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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 11
| Issue : 1 | Page : 23-33 |
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Does omeprazole, the proton-pump inhibitor, affects the structure of the kidney of male albino rats? Histological and laboratory study
Heba M Hussein, Nagwa El-Nefiawy, Haidy F Abdel Hamid, Marwa A Moneim
Department of Anatomy and Embryology, Faculty of Medicine, Ain Shams University, Abbasia, Cairo, Egypt
| Date of Submission | 29-Jan-2021 |
| Date of Decision | 01-Apr-2021 |
| Date of Acceptance | 17-Apr-2021 |
| Date of Web Publication | 04-Feb-2022 |
Correspondence Address:
Dr. Marwa A Moneim
Department of Anatomy and Embryology, Faculty of Medicine, Ain Shams University, P.O. Box 11381, Abbasia, Cairo
Egypt
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jmau.jmau_11_21
Introduction: This study was done to assess the injurious effects of omeprazole by an in vivo experimental study on rat kidneys. Materials and Methods: Forty-two adult male albino rats were divided into four groups: Control group (I) in which rats were not administrated any treatment. In Groups IIa, IIb, and IIc rats received daily oral omeprazole in dose of 0.75 mg per kg for 2, 4, and 6 weeks, respectively. At the end of the experiment, blood samples were collected for serum creatinine and blood urea nitrogen measurement. Then, animals were sacrificed, and kidney specimens were processed for paraffin blocks, sectioned and stained with H and E, Mallory trichrome and Periodic acid–Schiff, then examined by the light microscope. Stained sections and image analysis were used to count vacuolated cells, pyknotic nuclei, tubular casts, and area percent of collagen fiber deposition, and then, data were subjected to the statistical analysis. Results: Examination of omeprazole-treated groups showed injury of renal corpuscles, renal tubules, and vascular congestion with inflammatory cell infiltrate in renal interstitium. Thickening of basement membrane with deposition of collagen fibers was also detected. Statistically significant increase in the number of vacuolated cells, pyknotic nuclei, hyaline casts, and area percentage of collagen fiber deposition as compared with the control group was noticed, with deterioration of renal function tests. Conclusion: It was concluded that the long-term use of omeprazole resulted in structural damage of rat renal tissue associated with deterioration of renal function in a time-dependent manner.
Keywords: Kidney, omeprazole, pyknotic nuclei, serum creatinine, tubular casts
How to cite this article:
Hussein HM, El-Nefiawy N, Hamid HF, Moneim MA. Does omeprazole, the proton-pump inhibitor, affects the structure of the kidney of male albino rats? Histological and laboratory study. J Microsc Ultrastruct 2023;11:23-33 |
How to cite this URL:
Hussein HM, El-Nefiawy N, Hamid HF, Moneim MA. Does omeprazole, the proton-pump inhibitor, affects the structure of the kidney of male albino rats? Histological and laboratory study. J Microsc Ultrastruct [serial online] 2023 [cited 2023 Mar 31];11:23-33. Available from: https://www.jmau.org/text.asp?2023/11/1/23/337323 |
| Introduction | |  |
Proton pump inhibitors (PPIs) are widely used worldwide. They suppress gastric acid secretion. The standard clinical indications for the treatment with PPIs include gastroesophageal reflux disease, peptic ulcer disease, and erosive esophagitis. PPIs are also prescribed as a prophylaxis against peptic ulcer in combination with some medications such as steroids, nonsteroidal anti-inflammatory drugs, with antibiotics for eradicating Helicobacter pylori.[1]
PPIs inhibit gastric acid secretion through blocking gastric H, K-ATPase pump responsible for H + ions secretion into the gastric lumen. Several generations of PPIs are available in the market. All are similar in action with no evidence that one is more effective than another is. They differ in how they are broken-down by the liver and in their drug interactions, making the effect of some PPIs may last longer.[2]
PPIs have minimal side effects; however, high doses and long-term use may increase the risk of osteoporosis-related fractures of the hip, wrist, or spine; reduces absorption of vitamin B12 (cyanocobalamin); lower levels of magnesium with increased risk of heart attacks.[3]
Recent clinical observational studies claim that PPIs use is associated with an increased risk of acute kidney injury (AKI), incident of chronic kidney disease (CKD), CKD progression, or even end-stage renal disease (ESRD).[4]
The aim of this study was to show the effects of chronic use of the PPI “omeprazole” on the histological architecture and function of the kidney of male albino rats and to compare between the effects of omeprazole use after 2, 4, and 6 weeks in this regard.
| Materials and Methods | | |
Animals
Forty-two adult male albino rats 4–6 month old weighing about 200 g body weight were used in the study.
