Menoufia Medical Journal

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 30  |  Issue : 1  |  Page : 271--277

Light and electron microscopic studies of chronic renal failure using an adenine rat model


Fatma El-Nabawia A El-Safti, Shireen A Mohammed 
 Anatomy and Embryology department, Faculty of medicine, Menoufia University, Egypt

Correspondence Address:
Shireen A Mohammed
Anatomy and Embryology department, faculty of medicine, Menoufia University. 4 Talat Harb Street, Shibin el-koum, 32511, Menoufia
Egypt

Abstract

Objectives This work aimed to study chronic renal failure (CRF) in an adenine induced rat model using light microscopic, electron microscopic examinations in addition to morphometric studies. Past studies of adenine induced rat model of CRF did not emphasize on the electron microscopic changes that accompany CRF. Background Long term feeding of adenine to rats produced metabolic changes resembling chronic renal failure in humans. Among the changes happened were loss of weight and accumulation of uremic toxins (urea and creatinine). The pathological findings in the kidneys revealed glomerular and tubulo-interstitial damage. In the current work, we stressed on the changes that happen in the ultra-structural level. Methods Thirty adult male albino rats were divided into 2 groups: Group (I) ten rats, kept without any treatment, and sacrificed after 4 weeks. Group II (chronic renal failure induced group) twenty rats, received adenine powder daily (0.75% w/w for 4 weeks in food i.e., a daily adenine dose of 0.075 grams was added to 10 grams of food for each rat). Initial and final weights were recorded. Blood samples were collected for kidney function tests. Kidney sections were prepared for light and electron microscopic examination. Morphometric and statistical analysis were done. Results In comparison with the control group, the CRF induced group showed a significant decrease in their final body weight (P < 0.001), significant increase in the urea and creatinine levels (P < 0.001) and significant increase in collagen fiber surface area. Conclusion Long term feeding of adenine exerted a marked inflammatory, fibrotic and apoptotic accelerating effect on both the glomeruli and the tubules.



How to cite this article:
El-Safti FNA, Mohammed SA. Light and electron microscopic studies of chronic renal failure using an adenine rat model.Menoufia Med J 2017;30:271-277


How to cite this URL:
El-Safti FNA, Mohammed SA. Light and electron microscopic studies of chronic renal failure using an adenine rat model. Menoufia Med J [serial online] 2017 [cited 2019 Sep 17 ];30:271-277
Available from: http://www.mmj.eg.net/text.asp?2017/30/1/271/211477


Full Text



 Introduction



Chronic renal failure (CRF) is a global health problem; its incidence appears to be increasing. Estimates of the global burden of the diseases report that, kidney diseases are responsible for about 830 thousand deaths annually making them the 12th highest cause of death [1]. In most of the Middle East countries, the published population based studies for the incidence and prevalence of chronic renal failure are still lacking. However, Egypt has the 2nd highest prevalence of chronic renal failure estimated by 669 patients per million populations [2].

Animal models of renal failure are important tools to study pathophysiological events in kidney disease that allows translational studies aiming to improve management of CRF patients.

 Material



Adenine

A product of Sigma-Aldrich (Munich, Germany), was available in the form of white powder and was given to the rat in food at a concentration of 0.75% w/w [3],[4],[5] i.e., a daily adenine dose of 0.075 grams was added to 10 grams of food for each rat. A white sheet of paper was provided as bedding for each cage; any remaining food was collected and re-weighed and recorded.

Thirty young adult male albino rats of Sprague-Dawley strain, weighing between 150–180 gm. (6 months old) were obtained from Tanta. Egypt. They were fed standard diet. Ethical approval was obtained from Faculty of medicine, Menoufia University. Animals were divided into five main groups:

Group I (control) consisted of ten rats, kept without any treatment, sacrificed after 4 weeks.Group II (CRF induced) twenty rats, received adenine powder daily (0.75% w/w for 4 weeks in food) then sacrificed [4].

 Methods



Clinical assessment

Initial and final weights were recorded [5].

Final blood samples were taken for estimation of plasma urea and creatinine levels from the retro-orbital plexus [6],[7].

Light microscopic study

A small piece of the left kidney was fixed for 24 h in 10% neutral buffered formalin then dehydrated in ascending grades of alcohol, cleared and embedded in paraffin. After deparaffinizing, the 3–5 microns thick tissue sections were cut by microtome and were subjected to: Hx and E for routine histological examination, masson trichome stain for detection of collagen fiber deposition and toluidine blue for the semi-thin sections [8],[9].

Electron microscopic study

The right kidney was cut into transverse sections 2–4 mm in diameter. Ultrathin (70–80 nm) sections were stained with lead citrate and uranyl acetate [9] and were mounted in formvar-coated nickel grids then inspected and photographed with JEOL JEM-1230 electron microscope at Faculty of Science, Menoufia University.

Morphometric study

Ten different H and E and Masson's Trichome stained sections from ten different rats were examined in each group for the percentage of collagen fiber surface area (area stained blue) within the glomerulus, in the parietal layer of the Bowman's capsule and the peritubular area) [10].

