 1:400. This was followed by incubation with bio-tinylated goat anti-polyvalent antibody for 10 min at room temperature then washed in phosphate buffer. The sections were incubated with streptavidin peroxidase for 10 min and were then washed again in phosphate buffer. 3,3’-diaminobenzidine (DAB) plus chromogen was then added and left for 10 min, which was followed by counterstaining. Immunostaining was completed by the use of ultra-vision detection system anti-polyvalent, HRP/DAB (catalogue number TP-015-HD). Counter-staining was done using Mayer's hematoxylin (catalog number TA-060-MH). Ultra-vision detection system and Mayer's hematoxylin were purchased from Thermo Scientific Fischer, Chishire, UK. Positive immunoreactivity appeared as brown deposits.

Biochemical study

Blood samples, using capillary tubes, were drawn from retro-orbital veins for measurement of serum creatinine and CysC. Urine samples were collected to measure urinary Cys C. Serum and urinary CysC were measured using enzyme-linked immunosorbent assay (ELISA) technique. Rat CysC ELISA kit was purchased from MyoBioSource (catalogno. MBS763996, USA). Serum creatinine was quantitatively measured using commercially available assay kits.

Morphometric study

The area of CysC-positive immune staining in the sections from all kidney specimens was measured in all animals of the study. This was done in five non-overlapping fields at ×400 magnification in a field area of 7193.063 μm2 for every section using Leica Qwin 500C image analyzer computer system (England) present in Histology Department, Faculty of Medicine, Cairo University. Images were captured live on the screen from sections under a light microscope (Olympus BX-40, Olympus Optical Co. Ltd., Japan) with affixed video camera (Panasonic Color CCTV camera, Matsushita Communication Industrial Co. Ltd., Japan).


   Statistical Analysis Top


Data were presented as mean ± standard error mean. Differences between the variables in each group after 1, 6, and 24 h were compared by means of one-way ANOVA. A Tukey honestly significant difference (HSD) posthoc analysis was conducted to determine which of the means were significantly different from each other. Statistical significance was defined as P <0.05. Statistical analysis was performed with Statistical Package for the Social Sciences (SPSS) for Windows version 18.0 (SPSS Inc., Chicago, IL, USA). Data of area percent obtained from image analysis and levels of serum creatinine, serum, and urinary Cys C were analyzed.


   Results Top


Histopathological Results

Hematoxylin and eosin results

Examination of H and E-stained sections of Group II revealed the presence of cytoplasmic vacuoles involving the proximal tubular epithelium together with luminal tubular casts 1 h after IRI. This was persistent after 2 h associated with the presence of dark shrunken nuclei. Inflammatory cellular infiltration was also notable. After 4 h, cytoplasmic vacuoles became more evident. Multiple proximal tubules demonstrated the presence of casts in their lumina together with some sloughed epithelial cells, an appearance that was still evident 6 h after the IRI. Twenty-four hours after IRI, many proximal tubular epithelial cells possessed shrunken condensed nuclei and others were seen sloughed into the lumen [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d,[Figure 1]e.
Figure 1: Photomicrographs of kidney sections showing the histopathological changes in Group II IRI (a-e), Group III IRI + Dexa (f-j), Group III IRI + NAC (k-o), and Group IV IRI+ Theo. Arrow head – cytoplasmic vacuolization in proximal tubular epithelium, star – luminal casts, arrow – dark shrunken nuclei, wavy arrow – inflammatory cellular infiltration, curved arrow – sloughed epithelial cells, P – proximal convoluted tubules, G – glomerulus, C – congested capillaries, Thick arrow – sloughed epithelial cells and fragments in Bowman's space (p-t) Bars, 20 μm.

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Examination of kidney sections of Group III at 1 and 2 h demonstrated changes similar to those observed in the IRI group. After 4 and 6 h, most of the proximal tubules had relatively normal appearance where the lining cells had deeply acidophilic cytoplasm with large pale central nuclei. After 24h, the majority of the proximal convoluted tubules had relatively normal architecture [Figure 1]f,[Figure 1]g,[Figure 1]h,[Figure 1]i,[Figure 1]j.