Experimental groups
Rats were divided into two groups as follows:
Group I (control group)
Subsequently subdivided into four subgroups (6 rats/subgroup):
- Subgroup Ia: rats were not given any treatments and were allowed free access to food and water (ad libitum)
- Subgroups Ib, Ic, and Id: rats were not given any treatments but were given an amount of water equivalent to that was used to dissolve the drug in Group II (1 ml)[5] daily for 2, 4, and 6 weeks, respectively, by means of gastric gavage.
Group II (omeprazole group)
Subsequently subdivided into three subgroups (6 rats/subgroup):
- Subgroup IIa: rats were given daily oral omeprazole dissolved in 1 ml of water by gastric gavage for 2 weeks
- Subgroup IIb: rats were given daily oral omeprazole dissolved in 1 ml of water by gastric gavage for 4 weeks
- Subgroup IIc: rats were given daily oral omeprazole dissolved in 1 ml of water by gastric gavage for 6 weeks.
Omeprazole was used in the therapeutic clinical dose.[6] The dose was then converted using Nair and Jacob equation into rat's dose (0.75 mg/kg per day).[7] It was purchased as omeprazole 40 mg capsules (Alexandria Co. for Pharmaceuticals, Alexandria, Egypt).
Domestic rats live about 2–3.5 years. Two weeks of a rat's life almost equal 1 year in human. In addition, 4 weeks rat life is roughly equivalent to 2.5 human years.[8]
Kidney function tests
Retrobulbar blood samples were collected from animals of the study groups at the start and before sacrificing the rats: after 2 weeks for Groups (Ia, Ib, and IIa), 4 weeks for Groups (Ia, Ic, and IIb), and 6 weeks for Groups (Ia, Id, and IIc). Blood samples were used to measure serum creatinine and blood urea nitrogen (BUN) and computed calculate estimated glomerular filtration rate (eGFR) using Cockcroft–Gault equation that dependent upon body weight and serum creatinine, age, and sex.
Specimen collection
At the end of the experiment (2 weeks for Groups Ia, Ib, and IIa, 4 weeks for Groups Ia, Ic, and IIb and 6 weeks for Groups Ia, Id, and IIc), animals were sacrificed using cervical dislocation. Kidney specimens were excised and fixed in 10% neutral formalin for 48 h, then were processed as paraffin blocks.
Histological examination
Serial paraffin sections of 5 μm thickness were cut and stained with H and E, Periodic acid–Schiff (PAS), and Masson's Trichrome.[9] Sections were examined using the Olympus light microscope (CX31) in the Anatomy Department, Faculty of Medicine, Ain Shams University and were photographed.
Morphometric study and image analysis
H- and E-stained sections were used to assess the following parameters:
- Number of tubular cells with vacuolation
- Number of tubular cells with pyknotic nuclei
- Number of tubules with hyaline casts
- Percent area of collagen fibers deposition.
Computer image analysis
Image J” computer image analysis software version 1.40 g- Olympus Stream. OM Digital Solutions Americas, Inc. 306 S New Street, Suite 110 Bethlehem, PA, 18015 was used to measure all these parameters per microscopic field. For each of the previous entries, measurements were taken from six microscopic fields per slide, six slides per rat, and six rats per group.
Counting the number of tubular cells with vacuolation and tubular cells with pyknotic nuclei per microscopic field was performed using the (×40) objective lens. The number of blood vessels per microscopic field, tubules with hyaline cast, and area percent of collagen fibers were done using the (×10) objective lens.
For measurement of area percentage per microscopic field used for quantitation of collagen fibers, images were splitted into Red- Green- Blue (RGB) stacks and then the red stack was chosen and adjusted to gray scale threshold to mark the stained areas with a red-colored binary mask. Then, the percent of these areas in relation to the microscopic field was calculated.