Morphometric study was also done for assessment of the diameters of the renal corpuscle, the glomerulus and the Bowman's space.

The data were obtained using image analyzer software, Image J analyzer version 1.43o8, National Institutes of Health, USA and Digimizer version 4.3.5, MedCalc software. This was done in Anatomy and Embryology Department, Menoufia University.

Statistical analysis

Statistical Analysis was performed for the initial and final weights, final plasma urea and creatinine and for the morphometric results. Results were collected, tabulated and statistically analyzed by statistical package for social science (SPSS) version 20 on IBM compatible computer. Results were expressed as mean (x) ± standard deviation (SD). Analysis of variance (ANOVA) test for significance followed by Mann- Whitney test [11].

A value of P < 0.05 was considered significant. 6 rats were used for each.

 Results



General results

Comparing with control group, animals of chronic renal failure (CRF) induced group (Group II) showed marked decrease in their physical activity, decreased food intake and excessive wetting of the wood shaving. The manifestations started from the second week of the experiment and deteriorated till the fourth week.

Body weight

[Table 1] and [Histogram 1] shows the body weight changes among the studied groups. The CRF induced group showed significant (P < 0.001) decrease in their final body weight when compared to their initial ones.{Table 1}[INLINE:1]

Biochemical results

[Table 2] shows the kidney function tests result among the studied groups. When compared to the control group, CRF induced group showed significant (P < 0.001) increase in their final plasma urea and creatinine levels.{Table 2}

 Histological Results



1- Light microscopic study

Control group: The renal tissue consisted of an outer cortex and an inner medulla under a thin renal capsule. The renal cortex was formed of sparse globular masses; the renal corpuscles and different size renal tubules. The medulla consisted of only collecting tubules with thin wall and wide lumen.

The renal corpuscles consisted of globular lobulated capillary tuft; the glomerulus which projected into the glomerular capsule (Bowman's capsule). The outer layer of the capsule surrounded the glomerulus as the parietal layer (capsular epithelium), which then reflected into the glomerular capillaries as visceral layer of the Bowman's capsule (glomerular epithelium). The two layers were separated by the capsular space (Bowman's space). The parietal layer consisted of flat cells of simple squamous epithelium with flattened nuclei. The visceral layer consisted of the visceral epithelial cells, the podocytes. Masson Trichome stained kidney sections showed minimal amount of collagen fiber deposition under the capsule, in the renal corpuscles mainly within the glomerular basement membrane, parietal layer of the Bowman's capsule and in between the renal tubules [Figure 1],[Figure 2],[Figure 3].{Figure 1}{Figure 2}{Figure 3}

Chronic renal failure induced group: H and E stained kidney sections revealed major pathological changes affected mainly the renal cortex with minimal affection of the renal medulla. Distorted architecture of the renal cortex with multiple diffuse empty spaces within it was clearly noticed. Also, accumulated peritubular inflammatory cells areas of extensive hemorrhage could be seen in the cortex. Thick wall congested dilated blood vessels with vacuolation of their smooth muscle wall could be seen.

The renal cortex showed distorted renal corpuscles. Some corpuscles showed condensation, shrinkage with widening of their Bowman's space, while others showed complete obliteration of their Bowman's space. The lining epithelial cells of both proximal and distal convoluted tubules showed degenerative changes in the form of vacuolated cytoplasm (hydropic degeneration). Extruded cell debris into the tubular lumen and intraluminal cast were evident. Masson Trichome stained kidney sections showed extremely large amount of collagen fiber deposition beneath the capsule, large amount in the renal corpuscles mainly within the glomerular basement membrane, parietal layer of the Bowman's capsule, (capsular epithelium), large amount in between the renal tubules and huge amount within the wall of blood vessels which appeared as tense blue color [Figure 1],[Figure 2],[Figure 3].

Electron microscopic study

Control group: Podocytes had several cytoplasmic extensions (the primary processes) and numerous organelles, these processes wrapped around the glomerular capillaries giving rise to numerous secondary foot processes called pedicles. The lining endothelium was fenestrated. The proximal convoluted tubules had large oval or rounded euchromatic nucleus with peripheral heterochromatic nucleolus. Basally located closely packed numerous mitochondria could be noticed. They were of two shapes, elongated (rod shaped) mitochondria and rounded ones. Long closely packed apical microvilli projecting into the lumen forming a brush border could be seen. The distal convoluted tubule rested on an even basal lamina with unremarkable basal infoldings or striations (notched arrow). Their mitochondria appeared elongated and parallel to each other and to the longitudinal axis of the cell. The mitochondrial lamellar cristae could be clearly noticed. The nucleus appeared large, oval or rounded and euchromatic with central hyperchromatic nucleolus [Figure 4] and [Figure 5].{Figure 4}{Figure 5}

Chronic renal failure induced group: Ultra-thin sections revealed distortion and discontinuity of the primary processes of the podocyte. While the secondary foot processes (pedicles) lost their finger like projection shape and became fused together with effacement. The distal convoluted tubules showed apoptotic changes. The nuclei were fragmented with apoptotic body formation. The proximal convoluted tubules showed ballooning of its mitochondria with loss of their lamellar cristae. [Figure 4] and [Figure 5].

