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Interaction of Lactoferrin with Neutroplile Gelatinase Association Lipocalin in Treatment of Chronic Kidney Diseases Induced Experimentally by K. pneumonia

Shahad H. Alajeli¹, Majida A. Al-Qaiym^*2

, Lazim H. Altaie³

¹Department of X-ray, Medical Technical College, the University of Al-Zahraa for Women, Karbala -Iraq

²Department of Physiology, Biochemistry, and Pharmacology, College of Veterinary Medicine, University of Baghdad, Iraq.

³Departement of X-ray and sonar, Medical Technical College, Al Farahidi University, Baghdad-Iraq

Corresponding author: [email protected],

Available from: http://dx.doi.org/10.21931/RB/2023.08.03.26

ABSTRACT

Lactoferrin (Lf), among the critical components of the innate immune system, has excellent iron-carrying properties that provide valuable protection against all types of bacterial infections, inflammatory diseases and other forms of cellular stress. This study hypothesized an essential role for Lf in the treatment of chronic kidney disease (CKD) and the mechanism of neutrophil gelatinase association with lipocalin action through its role in iron sequestration and its effectiveness in reducing oxidative stress caused by Klebsiella pneumoniae in infected rats. Thirty adult female rats were divided into three equal groups; 1^st group was kept healthy as a control, and the ^{second and third} groups were infected with K. pneumoniae by the intraurethral route. Following 60 days of infection, the 3^rd group was treated for 30 days with Lf (0.5 gm/kg BW). Results of kidney function tests (urea, creatinine, and Kidney injury molecule-1 KIM-1) indicated the change of K. pneumoniae infection from acute to chronic. Lactoferrin-treated rats had reduced urea, creatinine, KIM-1, neutrophil gelatinase-associated Lipocalin (NGAL), erythropoietin (EPO), malondialdehyde acid (MDA), and interleukin-6 (IL-6), and elevated total antioxidant capacity (TAOC) in comparison with untreated rats. It can be postulated that Lf activity in improving kidney function and histopathological changes following chronic K. pneumoniae infection belongs to its antioxidant, anti-inflammatory, and bacteriostatic role.

Keywords: Lactoferrin, CKD, K. pneumoniae 16SrRNA, NGAL, Antioxidant

INTRODUCTION

Chronic kidney disease (CKD) is a global epidemic recognized as impairment of renal functions shown by a glomerular filtration rate less than usual, signs of kidney damage, or both, for at least three months^1.2. Renal failure due to acute kidney injury can be fatal³or might lead to progression to chronic kidney disease⁴. Direct infection of renal tissue by different bacteria induces urinary tract infection, which can progress to renal dysfunction in pyelonephritis by ascending infection or hematogenous dissemination⁵. Occasionally, renal injury may be due to the nephrotoxic effects of various antimicrobial agents used in management. Patients who are elderly, diabetic, pregnant, or immunocompromised are at an increased risk of acute injury, which has high mortality and morbidity⁶. As many as 20% of critically ill patients have irreversible renal damage due to acute cortical necrosis, and another 40% have incomplete renal recovery, leading to CKD⁷. It is not uncommon to observe that episodes of bacterial sepsis accelerate the rate of progression of pre-existing CKD by multiple mechanisms⁸. Because serum urea levels rise in acute and chronic renal disease, and serum creatinine is a late marker of acute kidney injury, estimating those biomarkers assesses renal function⁹. Kidney molecular injury-1 (KIM-1) and neutrophil gelatinase-associated Lipocalin (NGAL) are tubular injury biomarkers for acute kidney injury (AKI). Both may also add information on the persistence of renal injury in AKI and the risk of progression to CKD^10.11.

Klebsiella pneumoniae is a facultative anaerobic bacterium that is Gram-negative, non-motile, rod-shaped, lactose-fermenting and has a conspicuous polysaccharide capsule. Within the genus Klebsiella, K. pneumoniae is the most common human pathogen, causing a variety of infections in hospitals, long-term care facilities, and communities around the world, including diseases of the lungs, urinary tract, abdominal cavity, surgical sites, soft tissue infections, and even bacteremia ^12.13. This bacterium may be found in the natural flora of the mouth, skin, and gut and is the third most often isolated pathogen in blood culture patients with sepsis¹⁴. In addition, K. pneumoniae has been identified as a cause of severe and life-threatening infections, such as pyogenic liver abscesses, endophthalmitis, and meningitis, and is becoming a public health problem^15.16.