Statistical analysis
Statistical analysis was done using the SPSS software (Statistical Package for the Social Sciences software version 13.0, IBM® SPSS®-Egypt). One-way analysis of variance was employed to compare the means in different groups with each other. Bonferroni post hoc test was used to detect the significance between every two individual groups.
The significance of the data was determined by the probability (P. value). P > 0.05 was considered nonsignificant. P ≤ 0.05 was considered statistically significant and P ≤ 0.001 was considered highly significant.[10] Data were represented in tables and histograms, prepared by using MS Excel 2013.
Ethical consideration
All the experimental protocols were carried out in accordance with the guidelines approved by the Committee of Animal Research Ethics, Faculty of Medicine-Ain Shams University, which conforms to the Declaration of Helsinki, 2013.
| Results | | |
Histological results
Group I (control group)
Examination of sections of the rat kidney from the control group (Group I) showed normal renal architecture of the renal cortex made up of renal corpuscles and renal cortical tubules (proximal convoluted tubules [PCTs] and the distal convoluted tubules [DCTs]). The renal corpuscle was formed of many capillaries (containing red blood cells), their associated cells of the visceral layer of Bowman's capsule with distinct Bowman's space separating it from the parietal layer of Bowman's capsule, with mesangial cells and their matrix in between the capillary loops supporting it. The minimal amount of interstitial tissue is observed between the tubules [Figure 1]. | Figure 1: A photomicrograph of a section of the rat kidney from the control group (Group I) showing renal cortex with glomerulus (G) made up of many capillaries (containing red blood cells) and their associated cells of the visceral layer of Bowman's capsule (diamond arrow). The glomerulus is surrounded by distinct Bowman's space (arrow). The parietal layer of Bowman's capsule is made up of simple squamous epithelium (arrowhead). Notice the proximal convoluted tubule (P), the distal convoluted tubule (D) and peritubular blood vessels (V). Minimal amount of interstitial tissue is observed between the tubules (H and E, ×400)
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The renal medulla was divided into the outer and inner medulla (I. M). The outer medulla (O. M) made up of thin limb of loops of Henle lined by simple squamous epithelium, thick limb of loops of Henle lined by cuboidal epithelium, and collecting tubules lined by collecting tubule (CT). The I. M made up of the same structure with the absent of thick limbs of loop of Henle. The vasa recta, between medullary tubules, would be considered the continuation of efferent arterioles in the medulla [Figure 2]. | Figure 2: A photomicrograph of a section of the rat kidney from the control group (Group I) showing outer medulla with the thin limb of loops of Henle lined by simple squamous epithelium (double arrow), thick limb of loops of Henle lined by cuboidal epithelium (arrowhead). Collecting tubules lined by cuboidal epithelium (CT). Note the vasa recta, which represents continuation of efferent arterioles in the medulla (arrow) (H and E, ×400)
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With Masson trichrome staining, cortical renal architecture showed very thin blue color-stained collagen fibers at glomerular tuft basement membranes, parietal layer of Bowman's capsule, and around renal tubules; PCTs and DCTs [Figure 3]. | Figure 3: A photomicrograph of a cortical renal section of the rat from the control group (Group I) showing very thin blue color stained collagen fibers at; glomerular tuft basement membranes (g), parietal layer of Bowman's capsule (arrow) and around renal tubules; proximal convoluted tubule (double arrow) and distal convoluted tubule (arrowhead) (Masson trichrome, ×400)
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With PAS, the renal cortex showed the normal distribution of thin well-defined purple magenta color PAS reaction at the basement membrane surrounding renal corpuscle and cortical renal tubules (PCTs and DCTs) with stained brush border of PCTs. The glomerular basement membrane was thin well-defined formed by the endothelium of the capillary loops, their underlying basement membrane and the covering podocyte [Figure 4]. | Figure 4: A photomicrograph of a section of the rat kidney from the control group (Group I) showing renal cortex with the normal distribution of thin well-defined purple magenta color Periodic acid–Schiff reaction at basement membrane surrounding renal corpuscle and cortical renal tubules. Notice glomerular basement membrane (G), which is thin well-defined (arrow). Also, a thin well-defined basement membrane surround proximal convoluted tubules (arrowhead) with stained brush border (#) and distal convoluted tubules (double arrow) (Periodic acid–Schiff, ×400)
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Group IIa (2 week group)
