KI-67 Immunohistochemical Expression in Lung Tissue of Rats under Chronic Renal Failure

Abstract

Background

Chronic kidney disease (CKD) is associated with systemic complications, including pulmonary alterations due to kidney–lung crosstalk. This study evaluates K_i-67 immunohistochemical expression in rat lung tissue to assess cellular proliferative activity under experimental chronic renal failure (CRF) conditions, considering age-related variations.

Materials and Methods

Outbred male rats (n = 150, aged 6, 9, and 12 months) were divided into control and experimental groups. CRF was induced via repeated intramuscular injections of 50% glycerol (0.5 mL twice weekly for 1 month). Alveolar lung tissues were fixed, sectioned, and stained for K_i-67 using the 3,3′-Diaminobenzidine method. Quantitative analysis was performed with QuPath software (v0.4.0) to calculate proliferative indices. Data were analyzed using two-way ANOVA with post-hoc tests (p <0.05).

Results

In controls, K_i-67 proliferative indices declined with age: 55.86% at 6 months, 52.32% at 9 months, and 23.44% at 12 months. Under CRF, indices were significantly suppressed: 21.75, 11.06, and 2.32%, respectively (p <0.01 vs. controls), indicating impaired cellular renewal.

Conclusion

Experimental CRF exacerbates age-dependent reductions in lung cell proliferation, highlighting mechanisms, such as inflammation and hypoxia in pulmonary pathology. These findings underscore the need for targeted interventions in CKD-related lung dysfunction.

Keywords

lung tissue - regeneration - aging - cell proliferation - chronic renal failure - immunohistochemistry - K_i-67

Introduction

Chronic kidney disease (CKD) is a major global health issue, associated with high comorbidity and systemic complications.[1] [2] [3] Among extrarenal manifestations, pulmonary changes are prominent, including impaired cellular renewal, inflammation, and fibrosis.[4] [5] K_i-67, a nuclear marker expressed in active cell cycle phases, serves as a reliable indicator of proliferative activity in pathological conditions.[2] [6]

Research on lung dysfunction in CKD shows that reduced glomerular filtration rate causes fluid retention, leading to pulmonary congestion, edema, and restrictive spirometry, with prevalence rising in advanced stages. Hemodynamic alterations contribute to pulmonary hypertension, amplified by shared inflammatory pathways.[7]

In adenine-induced CKD mouse models, an adenine diet induces lung injury characterized by leukocyte infiltration, elevated inflammation and fibrosis scores, and oxidative stress markers, such as lipid peroxidation. Similar findings in rat adenine models demonstrate kidney–lung crosstalk mediated by systemic factors.[8] In rats, a 0.75% adenine diet attenuated ischemia–reperfusion lung injury ex vivo, with reduced septal thickening and neutrophil infiltration, although proliferation markers were not assessed. In ⅚ nephrectomy rats with high-salt diet, the lungs showed decreased angiotensin-converting enzyme 2 (ACE2) expression without vascular remodeling. Adenine-induced CKD in mice has also been linked to hyperphosphatemia-driven lung inflammation via IL-1β and CXCL2.[9]

In a ⅚ nephrectomy rat model combined with a high-salt diet to induce CKD-associated pulmonary hypertension, lung tissue exhibited no significant vascular remodeling, such as smooth muscle medial wall thickening or changes in vessel density, as confirmed by H&E, Masson's trichrome, and Van Gieson staining. However, immunofluorescence revealed decreased ACE2 expression in pulmonary vascular endothelial cells, suggesting altered vasoregulation without proliferative vascular changes. No specific proliferation markers like K_i-67 were evaluated in this context.[10]

Studies on adenine-induced CKD in mice have linked hyperphosphatemia to lung inflammation, with bronchoalveolar lavage fluid showing increased total cells and macrophages, and immunohistochemistry indicating elevated IL-1β and CXCL2 expression in bronchial epithelium. This suggests phosphate-mediated proinflammatory effects in the lungs, potentially applicable to rat models, though direct K_i-67 assessment was absent.[11]

Regarding K_i-67 as a proliferation marker in lung pathology, silver nanoparticle intoxication in mice led to elevated K_i-67 immunoreactivity in pulmonary epithelial cells, particularly bronchial cells, indicating its role as an indicator of inflammation and genotoxicity. These findings could parallel proliferative responses in CKD-related lung stress, although such effects have not been directly studied in chronic renal failure (CRF) models.[12]