Kidney sections of Group IV at 1 h had changes similar to those of Group II. Similar findings were observed after 2 and 4 h associated with peritubular as well as glomerular congestion. After 6 and 24h, tubular casts were evident in accordance to the previous observations [Figure 1]k,[Figure 1]l,[Figure 1]m,[Figure 1]n,[Figure 1]o.

Examination of kidney sections of Group V at 1, 2, and 4 h showed observations similar to those of the IRI group. By 6 h, similar findings were detected in addition to peri-tubular inflammatory cellular infiltration. Sloughed epithelial cells and fragments were also evident within the Bowman's space. Tubular epithelial vacuolization was evident after 24h, with some cells having shrunken nuclei. Epithelial sloughing was also detectable. Some of the proximal tubules demonstrated relatively normal architecture [Figure 1]p,[Figure 1]q,[Figure 1]r,[Figure 1]s,[Figure 1]t.

Immunohistochemical results

Kidney sections of Group I [Figure 2] and [Figure 3]a demonstrated cytoplasmic Cys C-positive immunostaining in the proximal convoluted tubules cells involving the whole cytoplasm.
Figure 2: Photomicrographs of kidney sections from sham group showing positive Cystatin C immuostaining (arrow) involving the cytoplasm of the epithelial cells of the proximal convoluted tubules (T). (Cystatin c immunostaining Bars [a] =50 μm, [b] =20 μm).

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Figure 3: Photomicrographs showing localization of cystatin C in kidneys by immunohistochemistry showing in: (a) Sham group: positive cystatin C immunostaining (arrow) involving the whole cytoplasm of the proximal tubular cells (T). (b) IRI group after 24 h: positive cystatin C immunostaining (arrow) involving only the apical portion of the proximal tubular cells. (c) Dexamethasone group after 24 h: apical localization of the positive immunostaining (arrow) as well as some reactions noted in the basal part of the cytoplasm (arrow head). (d) NAC group after 24 h: positive cystatin C immunostaining (arrow) involving the apical portion of the proximal tubular cells (T).

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In Group II [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d,[Figure 4]e, 1 h post IRI, the cytoplasmic immunostaining for Cys C was localized predominantly in the apical part of the cytoplasm, with some immune reaction observed in other parts of it. At 2 h post-IRI, the Cys C immunostaining was restricted to the PTC apices. The same localization was retained 4, 6, and 24h after the IRI [Figure 3]b. The least prevalence was at 24-h post-IRI where numerous tubules were negative for Cys C immuno-reactivity.
Figure 4: Photomicrographs showing localization of cystatin C in kidneys by immunohistochemistry in Group II IRI (a-e), Group III IRI + Dexa (f-j), Group III IRI + NAC (k-o), and Group IV IRI+ Theo (p-t). T proximal convoluted tubules, arrow – positive cystatin C immunostaining, G glomerulus, N tubules with negative immunostaining for cystatin C, star – tubules with minimal positive immunostaining, Bars, 50 μm.

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Examination of kidney sections from Group III (IRI + Dex) [Figure 4]f,[Figure 4]g,[Figure 4]h,[Figure 4]i,[Figure 4]j 1 and 2 h post-IRI revealed Cys C immunostaining mostly at the apex of the cytoplasm in addition to involving other parts of the cytoplasm in many of the proximal tubules. Starting 4 h after IRI + Dex treatment, the positive immunoreaction appeared almost exclusively at the apical border of the cells, an appearance that persisted till after 24 h [Figure 3]c.

Group IV (IRI + NAC) [Figure 4]k,[Figure 4]l,[Figure 4]m,[Figure 4]n,[Figure 4]o kidney sections showed apical localization of positive Cys C immunostaining in the PTCs throughout [Figure 3]d.