Examination of sections of the kidney from rats treated with omeprazole for 2 weeks (Group IIa) showed the renal corpuscles with complete obliteration of Bowman's space, which was apparent hyper-cellular due to vascular congestion and inflammatory cell infiltrate. Some tubules revealed marked flattening and necrosis of the tubular epithelial lining with pyknotic nuclei and karyorrhexis up to complete disintegration and widening of their lumen [Figure 5] and [Figure 6]. | Figure 5: A photomicrograph of a kidney section from rats treated with omeprazole for 2 weeks (Group lla) showing renal cortex with some tubules revealing marked widening of their lumen (T) and flattening of their lining epithelium (arrow). In addition, pyknotic nuclei of the tubular epithelial lining are seen (double arrow). Note the glomerulus (G) showing complete obliteration of Bowman's space with apparent hypercellularity due to vascular congestion and inflammatory cell infiltrate (star) (H and E, ×400)
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 | Figure 6: A photomicrograph of a kidney section from rats treated with omeprazole for 2 weeks (Group lla) showing renal cortex with many patches of tubular necrosis (T). Nuclei of tubular epithelial lining reveal nuclear pyknosis (arrowhead), Karyorrhexis (double arrowhead) and complete disintegration (arrow) (H and E, ×400)
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With Masson Trichrome staining, the renal cortex showed distorted renal corpuscle and its surrounding tubules, with blue-colored collagen fibers around glomerular capillaries, and markedly thickened parietal layer of Bowman's capsule with loss of Bowman's space at this region and in tubular basement membranes [Figure 7]. | Figure 7: A photomicrograph of a kidney section from rats treated with omeprazole for 2 weeks (Group lla) showing renal cortex with distorted renal corpuscle and surrounding renal tubules. Notice blue colored collagen fibers around glomerular capillaries (arrowhead), and markedly thickened parietal layer of Bowman's capsule with loss of Bowman's space at this region (arrow), and in tubular basement membranes (double arrow) (Masson Trichrome, ×400)
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With PAS, the renal cortex showed apparently thickened parietal layer of Bowman's capsule, marked increase of the mesangial matrix. There were apparently thickened tubular basement membranes with lost brush border in almost most PCT, and PAS stained renal interstitial deposits in a wide edematous interstitium [Figure 8]. | Figure 8: A photomicrograph of a section of renal cortex from rats treated with omeprazole for 4 weeks (Group llb) showing apparently thickened parietal layer of Bowman's capsule (arrowhead), marked increase of mesangial matrix (arrow). Notice also apparently thickened tubular basement membranes (double arrow), lost brush border (#) in almost most proximal convoluted tubule (T), and Periodic acid–Schiff stained renal interstitial deposits in a wide edematous interstitium (double arrowhead) (Periodic acid–Schiff, ×400)
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Group IIb (4 weeks group)
Examination of sections of the kidney from the rats treated with omeprazole for 4 weeks (Group IIb) showed renal cortex with diffuse damage of renal corpuscles, some renal corpuscles were distorted, with lost Bowman's space and tight apposition of glomerular capillaries with their covering visceral layer of Bowman's capsule to the irregular outer parietal layer and apparent hypercellularity. Furthermore, abnormally shrunken glomerular capillary tuft could be seen in other glomeruli [Figure 9]. | Figure 9: A photomicrograph of a section of renal cortex from rats treated with omeprazole for 4 weeks (Group llb) showing renal cortex with diffuse damage of renal corpuscles (arrow). Some renal corpuscles are distorted with lost Bowman's space and tight apposition of glomerular capillaries with their covering visceral layer of Bowman's capsule to the parietal layer, irregular outer parietal layer and apparent hypercellularity. Also abnormally shrunken glomerular capillary tuft (arrowhead) is seen in other glomeruli (H and E, ×400)
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The renal medulla showed edema manifested by widening of the interstitial space with mononuclear cell infiltrate in the vicinity with many congested blood vessels in the renal interstitium [Figure 10]. | Figure 10: A photomicrograph of a section of renal medulla from rats treated with omeprazole for 4 weeks (Group llb) showing edema manifested by widening of the interstitial space with mononuclear cell infiltrate in the vicinity (star) with many congested blood vessels (V) the renal interstitium (H and E, ×400)