Morphometric results

Percentage of surface area of collagen fiber deposition

Comparing to the control group, the CRF induced group showed a significant increase (P < 0.001) in percentage of surface area of collagen fiber deposition.

The protected group (Group IV) showed a non-significant increase (P > 0.05) in the percentage of surface area of collagen fiber deposition when compared to the control group.

The treated group (Group V) showed a highly significant increase (P < 0.001) in the surface area percentage when compared to the control group [Histogram 2].[INLINE:2]

Renal corpuscle measurements

Comparing with the control group, the CRF induced group (Group II) showed a significant decrease (P < 0.05) in the diameter of the renal corpuscle (P < 0.001). The renal glomerulus showed either a significant decrease (Labeled as group III in the table) (P < 0.001) or a non-significant decrease (P > 0.05) (Labeled as group III in the table). However, Bowman's space showed a significant increase (Labeled as group III in the table) or decrease in its diameter (Labeled as group III in the table) [Table 3].{Table 3}

 Discussion



Long term feeding with adenine provides an animal model of uremia, from which, phases and various mechanisms of chronic renal failure can be assessed [11].

The model of adenine-induced CRF was originally developed by Yokozawa

[12] who discovered that when adenine was present in excess, it became a significant substrate for xanthine dehydrogenase, which could oxidize adenine to 2, 8 dihydroxyadenine (DHA). Both adenine and DHA had very low solubility; they precipitated in the renal tubules and led to tubular occlusion.

In the present study, the elevated plasma urea and creatinine levels of the CRF induced group judged the occurrence of CRF as mentioned by Peng et al. [13]. These uremic toxins might be the cause of the significant reduction of body weight, in the CRF group, or due to loss of appetite as mentioned by Burns et al. [11].

In the present work, two forms of mitochondrial damage were observed in PCTs of the CRF induced group. The ballooned as well as the destructed dense shrunken mitochondria, the former might be due to failure of energy (due to impaired oxidative phosphorylation) dependent ion and cation pump respectively [14]. This might lead to accumulation of sodium with subsequent entry of water [15],[16]. The latter might be due to condensation of matrix proteins [17].

The current study showed the presence of interstitial fibrosis, this was evidenced by the significant increase of collagen fiber deposition in the interstitium. Fibrosis might suggest the presence of chronic inflammation which was emphasized by the presence of peritubular inflammatory cells and fibroblasts. This was in accordance with Helmy et al.[16] who added that fibroblasts secreted excess extracellular matrix and this in turn, destroyed the normal interstitial architecture of the kidney.

Morover, the significant increase of collagen fiber deposition within the glomerulus indicated the occurance of glomerulosclerosis due to chronic inflammation which was emphasized by the mesangial matrix hypercellularity with overproduction of extracellular matrix. Elenkov et al. [17] confirmed the inflammatory theory in CRF by the significant increase in lipid peroxidation end product MDA (Malondialdehyde) and C- reactive protein (CRP) inflammatory marker.

In this study, intraluminal exfoliation of renal tubular epithelial cells might be due to ischemia which was also induced by oxygen free radicals [18]. This was consistent with destruction of the apical microvilli of PCTs [4]. The destructed microvilli might be due to lipid peroxidation and inflammation [19]. The intraluminal casts might be due to destructed microvilli in the form of clusters or the exfoliated necrotic epithelial cells which provided the suitable matrix for cast formation. This was in agreement with Kays et al. [20].

The present study showed an evidence of apoptosis detected in CRF induced group. Wiegele et al. [21] attributed the apoptosis to ischemia caused by glomerulosclerosis. However, Goilav [22] explained that apoptosis was triggered inside the mitochondria by activation of the caspase cascade pathway. While Constantinou et al. [23] postulated a caspase independent pathway as he found that the mitochondria released apoptosis inducing factor which triggered apoptosis resulting in subsequent tubular atrophy.

The present work showed that the Bowman's space was either widened or obliterated; this indicated early and late stage of the disease consecutively widening of the capsular space might be due to shrinkage of the glomerular capillary tuft. Obliteration of the space could be due to mesangial cell hypercellularity with subsequent sclerosis [19].

In CRF induced group, we could prove the occurrence of two forms of cell death; necrosis and apoptosis. The former was represented by shrinkage, peripheral migration of chromatin and increased heterochromatin while the latter was by nuclear fragmentation and formation of apoptotic bodies as described by Helmy et al. [16].

 Conclusion



From the present study, we concluded that; chronic renal failure resulted in two forms of cell death, necrosis and apoptosis. Adenine feeding caused glomerular and tubule-interstitial damage, these changes mimic that happen with CRF in humans and this provide translational studies aiming to improve management of CRF patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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