Examination of the kidney sections of Group V (IRI + Theo) [Figure 4]p,[Figure 4]q,[Figure 4]r,[Figure 4]s,[Figure 4]t revealed a positive Cys C immunostaining mainly in the apices of the PTCs at 1 h. Subsequently, there was a decline in the prevalence after 2, 4 and 6 h reaching a point where only few tubules demonstrated positive reaction after 24-h. Occasionally positive immunostaining for Cys C was noted to involve the glomerulus especially after the 1sth.

Morphometric results [Table 1] and [Figure 5]
Figure 5: Charts showing the means of area percent of positive Cys C immunostaining, serum Cys C, urinary Cys C, and urinary Cys C after 1, 2, 4, 6, and 24 h from IRI in all the study groups.

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Table 1: Mean area % of cystatin C immune-histochemical staining, serum cystatin C, serum creatinine, and urinary cystatin C after 1, 2, and 6 h in all groups.

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There was a statistically significant difference in the expression of Cys C in the kidneys among the five groups 1 h after the IRI, F(4,20) = 3.921, P=0.017. Posthoc comparisons using the Tukey HSD test revealed that the mean area % for the IRI group [M = 8.92, standard deviation (SD) = 3.35, 95% confidence interval (CI) = 4.76, 13.07] was significantly lower than the Theo group (M = 15.29, SD = 4.75, 95% CI = 9.38, 21.19) P= 0.016. The mean area % across the other groups was not significantly different.

Six hours after the IRI, there was a significant difference between the groups; F(4,20) = 14.36, P <0.001. Posthoc analysis revealed that the mean area % for the IRI group (M = 3.7, SD = 0.65, 95% CI = 2.89, 4.5) was significantly lower than that of the Dexa group (M = 10.27, SD = 1.69, 95% CI = 8.16, 12.38), the NAC group (M = 10.4, SD = 3.09, 95% CI = 6.2,13.87), and the control group (M = 11.05, SD = 1.5, 95% CI = 9.19, 12.9). The mean area % was not significantly different between the IRI and the Theo group (M = 7.07, SD = 0.97, 95% CI = 5.86, 8.27) (P = 0.52). However, the mean area % of the Theo group was significantly lower than the control group (P = 0.016). The rest of the comparisons of the mean area % at 6 h across all the groups were not significant.

At 24h, there was a statistically significant difference between the groups F(4,20) = 31.269, P = 0.000. Post hoc analysis revealed the mean area percent of Cys C staining was significantly higher in the Dex, NAC, and control groups compared to the IRI and the Theo groups. There was no significant difference in the mean area percent between the Theo and the IRI groups and, on the other side, there was no significant difference between the means in the three other groups (Dex, NAC, and control).

Biochemical Results [Table 1], [Table 2] and [Figure 5]
Table 2: Result of the ANOVA in the four variables (Cystatin C immunostaining area, serum Cystatin C, serum creatinine, and urinary Cystatin) among the five groups.

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Urinary cystatin C

Regarding the urinary Cyst C levels, there was a significant difference among the groups at 1 h. Post hoc analysis revealed that the levels were higher in the IRI group compared to the other groups. The mean levels of the NAC and the control groups were comparable and both were significantly higher than those of the Dex and Theo groups.

At 6 h, urinary Cyst C levels were significantly higher in the IRI compared to the other groups. The levels were comparable among the three drug groups (Dex, NAC, and Theo) and all were significantly higher than the control group but significantly lower than the IRI group.

At 24 h, the levels were significantly higher in the IRI group compared to the other groups. The mean level of the Dex group was significantly higher than the NAC group (Dex mean = 2.3 ± 0.187, NAC mean = 1.78 ± 0.259,

P = 0.03). However, it was not significantly higher than that of the Theo and control groups (Theo mean = 2.05 ± 0.218, control mean = 1.84 ± 0.114, P =0.54 and 0.065, respectively). The mean levels were comparable among the NAC, Theo, and control groups.