In a rat silicosis model, K_i-67 immunohistochemical (IHC) staining showed positive labeling in spindle cells within fibrotic lung lesions, highlighting proliferative activity associated with epithelial–mesenchymal transition processes. These observations may inform similar remodeling mechanisms in CRF-induced pulmonary fibrosis, despite differing etiologies.[13]

Previous studies have described morphological changes in renal tissue during CRF; however, data on the proliferative status of lung cells under chronic kidney injury remain limited. Moreover, age-related differences in proliferative potential and the impact of CRF on lung regeneration in experimental models are not fully elucidated.[14] [15] [16]

Data on lung proliferative status in kidney injury are limited, with respect to age-related differences. This study aimed to evaluate K_i-67 expression in lung tissue of outbred rats aged 6, 9, and 12 months under acute kidney injury conditions, hypothesizing an age-dependent suppression of proliferative activity.

Materials and Methods

The study utilized outbred (hybrid) male rats aged 6, 9, and 12 months, sourced from a certified laboratory animal facility. Animals were housed under standard conditions with a 12-hour light/dark cycle, ad libitum access to food and water, and maintained in accordance with institutional ethical guidelines for animal research. Rats were randomly divided into two main groups: a control group (receiving saline injections) and a CRF experimental group. For each age category (6, 9, and 12 months), both the control and experimental groups consisted of 25 rats each, resulting in a total of 150 animals (75 per main group).

CRF was induced using a modified method based on G. Greven's protocol, involving intramuscular injections of a 50% glycerol solution at a dose of 0.5 mL twice a week for 1 month. Control animals received an equivalent volume of sterile saline on the same schedule. The induction was performed under aseptic conditions, and animals were monitored daily for signs of distress or complications. Renal function was confirmed via serum creatinine and urea levels at the end of the induction period, prior to tissue collection.

Tissue collection and processing: At the end of the experimental period, rats were euthanized humanely using an overdose of sodium pentobarbital (100 mg/kg, intraperitoneal). Lung tissues were immediately excised, with the alveolar regions specifically selected for analysis to focus on parenchymal changes. Samples were fixed in 10% neutral buffered formalin for 24 to 48 hours at room temperature. Following fixation, tissues were dehydrated through a graded series of alcohols, cleared in xylene, and embedded in paraffin wax using standard histological protocols.

Histological and IHC staining: Paraffin-embedded blocks were sectioned at 4 to 5 μm thickness using a rotary microtome. For general morphological assessment, sections were stained with hematoxylin and eosin (H&E) and examined under light microscopy to evaluate structural alterations such as alveolar integrity, inflammation, and fibrosis.

For IHC detection of K_i-67, deparaffinized and rehydrated sections underwent antigen retrieval using heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide, followed by incubation with a primary monoclonal antibody against K_i-67 (clone MIB-1, dilution 1:100) overnight at 4°C. Visualization was achieved using a secondary biotinylated antibody and the 3,3′-diaminobenzidine (DAB) chromogen method, with hematoxylin counterstaining. Negative controls were included by omitting the primary antibody to ensure specificity.

Microscopic examination and quantitative analysis: All stained sections were examined using a light microscope at ×400 magnification. For IHC quantification, the alveolar regions were prioritized, and images were captured from at least 10 non-overlapping fields per sample using a digital camera attached to the microscope.

Image analysis was performed with QuPath software (version 0.4.0). Positive (brown-stained nuclei) and negative cells were counted within defined tissue areas (expressed in pixels2) using automated cell detection algorithms. The proliferative index was calculated as the percentage of K_i-67-positive cells relative to the total number of identified cells in each field, averaged across all fields per sample.

Statistical Analysis

Data were presented as mean values (M) ± standard error of the mean (m). Differences between control and experimental groups, as well as across age categories, were assessed using Student's t-test for unpaired samples. Statistical significance was defined as p < 0.05. All analyses were conducted using GraphPad Prism software (version 9.0).

Antibody details: The primary antibody used for K_i-67 IHC staining was a mouse monoclonal antibody, clone MIB-1, manufactured by Agilent Technologies (Dako). This clone is widely recognized for its specificity in detecting the K_i-67 antigen in proliferating cells. The antibody was diluted at a ratio of 1:100 in phosphate-buffered saline prior to use. Incubation was performed overnight (approximately 12–16 hours) at 4°C to ensure optimal binding while minimizing non-specific reactions.