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With Masson Trichrome staining, the renal cortex showed blue-colored collagen fiber deposition around glomerular capillaries, at the parietal layer of Bowman's capsule, at tubular basement membranes with markedly widened renal interstitial space showing a large amount of collagen fibers deposited particularly around blood vessels [Figure 11]. | Figure 11: A photomicrograph of a renal cortical section from rats treated with omeprazole for 4 weeks (Group llb) showing blue colored collagen fiber deposition around glomerular capillaries (arrowhead), at the parietal layer of Bowman's capsule (double arrow), at tubular basement membranes (arrow). Note markedly widened renal interstitial space with a large amount of collagen fibers deposited (star) particularly around blood vessels (V) (Masson Trichrome, ×400)
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With PAS, the renal cortex showed apparently thickened parietal layer of Bowman's capsule, increase of the mesangial matrix. There were apparently thickened tubular basement membranes with lost brush border of most PCTs and PAS-stained renal interstitial deposits in a wide edematous interstitium [Figure 12]. | Figure 12: A photomicrograph of a section of renal cortex from rats treated with omeprazole for 4 weeks (Group llb) showing apparently thickened parietal layer of Bowman's capsule (arrowhead), increase of mesangial matrix (arrow). Notice also apparently thickened tubular basement membranes (double arrow), lost brush border (#) in most proximal convoluted tubules, and Periodic acid–Schiff stained renal interstitial deposits in a wide edematous interstitium (double arrowhead) (Periodic acid–Schiff, ×400)
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Group IIc (6 weeks group)
Examination of sections of the kidney from the rats treated with omeprazole for 6 weeks (Group IIc) showed renal cortex with diffuse damage and distortion of renal corpuscles in the form of lost Bowman's space with adherence of glomerular capillary tuft to ill-defined, irregular parietal layer of Bowman's capsule with apparent hypercellularity, and vascular congestion of capillary tuft [Figure 13]. Some tubules demonstrated vacuolated lining epithelium with their nuclei revealed nuclear pyknosis, Karyorrhexis, and intra-luminary homogenous pink material (hyaline casts) [Figure 14]. | Figure 13: A photomicrograph of a kidney section from rats treated with omeprazole for 6 weeks (Group llc) showing renal cortex with diffuse damage and distortion of renal corpuscles (arrow) showing lost Bowman's space with adherence of glomerular capillary tuft to parietal layer of Bowman's capsule. Note ill-defined, irregular outer boundary of the renal corpuscle and apparent hypercellularity and vascular congestion of capillary tuft. Renal tubules look normal (H and E, ×400)
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 | Figure 14: A photomicrograph of a renal cortical section from rats treated with omeprazole for 6 weeks (Group llc) showing homogenous pink material (hyaline casts) filling the lumen of many tubules (arrow). Some tubules demonstrate vacuolated lining epithelium (arrowhead). Nuclei of tubular epithelial lining reveal nuclear pyknosis (curved arrow), Karyorrhexis (double arrowhead) (H and E, ×400)
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With Masson Trichrome staining, the renal cortex showed increased blue-colored collagen fibers deposition at glomerular capillaries, at the parietal layer of Bowman's capsule, and in renal interstitium [Figure 15]. | Figure 15: A photomicrograph of a renal cortical section from rats treated with omeprazole for 6 weeks (Group llc) showing increased collagen fibers deposition at glomerular capillaries (arrow), at parietal layer of Bowman's capsule (arrowhead), and in renal interstitium (double arrow) (Masson Trichrome, ×400)
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With PAS, the renal cortex showed obviously thickened parietal layer of Bowman's capsule, marked increase of PAS-positive mesangial matrix. There were apparently thickened tubular basement membranes with lost brush border in the majority of PCTs [Figure 16]. | Figure 16: A photomicrograph of a section of renal cortex from rats treated with omeprazole for 6 weeks (Group llc) showing obviously thickened parietal layer of Bowman's capsule (arrowhead), marked increase of Periodic acid–Schiff positive mesangial matrix (arrow). Notice also apparently thickened tubular basement membranes (double arrow) and lost brush border (#) in the majority of proximal convoluted tubules (Periodic acid–Schiff, ×400)
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Morphometric results and statistics
Estimation of the number of the tubular cells with vacuolations
There was a highly statistically significant increase in the number of tubular cells with vacuolation in Groups IIa (2 weeks), IIb (4 weeks), and IIc (6 weeks) compared with the control group with P < 0.001. Groups IIb (4 weeks) and IIc (6 weeks) revealed highly significant increase in the number of tubular cells with vacuolation compared with Group IIa (2 weeks). Similarly, 6 weeks Group (IIc) demonstrated a highly significant increase compared with 4 weeks Group (IIb). Therefore, this statistical significance was time dependent [Table 1] and [Histogram 1].