Serum creatinine

One hour after the IRI, the mean serum creatinine was significantly lower in the NAC group compared to the IRI, Dex, and Theo groups. There was no significant difference between control and the NAC groups (NAC mean = 0.116 ± 0.011, control mean = 0.156 ± 0.015, P = 0.139). The mean serum creatinine for the IRI group was comparable to that of the control (IRI mean = 0.186 ± 0.051, P = 0.375).

After 6 h, mean creatinine was significantly higher in the IRI group compared to all the other groups. The mean was significantly lower in the Dex group compared to the NAC and Theo groups, but it was still significantly higher than that of the control group. The mean creatinine levels of the Theo and NAC groups were comparable.

After 24 h, the mean serum creatinine was significantly higher in the IRI group (IRI mean = 1.07 ± 0.083) compared to all the other groups (P = 0.000 compared to each group separately). The mean was significantly lower in the Dex group compared to the NAC and Theo groups, but it was still significantly higher than that of the control group (Dex mean = 0.388 ± 0.016, NAC mean = 0.59 ± 0.016, Theo mean = 0.64 ± 0.016, and control mean = 0.156 ± 0.015, P= 0.000 compared to each group separately). The mean serum creatinine of the Theo and NAC groups was comparable (P = 0.312).

Serum cystatin C

One hour after the IR injury, the mean serum Cys C level was significantly higher in the IRI group compared to all the other groups. This was followed by the mean serum Cys C level of the Dex group, then the NAC group. The mean of the Theo group was the lowest and was comparable to that of the control group.

Six hours after the injury, Cys C was highest for the IRI group compared to all the other groups. The mean of each of the drug groups was significantly higher than that of the control group. There was a significant difference in the means of the drugs (Dex> NAC>Theo).

Twenty-four hours after the injury, the mean Cys C level of the IRI group was significantly higher than that of all the other groups (IRI mean = 12.92 ± 0.13). Post hoc analysis against each of the other groups revealed a significant difference (P = 0.000 in each case). The 2nd highest Cys C levels were observed in the NAC group (mean = 8.06 ± 0.114), which also was significantly higher when compared to each of the other groups namely the Theo, Dex, and control groups) separately (P = 0.000 in each case). The Theo group ranked 3rd (mean Cys C = 6.96 ± 0.27), which was significantly higher than the means of the Dex and control groups. Dex group was the one with the lowest Cys C levels (mean = 5.92 ± 0.19), but it was significantly higher than the mean of the control group (2.03 ± 0.033, P = 0.000).