Analysis Method

The quantitative analysis of K_i-67 expression was conducted using QuPath software (version 0.4.0), which employed automated cell detection algorithms for identifying and classifying nuclei. While the core process was fully automated—leveraging machine learning-based segmentation for nucleus detection and intensity measurement—it included manual oversight for quality control, such as verifying region annotations and excluding artifacts (e.g., necrotic areas or staining inconsistencies) to refine accuracy. This semi-automated approach helped maintain reliability across samples.

For determining “positive staining,” thresholds were set based on optical density (OD) measurements of the DAB chromogen. Specifically, a nucleus was classified as K_i-67-positive if its mean DAB OD exceeded 0.15, with a range typically between 0.1 and 0.2 to distinguish weak-to-strong staining from background noise. These values were calibrated empirically during initial script optimization in QuPath, using positive and negative control slides to balance sensitivity and specificity, avoiding over- or undercounting of proliferative cells.

Results

K_i-67 IHC Expression in Control Group Rats

In the control group, which consisted of healthy outbred rats without induced CRF, K_i-67 expression levels in lung alveolar tissue varied notably with age, reflecting natural physiological changes over time. For the 6-month-old rats, analysis of a tissue area measuring 529,856 px2 revealed a total of 1,769 cells, with 988 showing positive K_i-67 staining (brown nuclei) and 781 negative. This resulted in a proliferative index of 55.86%, indicating robust cellular proliferation typical of younger animals, where alveolar epithelial and stromal cells maintain high regenerative capacity ([Fig. 1]).

Fig. 1 Expression of the immunohistochemical marker K_i-67 in the lung tissue of 6-month-old outbred control rats, stained using the DAB chromogen method. Image magnified ×400. The slides were scanned and expression levels were determined using QuPath 0.4.0 software. Expressed cells are highlighted in red. DAB, 3,3′-diaminobenzidine.

As age increased to 9 months, proliferative activity showed a slight decline. Over a larger tissue area of 1,002,852 px2, 1,977 cells were identified, including 942 Ki-67-positive and 1,035 K_i-67-negative cells. The expression level was calculated at 52.32%, suggesting that although proliferation remains relatively strong, early signs of age-related slowdown in lung tissue renewal are evident compared with the younger cohort.

By 12 months, a more pronounced reduction was observed. In a 102,500 px2 area, only 350 cells were counted, of which 82 were Ki-67-positive and 268 negative, yielding a proliferative index of 23.44%. This sharp drop highlights diminished cellular division and regenerative potential in older rats, consistent with involutive processes in aging lung parenchyma ([Fig. 2]).

Fig. 2 Immunohistochemical expression of the K_i-67 marker in the lung tissue of a 12-month-old white outbred rats from the control group, stained using the DAB chromogen method. Image magnified ×400. The slides were scanned and analyzed using the QuPath-0.4.0.ink software to determine the level of expression. The positively expressed cells are shown in red. DAB, 3,3′-diaminobenzidine.

K_i-67 Expression in Experimental Group Rats with Induced CRF

In the experimental group, where CRF was induced via glycerol injection, K_i-67 expression was markedly suppressed across all age groups, underscoring the detrimental impact of renal dysfunction on pulmonary cellular proliferation. For 6-month-old rats, examination of a 524,340 px2 tissue section identified 979 cells, of which 213 were K_i-67 positive and 766 negative, resulting in a proliferative index of 21.75%. This represents a substantial decrease from the control group's 55.86% at the same age, indicating impaired tissue renewal likely due to systemic inflammation and oxidative stress from CRF ([Fig. 3]).

Fig. 3 Expression of the immunohistochemical marker K_i-67 in the lung tissue of 6-month-old outbred rats from the experimental group, stained using the DAB chromogen method. Image magnified ×400. The slides were scanned and expression levels were determined using QuPath 0.4.0 software. Expressed cells are shown in red. DAB, 3,3′-diaminobenzidine.

The 9-month-old experimental rats displayed even lower activity: in a 1,002,500 px2 area, 235 cells were detected, with just 26 positive and 209 negative, equating to an 11.06% expression level. This further decline suggests deepening regenerative deficits as CRF progresses in middle-aged animals.