Lactoferrin (lactotransferrin; Lf) is a globular glycoprotein with a strong affinity for metal ions, particularly iron, copper, zinc, and manganese¹⁷. Recent research shows that Lf treatment can protect against AKI and early renal fibrosis while restoring kidney function¹⁸. Lf shows protective effects against AKI and CKD; this reduces markers related to inflammation, oxidative stress, apoptosis, and kidney fibrosis and induces autophagy and mitochondrial biogenesis in the kidney¹⁹. LF has a variety of pharmacological effects that are mediated through a variety of receptors, including antibacterial, antifungal, antiviral, anti-inflammatory, and antioxidative activities, as well as the ability to regulate the amount of iron absorbed in the gut and can also chelate iron, either directly or indirectly, through its involvement in iron transport and modulates immunological responses²⁰.

Neutrophil gelatinase-associated lipocalin (NGAL) is one of the proteins that are expressed in damaged kidneys and has endogenous antimicrobial and iron scavenging activity²¹; therefore, it can be considered as a tissue injury marker and a marker for the gradual loss of nephron function and CKD progression^22.23. Lactoferrin (Lf) and neutrophil gelatinase-associated lipocalin (NGAL) are among the critical components of the innate immune system co-localized in specific (secondary) granules of human neutrophils, Lf and NGAL are released as acute phase proteins following neutrophil activation and degranulation^24.25. Because their excellent iron-carrying properties provide valuable protection against all types of bacterial infection, inflammatory disease and other cellular stress, they have been linked to high affinity for Fe^3+. By removing iron from the site of injury, Lf and NGAL may limit iron-mediated toxicity, although the mechanisms employed by each differ. Antibiotic resistance makes infections caused by K. pneumoniae increasingly challenging to treat and directly threaten health²⁸. Thus, this study aimed to investigate the role of lactoferrin as a new therapeutic for CKD to limit K. pneumoniae infections via modulating the anti-inflammatory, antioxidant, and tissue repair responses.

MATERIALS AND METHODS

Bacterial isolation and identification

Klebsiella pneumonia was obtained from Medical City Hospital, Baghdad, Iraq, isolated from a sputum swap from a Covid-19 patient. Firstly, the trade was transported into a tube containing 3 ml of brain_heart infusion broth and incubated at 37ºC for 48 hours. A slant of brain-heart infusion agar was used to maintain the bacterial isolate. To improve bacterial identification, selected colonies were cultured on blood, congo red, and MacConkey's agars. The VITEK®2 system did biochemical identification of the bacteria. Then, the isolate was confirmed at the molecular level using polymerase chain reaction (PCR) targeting the 16S rRNA gene. The bacterial DNA was extracted from the bacterial growth according to the protocol of ABIO pure. The extracted DNA was quantified by Quantus Fluorometer, briefly mixing 1 μl of DNA with 200 μl of diluted Quantifluor Dye. The DNA concentration values were detected after 5 min incubation at room temperature; for the molecular identification of K. pneumoniae, 27 F and 1492 R primers (supplied by the Macrogen Company) for the 16S rRNA gene were used to amplify the DNA sequence. After PCR amplification, agarose gel electrophoresis was adopted to confirm the presence of amplification. Finally, the PCR products were sent for Sanger sequencing using ABI3730XL, an automated DNA sequencer, by Macrogen Corporation – Korea. The results were received by email and then analyzed using Genius software.

Table 1. Primers and their sequences for K. pneumoniae molecular identification

Experimental Design

The thirty adult female Albino Wister Rats' body weight ranged between 150 and 200 gm and were randomly divided into two groups. The first group was considered the control. The second group was infected with K. pneumoniae using an experimental model of ascending pyelonephritis²⁹, in which 0.5 ml of the bacteria suspension (10⁵–10⁷ bacteria/ ml) was introduced within 5 seconds via the urethra into the urinary bladder. The infection was repeated after 20 days to ensure condition. Following 60 days of disease, the second group was divided into two subgroups; the first sub-group continued with infection, and the second sub-group was administered lactoferrin orally at a dose of 0.5 g/kg BW for 30 days³⁰. Blood samples were collected from all groups of anesthetized rats at 60 days of infection and after 30 days of treatment. This study was carried out according to the approval of the scientific committee in the Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of Baghdad, following the ethical animal welfare standards.