   Discussion Top


In our study, there was a notable rise in the levels of serum creatinine, Cys C, and urinary Cys C in the IRI group along with a decrease in the area % of Cys C immunostaining following the ischemia–reperfusion insult. The change in serum creatinine at 1 h after the IRI was less noticeable in the IRI group compared to the control group. However, the rise in serum and urinary Cys C occurred early at 1 h and was significant compared to the sham group. The levels of serum creatinine and Cyst C and urinary Cyst C were significantly higher in the IRI group at 6 and 24 h compared to the sham group. These results highlight the notion that serum Cys C is a more sensitive marker of the change occurring in the GFR compared to serum creatinine. They also demonstrate that the biochemical effect of the damage occurring in the PTCs –namely the rise in the level of urinary Cys C– appears early on after the IR insult. In our study, we used the crude levels of urinary Cys C to avoid the possibly misleading results of urinary Cys C/creatinine ratio as many studies have pointed out that the use of urinary creatinine during acute kidney injury (AKI) is not prudent as it is continuously changing.[16],[17] In order to assimilate the changes occurring in the distribution of the Cys C immunostaining after the IR insult, a review of the renal handling of this protein is warranted. Cys C is normally reabsorbed by the brush border of the PTCs, thus rendering its level in urine under normal circumstances minimal. In case of damage to PTCs, this reabsorption is impaired and consequently its urinary level rises. These findings are further corroborated by other studies.[7],[18],[19] The urinary levels of Cys C were found in one study to be high in a glomerular disease – anti-GBM glomerulonephritis – in rats.[20] Furthermore, our results show a significant difference in the area % denoting the immune staining for Cys C in the IRI group at 6 and 24 h compared to the control group. The immune staining for Cys C was much lower in the IRI group compared to the sham group. In other words, the more damage there is to the PTCs, the less likely they are able to reabsorb the Cys C and as a result Cys C is less likely to show-up inside the PTCs cytoplasm. Interestingly, there was a difference in the site of the Cyst C deposits inside the PTC cytoplasm. While in the sham group the deposits were located allover the cells denoting successful reabsorption and transport, they were more confined to the area of the brush border in the IRI group, possibly denoting a halt in the mechanism responsible for its intracellular transport. It is known that the endocytic apparatus of the PTCs formed by the megalin and cubulin receptors is responsible for the reabsorption of many of the filtered proteins including vitamins, hormones, albumin, and Cys C.[21],[22] Cubilin is a peripheral membrane protein that is similar to the intrinsic factor-B12 receptor of the small intestine.[23] It has been suggested that megalin mediates the endocytosis and transport of cubilin because it has been demonstrated that in megalin knockout mice, transferrin accumulates at the luminal border and is not internalized.[24] However, though the histological localization of transferrin in the latter study matches that of Cys C in our results – which would imply a role for megalin in intracellular trafficking, the case for Cys C is rather more complicated. In a study that measured the levels of urinary Cys C after IRI, the binding of purified human Cys C to megalin and cubilin using surface Plasmon resonance was confirmed. Megalin-deficient mice not exposed to IRI showed high urinary levels of Cys C. Urinary excretion of Cys C was increased following the IRI, however histological expression of megalin using immunohistochemistry was not reduced.[25] This finding may suggest that the function of megalin or the turnover of internalized vesicles is negatively affected in the case of IRI rather than the physical expression of megalin itself.

As previously outlined, the mechanism underlying IRI involves the damaging effect of the high amounts of ROS released, the intense vasoconstriction that follows, and the inflammatory cascade that results in cell death by several pathways. Each of these mechanisms was separately targeted by a drug in our study.

In our study, the use of NAC immediately following the IRI resulted in a milder impairment of GFR. This is evidenced by a significantly lower rise in the levels of serum creatinine and Cys C in the NAC group compared to the IRI group at 1, 6, and 24 h (P <0.05). The negative effect that IRI had on the PTC function was milder as reflected by the lower levels of urinary Cys C in the NAC group compared to the IRI group at 1, 6,and 24 h (P <0.05). The immunostaining for Cys C was significantly more evident in the NAC group compared to the IRI group at 6 and 24 h. It is worth noting that in this group more vascular congestion was seen in the sections examined.

NAC is known to have anti-oxidant properties because it increases the level of endogenous glutathione (GSH). The latter is a substrate for the ROS scavenging enzymes and therefore, has a role in the regulation of apoptosis.[9] It has been suggested that NAC also has an anti-inflammatory role because it inhibits the transcription factors activator-protein1 and nuclear factor (NF)-κB which are known pro-inflammatory mediators.[25] NAC also has a vasodilatory effect probably mediated by its action on voltage-gated potassium channels and by affecting the level of intracellular calcium.[26],[27] The latter effect could explain the vascular congestion revealed in the NAC group in our study.