Estimation of the number of the pyknotic nuclei in the tubular cells
Assessment of the number of pyknotic nuclei in tubular cells showed that there was highly statistically significant increase in the number of tubular cells with pyknotic nuclei in Groups IIa (2 weeks), IIb (4 weeks), and IIc (6 weeks) compared with the control group with P < 0.001. Moreover, Groups IIb (4 weeks) and IIc (6 weeks) revealed highly significant increase in the number of tubular cells with pyknotic nuclei compared with Group IIa (2 weeks). However, 6 weeks Group (IIc) demonstrated only a significant increase compared with Groups IIb (4 weeks) [Table 1] and [Histogram 2].
Estimation of the number of the hyaline cast in tubular lumen
Assessment of the number of the hyaline cast in tubular lumen showed that there was nonsignificant statistically increase in the number of tubules with intra-luminary hyaline casts in Group IIa (2 weeks) compared with the control group. However, there was highly statistically significant increase in the number of tubules with intra-luminary hyaline casts in Groups IIb (4 weeks), and IIc (6 weeks) compared with the control group with P value 0.76, 0.0002, and 0.000001, respectively.
Moreover, Groups IIb (4 weeks) and IIc (6 weeks) revealed a statistically significant increase in the number of tubules with intraluminary hyaline casts as compared with Group IIa (2 weeks). Similarly, 6 weeks Group (IIc) demonstrated a statistically significant increase as compared with 4 weeks Group (IIb) [Table 1] and [Histogram 3].
Estimation of the area percentages of interstitial collagen fibers deposition
Assessment of area percentages of interstitial collagen fibers deposition showed that there was highly statistically significant increase in the area percentages of interstitial collagen fibers deposition in Groups IIa (2 weeks), IIb (4 weeks), and IIc (6 weeks) compared with the control group with P < 0.001. Moreover, Groups IIb (4 weeks) and IIc (6 weeks) revealed a significant increase in area percentages of interstitial collagen fibers deposition as compared with Group IIa (2 weeks). Similarly, 6 weeks Group (IIc) demonstrated significant increase as compared with 4 weeks Group (IIb) [Table 1] and [Histogram 4].
Laboratory results
Estimation of blood urea nitrogen level
Comparison of the means of the BUN level among the groups of the study revealed a significant increase in 2 weeks (IIa) compared with the control group. The 6 weeks Group (IIc) showed highly statistically significant increase compared with the control group. Four weeks group showed a significant increase in means BUN level in a time-dependent manner [Table 2] and [Histogram 5]. | Table 2: Mean standard deviation of blood urea and serum creatinine of all the study groups
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Estimation of serum creatinine level
Both four (IIb) and 6 weeks (IIc) groups demonstrated highly statistically significant increase in the serum creatinine level compared with the control group. The 2 weeks (IIa) group did not reveal statistical difference [Table 2] and [Histogram 6].