Kidney function markers: Serum urea and creatinine were determined using a commercial kit (Linear Chemicals, SLU/Spain) by an enzymatic colorimetric method. Kidney injury molecule-1 (KIM-1) was measured using a commercial kit (Al-Shkairate / Jordan). Serum Neutrophil Gelatinase Associated Lipocalin (NGAL) was determined using the Rat Neutrophil Gelatinase Associated Lipocalin ELISA Kit (MyBioSource/USA).

Anti-inflammatory and antioxidant tests.

Serum malondialdehyde (MDA) and Total Antioxidant Capacity (TAOC) were determined by a colorimetric assay kit (MyBioSource/USA). Furthermore, serum Interleukin-6 (IL-6) was determined by the Rat Interleukin-6 ELISA Kit (MyBioSource/USA).

Statistical analysis

Data presented as a mean ±SE. Statistical analysis of data was performed by two-way analysis using SAS (Statistical Analysis System) and Microsoft Office Excel for Windows (2010). Least significant differences (LSD) were performed multiple (multiple comparisons) to evaluate significant differences at p ≤ 0.05³¹.

RESULTS

Bacterial identification and confirmation.

Klebsiella pneumoniae grew successfully on selective agar media, giving its characterized feature on MacConkey's agar, i.e., mucoid pink color, due to lactose fermentation; they also showed gray-white, large mucoid translucent and non-hemolytic colonies on blood agar, while on Congo red agar they were black due to biofilm formation. The bacterial identification was confirmed at 99% using the VITEK2 system. The PCR amplification fractionated on agarose gel electrophoresis showed a band of roughly 1500 bp representing 16 S rRNA of K. pneumoniae (Figure 1). The above bacterial gene sequence analysis using the BLAST program in NCBI confirmed that the 16S rRNA gene belonged to K. pneumoniae.

Figure 1: 1.5% Agarose gel electrophoresis shows a band of approximately 1500 bp of the 16S rRNA gene expected to belong to K. pneumoniae. M: 100bp DNA size marker, 1: PCR amplicon.

Kidney function markers

Treatment of rats with lactoferrin for 30 days reduced significantly (p< 0.05), the elevated serum urea, creatinine, KIM-1, and NGAL of rats with CKD caused by K. pneumoniae (Tables 2,3,4,5). Erythropoietin showed a significant reduction in K. pneumoniae-infected rats before treatment, but lactoferrin pushed it up to the normal range of the control group (Table 6).

Kidney histopathological changes

Histopathological analysis of the light microscopic image of the kidney section of control rats revealed normal tissue (Figure 2A,2B). K. pneumoniae infection with no treatment for about 90 days revealed severe destruction of the glomerular in the cortical region of the kidney with loss of parenchyma, multifocal mononuclear cells (MNCs), Tubular dilation, interstitial MNCs infiltration forming granuloma-like lesion with giant cell formation (figure 2C), all these changes indicated the chronic changes due to bacterial infection. The medullary section of the kidney from infected rats showed few hyaline casts with cystic tubular dilation (Figure 2D). The results of histopathological changes showed that treatment for 30 days with lactoferrin produced no apparent pathological changes in the cortical. Although, there was prevascular mononuclear cell aggregation, indicating a delayed hypersensitivity reaction (Figure 2E). The medullary region showed that lactoferrin treatment produced nodular granulomatous lesions composed mainly of lymphocytes and plasma (Figure 2F).

Antioxidant and anti-inflammatory markers

Table 6 shows that MDA, an oxidative stress marker, was significantly (P ≤ 0.05) high before treatment in infected groups, but after 30 days of treatment with Lf, its serum levels significantly decreased (P ≤ 0.05). The serum TAOC was significantly increased in the K. pneumoniae infection treated with Lf for 30 days. The results also showed that the serum level of IL-6, a proinflammatory cytokine, was significantly (P ≤ 0.05) higher in the groups of K. pneumoniae-infected female rats before treatment compared with control female rats. After 30 days of treatment, with Lf, IL-6 levels reduced significantly (P ≤ 0.05) compared with the non-treated group (Table 7,8).