In the hope that these properties could be of clinical benefit, numerous studies have examined the use of NAC to prevent or ameliorate contrast-induced AKI (CI-AKI) both in rats and humans.[28],[29] However, there was a great heterogeneity in the results of these studies. Recent systematic reviews that evaluated trials addressing this question yielded inconsistent results and none ranked NAC high up on the list of drugs that were successful in preventing CI-AKI.[30],[31] The KDIGO AKI guidelines (2012) recommend the use of oral NAC for the prevention of CI-AKI on a level 2D evidence.[32] Experimental studies done in rats that used NAC to ameliorate AKI caused by IRI differ from ours in some points. In the study done by Cusumano et al in 2015, the groups used were compared regarding the effect the drugs – namely NAC, atorvastatin, and atorvastatin + NAC had on the levels of the enzymes myeloperoxidase, catalase, glutathione peroxidase, and superoxide dismutase along with the degree of tubular damage evident histologically.[33] NAC increased the levels of glutathione peroxidase and superoxide dismutase but failed to prevent tubular damage brought about by the IRI. That study focused on the analysis of the antioxidant enzymes involved and not on the functional outcome of the kidney. In the study done by Sen et al in 2014 which also used NAC in rat models of IRI, the best functional outcome estimated by the levels of serum creatinine, tumor necrosis factor (TNF)-alpha, and NGAL was seen when both NAC and dexpanthenol were used. However, in that same study, the use of NAC alone had a positive impact compared to the IRI group.[34] Another similar study done by Zhang et al in 2014 revealed comparable results.[35] In that study, the use of NAC not only resulted in lower serum creatinine levels but also lower levels of malondialdehyde – the end product of lipid peroxidation – and of apoptotic cells detected by TUNEL staining [terminal deoxy-nucleotidyl transferase (TdT) dUTP nick-end labeling assay]. Though these studies agree with ours on the benefit of using NAC in preventing renal IRI, all those studied rats were pretreated with NAC, while in ours, it was given post IRI. Our study revealed that NAC still has some benefit if given immediately post IRI, which means it can be used in the case of an unplanned IRI, for example, post CPR.

Theo was the second drug used in our study. In the Theo group, the rise in the levels of serum and urinary Cys C at 1, 6, and 24 h was significantly less than that occurring in the IRI group (P <0.05). This positive effect for the use of Theo was also reflected in the lower levels of serum creatinine in this group at 6 and 24-h compared to the IRI. Results of the immunostaining for Cys C concurred with the biochemical values to some extent where the mean area % was significantly higher than that of the IRI group at 1 h (P = 0.016) and at the same time the area % was significantly lower than the sham group at 6 and 24 h (P = 0.016 and P <0.001, respectively). The mean area % of the Theo and IRI groups was comparable at 6 and 24 h. The hypothesis behind the use of the phosphodiesterase inhibitor – Theo – is that it has been known for decades to inhibit tubulo-glomerular feedback response to changes in NaCl concentration. This is mediated by blocking the A1 adenosine receptors.[10] This action means that it can counteract the intense vasoconstriction occurring in cases of IRI. The presumed benefits of Theo were tested in several trials, for example, to prevent CI-AKI in humans with mixed results.[30],[36] In a study that explored the use of Theo to safeguard against renal injury in case of pneumoperitoneum in an experimental rat model, the results were not encouraging. In that study, while the levels of serum Cys C and apoptotic index by TUNEL were lower in the Theo group, the creatinine levels, the total antioxidant status, and total oxidant status levels were higher in the Theo group.[15] In another similar experimental study, the creatinine levels along with the levels of other markers for injury (NF-κB, TNF-α, monocyte chemo-attractant protein-1, macro-phage inflammatory protein-2, and intercellular adhesion molecule-1 mRNA) were all lower in the Theo group compared to IRI group.[37]