Estimation of estimated glomerular filtration rate
Two weeks group (IIa) did not show statistical significance, whereas the 4 weeks Group (IIc) demonstrated a statistically significant decrease of eGFR as compared with the control group. The 6 weeks Group (IIc) showed high statistically significant decrease of eGFR compared with the control group [Table 3] and [Histogram 7].
| Discussion | | |
The aim of the present study was to show the effects of the chronic use of the PPI (omeprazole) on the histological architecture and function of the kidney of male albino rats and to compare between these effects after 2, 4, and 6 weeks of administration.
The kidney of the control group was composed of outer darkly stained cortex and inner lightly stained medulla, which furthermore, was divided into O. M near the cortex, and I. M away from the cortex. The nephron was formed of renal corpuscles, PCTs, loop of Henle, and DCTs. This was in accordance with Bonventre and Yang.[11]
The renal medulla is divided into outer and IM according to their proximity from the cortex. The O. M consisted of the thin limb of loops of Henle lined by simple squamous epithelium, thick limb of loops of Henle lined by cuboidal epithelium and collecting tubules lined by cuboidal epithelium, which agreed with the description of Song and Yosypiv.[12] The interstitial space between the renal tubules was very narrow with a visible small number of fibroblastic cells between them which goes with Katz et al.[13]
In Masson trichrome-stained sections, the cortical and medullary renal architecture showed thin blue-stained collagen fibers at renal corpuscle; glomerular tuft basement membranes, parietal layer of Bowman's capsule and mesangial cells, around the cortical and medullary tubular basement membranes and in the renal interstitium. This was in accordance with Clin.[14]
In PAS-stained sections, the renal cortex showed a positive magenta color PAS reaction (thin well-defined magenta) at the basement membrane surrounding renal corpuscle, renal cortical and medullary tubules, and brush border of PCT. This was in accordance with Ross and Pawlina.[15]
On the other hand, Group (IIa) (2 weeks of omeprazole administration) showed few distorted glomeruli. Some of them were shrunken and atrophic; others were congested with tight apposition of the glomerular capillaries with their covering visceral layer of Bowman's space and the irregular parietal layer and loss of Bowman's space, others filled with extravasted blood and mononuclear inflammatory cells. This was similar to the findings stated by Javed et al.,[16] who added that the incidence of these toxic effects increase as the dose of the drug was increased. Moreover, both the cortical and medullary tubules showed marked damage, which was more obvious in the cortical tubules. This agreed with Basile et al.,[17] who stated that drug-induced kidney injury affected the cortex more than the medulla owing to a higher concentration of the drug present in the cortex which decreased gradually till reaching the medulla due to absorption process. Multiple vacuolation of cells lining the renal tubules with loss of the epithelial lining in some areas were observed. The nuclei of the tubular cells showed pyknosis, Karyorrhexis up to complete disintegration. This was in accordance with Samuel[18] who observed vacuolated cytoplasm with darkly stained pyknotic nuclei as a histopathological finding after treating male albino rats with PPI for 4 weeks.
The present study showed the accumulation of a large number of mononuclear inflammatory cells with perivascular congestion in the interstitial tissues. This coincided with Praga and González[19] who correlated between omeprazole and interstitial nephritis and stated that patchy or diffuse inflammatory cells and interstitial edema were the most significant findings.
Furthermore, the apparent increase in the collagen fiber deposition appeared surrounding glomerular capillaries at the parietal layer of Bowman's capsule and surrounding renal tubular basement membrane. Zhong et al.[20] considered fibrous tissue deposition the second step in chronic kidney injury and described it as misdirected defensive body mechanism to limit the problem. Examination of PAS-stained renal sections showed positive PAS reaction (purple-magenta color) at renal corpuscles and basement membrane of cortical and medullary renal tubules. There was the loss of brush border in most of PCT. Vleming et al.[21] stated that quantitative estimates of the glomerular deposition of PAS positive extracellular matrix seemed to be the most important structural correlate of renal function. This study demonstrated that the severity of renal insufficiency in a variety of renal diseases correlates with the severity of glomerular pathology when quantitative scoring is applied.
There were patches of PAS-stained interstitial deposits. This was similar to Samuel[18] who observed focal interruption of PAS reaction in the brush border of PCT after treating male albino rats with PPI (esmoprazole) for 4 weeks.