Table 2. Serum urea (mg/dl) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

Table 3. Serum creatinine (mg/dl) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

Table 4. Serum Kim1 (pg/ml) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

Table 5. Serum NGAL(pg/ml) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

Table 6. Serum MDA (nmol/ml) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

Table 7. Serum TAOC(U/ml) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

Table 8. Serum Il-6 (pg/ml) at day 60 days of K. pneumoniae infection and after 30 days of lactoferrin treatment. Means ± SE, n= 6

ular mononuclear cell

Figure 2. Light microscopic image for kidney tissues for A: cortical section of the control group (10x), B: kidney medulla section (40x), C: cortical area of K- pneumoniae infected (10x), D: medulla sections of K- pneumoniae infected (10x), E: cortical section of K- pneumoniae infected with Lf treatment (40x), E: medulla section of K- pneumoniae infected with Lf treatment (10x).

DISCUSSION

Animal models have been extensively used to clarify the pathophysiology and processes behind kidney failure. Rats are quickly produced and can be made to develop classic kidney damage in various ways, including surgery and the injection of medicines or poisons³². In the present study, the elevation of urea and creatinine to more than 50 %, according to KDIGO, indicates the transition of infection from acute to chronic, resulting in chronic kidney disease (CKD). Chronic kidney disease has been induced by an ascending method for kidney infection with K. pneumoniae²⁹. K. pneumoniae is a highly virulent and antibiotic-resistant bacteria involved in CKD³³. The pathogenicity of K. pneumoniae has been acquired through the activity of different virulence factors, such as capsular polysaccharide (CPS) and siderophores³⁴. The mechanism by which rats infected with K. pneumoniae developed CKD could result in inflammation accompanied by oxidative stress and renal dysfunction³⁵. As a primary histological hallmark of acute kidney damage, the ascending manner of bacterial infection results in biological processes, such as tubular cell death, necrosis, inflammation, and apoptosis³⁶. Fibrosis, vascular disruption, tubular loss, glomerulosclerosis, and persistent interstitial inflammation are all pathological changes of chronic kidney disease³⁷. Siderophores secreted by K. pneumoniae induce stabilization of the transcription factor HIF-1α, increasing the expression of proinflammatory cytokines³⁸. Various CKDs have been linked to multiple diseases, including chronic inflammation and renal fibrosis³⁹. The interaction of bacterial siderophores with host cells further promotes the pathogenicity of K. pneumoniae in different mechanisms⁴⁰ and is likely relevant for other pathogens that also secrete siderophores in the host⁴¹. Because serum urea levels rise in acute and chronic renal diseases, estimating those biomarkers assesses renal function and indicates a defect in glomerular filtration^42.43.9. Although many biomarkers in the blood can be measured to diagnose kidney diseases and are linked to kidney function, the kidney injury molecule-1 (KIM-1) is the most closely linked.

Kidney molecular injury and neutrophil gelatinase-associated lipocalin (NGAL) are tubular injury indicators for acute kidney injury (AKI), the persistence of renal injury, and the risk of progression to CKD⁴⁵. The persistent serum NGAL and KIM above the control level for more than 90 days of infection approved the transition of acute to chronic disease; they improved as biomarkers of CKD progression of end-stage renal disease⁴⁶. NGAL extensively impacts immune processes and has a direct bacteriostatic effect by blocking the siderophores on the Gram-negative bacterial wall⁴⁷. Decrease in endogenous erythropoietin (EPO) production⁴⁸. Erythropoietin, primarily generated by the kidney's fibroblast-like interstitial pericytes cells, surrounds pre-tubular capillaries and is considered a kidney biomarker. As shown by histopathological changes of kidney tissue in the present model of CKD, the kidney's interstitial showed young proliferated fibroblast cells, and differentiation of these pericytes or fibroblast to myofibroblasts cause interstitial fibrosis in CKD is indicative of long-standing kidney disease⁴⁹ subsequently the ability of EPO production decreases^50.51. Furthermore, EPO production inhibition or EPO resistance is affected by some inflammatory cytokines⁵². It is generally understood that CKD increases inflammation and immune activation molecules, which would impede hypoxia-induced EPO synthesis⁵³.