In the Dex group, our results show an advantage for its use with this group having lower serum and urinary Cys C levels and serum creatinine levels than the IRI group. The findings are corroborated by the Cys C immunostaining where the values for the area % were significantly higher than those of the IRI group and were comparable to the sham group at 6 and 24 h (sham at 6 = 11.05 ± 1.49, Dex at 6 = 10.27 ± 1.69 P = 0.956, and Dex at 24 = 9.36 ± 1.26, P = 0.244). Dex was selected in this study because this glucocorticoid has the highest anti-inflammatory and longest duration of action. The actions of glucocorticoids are mediated by the glucocorticoid receptor which is acytoplasmic protein that consists of a central DNA binding domain, a N-terminal domain, and a C-terminal ligand binding domain that can bind co-regulators. Binding of the hormone to the receptor allows it to bind chromatin inducing the expression or suppression of target genes.[8] All subtypes of T cells are affected by glucocorticoids. Glucocorticoids also affect the cytokines that guide the differentiation of T-cells into their different types. Glucocorticoids induce apoptosis of T lymphocytes by both the intrinsic and the extrinsic pathways. The earlier is usually triggered by pro-apoptotic proteins, for example, Bid, Bad, and Blk that activate the pro-apoptotic Bak and bax and inhibit the anti-apoptotic Bcl-2, Mcl, and Bcl-X. In the latter pathway, caspase-8 is directly recruited followed by caspase-3, and this triggers apoptosis.[38] Glucocorticoids also affect the action of many kinases and consequently the function of the lymphocytes. Regarding the glucocorticoid action on macrophages, they interfere with the intracellular signaling responsible for the transcription of pro-inflammatory cyto-kines genes. Glucocorticoids affect the MAP kinases; they suppress the phosphorylation of JNK and p38 MAP.[39] The production of pro-inflammatory interferon gamma, interleukin (IL)-alpha, and IL-beta is suppressed both at the mRNA and the protein level.[40] Contrary to their actions on macrophages and lymphocytes, glucocorticoids tend to prevent the apoptosis of neutrophils and to prolong their survival also by modulating the transcription of Bcl-2 family of proteins.[41]

In an experimental study that tried to explore the Dex effect in IRI, it was found that it probably mediates its protective effect in this case by inhibiting the MAPK-ERK activation (mitogen-activated protein kinase-extracellular signal-regulated kinase), hence causing down-regulation of the NF-κB mediated inflammation. Dex was also found to lower the IRI-caused rise in the levels of TNF-αlpha, IL-6, and IL-1β.[42]In another study, Dex was found to up-regulate BcL-xl, downregulate Bax, inhibit caspase 9 and 3 activation, and consequently inhibit PTCs apoptosis.[13] A third study in rat models proved the beneficial effect of Dex in IRI and suggested that its beneficial effect could be due to the attenuation of PI3K/AKT (phosphoinositide 3-kinase/serine-threonine kinase AkT) signaling.[43]

Our study is unique in that it compared three drugs with proven benefits in IRI and further elaborated the results by Cys C immuno-staining. Dex was superior to both NAC and Theo at 24 h as the mean level of serum creatinine was significantly lower in this group compared to the NAC, IRI, and Theo groups (P <0.001). The urinary Cys C level at 24 h for Dex was significantly higher than that of the NAC group but comparable to that of the Theo group (Dex 2.3 ± 0.18, Theo 2.05 ± 0.21 P = 0.54) (Dex 2.3 ± 1.8, NAC 1.7 ± 0.25 P = 0.03). Caution should be exercised while interpreting the urinary Cys C values as it was measured in random samples and the urine output in these cases is affected by the GFR. The area % of immunostaining for Cys C was more in the Dex group at 24 h compared to the Theo group (Dex 9.36 ± 1.2, Theo 5.39 ± 1.3, P = 0.001), but it was similar to that of the NAC group (NAC 9.15 ± 1.1, Dex 9.36 ± 1.2, P = 0.999).

In conclusion, our study revealed a clear benefit for the use of Dex to ameliorate IRI over NAC and Theo if used immediately following the insult. The effect is evident 24 h after the insult. The damage occurring in renal IRI is more evident in the PTCs and it affects both structure and function of these cells. The role of serum Cys C as an early marker of AKI compared to serum creatinine is highlighted.


   Statement of Ethics Top


The study conforms with international standards guiding the use of animals and has followed the guidelines of the animal house of faculty of medicine, Cairo University.

Conflict of interest: None declared.



 
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Correspondence Address:
Ahmed Salaheldin Sayed Zeid
Department of Pediatrics, Pediatric Nephrology Unit, Faculty of Medicine, Cairo University, Cairo 12613
Egypt
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DOI: 10.4103/1319-2442.301203

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    Abstract
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   Statement of Ethics
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