The interstitial tissues became wide indicating interstitial edema. Furthermore, massive interstitial hemorrhage and intraluminal hyaline casts were prominent This goes with Javed et al.[16] who observed hemorrhage and edema in the renal interstitium in the kidney sections from rats treated with omeprazole for 6 weeks.
Regarding Group (IIc) (after 6 weeks of omeprazole administration) results revealed increasing medullary damaged in the form of lost tubular epithelial lining, obliterated lumen of many medullary tubules, and presence of pink interstitial medullary exudate. The interstitial tissues showed an accumulation of a large number of mononuclear inflammatory cells with perivascular congestion. This goes with Javed et al.[16] who observed hemorrhage and edema in the renal interstitium in kidney sections from the rats treated with omeprazole for 6 weeks.
The histomorphometric statistical analysis demonstrated highly statistically significant increase in these parameters; number of vacuolated tubular cells, number of tubular cells with pyknotic nuclei, area percentage of interstitial collagen fiber and intraluminar hyaline casts. These occurred for Groups IIa (2 weeks group), IIb (4 weeks group), and IIc (6 weeks group) in comparison with control. This indicates an increased risk of kidney damage is directly proportional to increased duration of omeprazole administration, which agrees with Javed et al.[16] who added increase toxic effect as the dose of drug increased.
As for the level of serum creatinine, an increase which was nonsignificant in Group IIa (treated with omeprazole for 2 weeks) compared with Group l (control group) was noticed. This increase became highly significant in Groups IIb. Serum creatinine did not reveal the statistical difference between Group IIb (4 weeks group) and Group IIc (6 weeks group).
BUN showed a statistically significant increase in Group IIa (2 weeks) in comparison with the control group (Group l). On the other hand, there was a significant decrease in 4 weeks group (Group IIb) in comparison with the control group (Group l) and as compared to Group IIa (2 weeks). However, 6 weeks group (Group IIc) showed a highly significant statistical increase in comparison with Group 1 (control group), Group IIa (2 weeks group), and Group IIb (4 weeks group).
This was in accordance with Gounden et al.[22] who illustrated a fluctuation of BUN in serum due to affection of multiple factors which can decrease it (like fluid excess) and elevated with conditions not related to the kidney. The elevation of BUN might be due to physiological or pathological conditions such as; increased protein intake in diet, intestinal bleeding.
The eGFR in Group IIa (2 weeks) did not show any statistically significance difference from control group (l). There was a statistically significant decrease in Group IIb (4 weeks) as compared with the control group. This decrease was highly significant in Group IIc (6 weeks) compared with the control.
In Meta-analysis interpreted by Yang et al.[23] for five large population-based cohort studies with thousands of patients participant for about 5 years, and by follow up of kidney function tests (s. creatinine and BUN), there was elevation of kidney functions and deterioration of eGFR after different duration of PPIs administrated varied according to many patient-related factors.
Diagen[24] stated that no relationship was found between the duration or dosage of PPIs therapy and the onset of AKI. It might occur after a short or long duration of therapy but suggested at least 1 week of administration of PPIs.
This was similar to Klatte et al.[5] who considered eGFR as a golden standard for evaluation of kidney function and defined kidney injury when doubling of creatinine level or decrease in eGFR more than 30%. If this prolonged for 3 months or more it meant progression to chronic kidney injury. It was declared the PPIs as a risk factor for kidney injury, either acute or chronic and might progress to ESRD.
Lazarus et al.[25] and Xie et al.[26] conducted three cohort studies and found 1.22- to 1.50-fold increased risk of CKD for PPI users versus non-PPI users. Kieboom et al.[27] explained that PPIs were reported to be associated with hypomagnesaemia which could cause endothelial dysfunction, oxidative stress, and inflammation. This related to interstitial tubular injury and caused decline in renal function.
| Conclusion | | |
It is concluded that the long-term use of omeprazole resulted in structural damage of rat renal tissue associated with deterioration of renal function in a time-dependent manner.
Extreme caution should be taken with clinical prescription of omeprazole and close monitoring of renal function is highly recommended.
Financial support and sponsorship
This study was financially supported by Alexandria Co. for Pharmaceuticals, Alexandria, Egypt.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16]
[Table 1], [Table 2], [Table 3]
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