During the host cell-pathogen interaction, a cytokine burst occurs to recruit the cells of the innate immune system and enhance the defense against pathogens. Mostly, interIeukin-6^54.55.56. Serum NGAL reflects renal impairment and presents a solid and independent risk marker for the progression of end-stage renal disease. Neutrophil gelatinase-associated lipocalin is a member of lipocalin proteins, which are extracellular proteins that bind small and hydrophobic molecules and play a role in cellular homeostasis NGAL has been used as a diagnostic marker in cases of glomerular and tubular dysfunction^57.58. Both NGAL and IL-6 are biomarkers for UTI infection⁵⁹. Lactoferrin has recently become the subject of significant investigation. Lactoferrin has a considerable potential pharmacological role in treating kidney diseases.

Upregulated innate lymphocyte activation and cytokine production from injured epithelial cells leads to kidney disease⁶⁰. Innate lymphoid cells (ILCs) have emerged as essential mediators of mucosal surface protection and repair during infection⁶¹. The innate immune system is capable of guarding against K. pneumoniae infection^62.63. Both inflammation and oxidative stress can contribute to the development of CKD and its various complications. Permanent oxidative stress occurs in patients with chronic kidney disease. Increased ROS production and/or insufficient antioxidant system performance (TAOC) can lead to oxidative stress, which is associated with the damage or oxidative modification of vital molecules such as nucleic acid, proteins (enzymes), and lipids, producing malondialdehyde (MDA). In CKD patients, oxidative stress causes lipid and protein peroxidation^64.65. Most kidney failure indices, oxidative stress and inflammation measured in plasma were improved in rats given lactoferrin, suggesting different mechanisms of K. pneumoniae-induced CKD. Lactoferrin acts as an antioxidant, the principal component of immune homeostasis. It may decrease intracellular ROS levels and oxidative stress-induced apoptosis. Transitional metals such as iron are the leading oxidative stress cause, which controls ROS production. Lactoferrin can inhibit lipid peroxidation due to iron sequestration⁶⁶.

The results indicate that Lf ingestion effectively treated kidney tubular oxidative damage by protecting kidney tissue from ferric nitrilotriacetate-induced kidney tubular oxidative injury in rat models. This antioxidant activity is mediated by diminishing H₂O₂-induced apoptosis by inducing autophagy⁶⁹. Treatment with Lf restored kidney function and prevented fibrosis by inhibiting apoptosis and augmenting autophagy. The antifibrotic activity of lactoferrin in the treated group results from the role of lactoferrin in down-regulation of tissue growth factor β1 gene expression¹⁸. As an anti-inflammatory, lactoferrin has been used successfully against kidney inflammation⁷⁰. The anti-inflammatory mechanism of lactoferrin could be through iron metabolism. Lf can reduce the damage of excessive inflammatory responses by sequestering free iron and directing reactive oxygen intermediates⁷¹ or by reducing hyperoxia-mediated kidney inflammation and kidney injury in mice models. The PCR technique in the medical field was applied in diagnosing pathogenic microorganisms^72-83 and genetic-related diseases ^84-88.

CONCLUSION

The results of lowering inflammatory biomarkers indicated lactoferrin's multifactorial activity in CKD caused by K. pneumoniae. In the current work, lactoferrin improved kidney tissue by acting as an antioxidant and antifibrotic; when Lf is administered as an iron sequestration molecule, which sequesters and conjugates bacterial siderophores and inhibits bacterial absorption, this conjugation of lactoferrin to bacterial siderophores are improving the therapy of CKD and renal repair, which lowers bacterial resistance and has anti-inflammatory properties.

Funding: Self-Funding.

Acknowledgments: The authors thank everyone who helped with this work.

Conflicts of Interest: No conflict of interest.

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Received: 28 May 2023/ Accepted: 15 July 2023 / Published:15 September 2023

Citation: AlajeliS H., Al-Qaiym M A., AltaieL H. Interaction of Lactoferrin with Neutroplile Gelatinase Association Lipocalin in Treatment of Chronic Kidney Diseases Induced Experimentally by K. pneumonia. Revis Bionatura 2023;8 (3) 26. http://dx.doi.org/10.21931/RB/2023.08.03.26