In the 12-month-old group, proliferation was nearly absent. Over 523,369 px2, 234 cells were counted, including only seven positive and 227 negative, yielding a mere 2.32% proliferative index. These findings point to severe degenerative changes, such as fibrosis, dominating aged lungs under CRF conditions, with cellular proliferation effectively halted ([Fig. 4]).

Fig. 4 Immunohistochemical expression of the K_i-67 marker in the lung tissue of a 12-month-old white outbred rats from the experimental group, stained using the DAB chromogen method. Image magnified ×400. The slides were scanned and analyzed using the QuPath-0.4.0.ink software to determine the level of expression. The positively expressed cells are shown in red. DAB, 3,3′-diaminobenzidine.

Discussion

The findings of this study demonstrate that cellular proliferative activity in lung tissue is strongly influenced by both aging and CRF. In the control group, a gradual decline in K_i-67 expression with age was observed, consistent with physiological senescence and reduced regenerative potential.

In contrast, CRF caused a profound suppression of cellular renewal at all studied ages.[17] [18] The reduction in K_i-67 expression suggests impaired proliferative capacity of alveolar epithelium and stromal elements, which likely contributes to degenerative and fibrotic changes in the lung parenchyma. This is consistent with earlier reports linking systemic renal pathology to pulmonary remodeling and endothelial dysfunction.[19]

The present study demonstrates a significant age-dependent reduction in K_i-67 IHC expression in the alveolar regions of rat lung tissue under conditions of experimentally induced CRF. In control animals, proliferative activity declined progressively with age, from 55.86% at 6 months to 23.44% at 12 months, reflecting natural senescence-related slowdown in cellular renewal. However, CRF exacerbated this decline dramatically, dropping to 21.75, 11.06, and 2.32% across the respective age groups, indicating severe impairment in pulmonary cell proliferation.

The observed decrease in K_i-67 expression in CRF lungs can be attributed to several interconnected pathophysiological mechanisms, including hypoxia, inflammation, and hormonal alterations. Hypoxia, a hallmark of CRF, arises from anemia due to reduced erythropoietin production and impaired renal oxygenation, leading to systemic oxygen deprivation that suppresses cell cycle progression and the expression of proliferative markers, such as K_i-67 in distant organs, including the lungs. This is compounded by chronic inflammation, where elevated proinflammatory cytokines (e.g., IL-1β and TNF-α) and oxidative stress in CKD inhibit cellular proliferation by promoting apoptosis and cell cycle arrest, directly reducing K_i-67-positive cells in pulmonary tissues. Hormonal changes, such as disruptions in the renin-angiotensin-aldosterone system or glucocorticoid imbalances, further contribute by altering metabolic pathways that favor fibrosis over regeneration, limiting the entry of cells into active phases of the cell cycle. These mechanisms align with broader kidney–lung cross-talk, where renal dysfunction triggers pulmonary vascular and parenchymal changes via shared inflammatory and hypoxic pathways.[8] [20]

Our findings are consistent with prior research on renal–pulmonary interactions in experimental models. For instance, studies in adenine-induced CKD rodents have reported heightened lung inflammation and fibrosis, with reduced proliferative responses in alveolar cells, mirroring the suppressed K_i-67 we observed. Similarly, investigations into toxin-mediated lung injury have linked decreased K_i-67 to inflammatory processes, supporting the role of systemic CKD effects in pulmonary remodeling.[7] [12]

Clinically, these results have implications for CRF patients, who often exhibit pulmonary complications such as fibrosis and impaired gas exchange. The marked reduction in proliferative activity parallels the diffuse alveolar damage and fibrotic scarring seen in CKD-associated lung disease, where chronic hypoxia and inflammation lead to thickened alveolar walls, reduced diffusion capacity for carbon monoxide, and overall gas exchange deficits. In idiopathic pulmonary fibrosis comorbid with CKD, lower estimated glomerular filtration rates correlate with poorer survival and exacerbated fibrosis, suggesting that diminished cellular renewal—as indicated by low K_i-67—contributes to progressive lung deterioration and respiratory failure in these patients. Furthermore, metastatic calcifications in CRF lungs can impair gas exchange and promote fibrosis, aligning with our model's degenerative changes. Targeting proliferative pathways could offer therapeutic avenues to preserve lung function in CKD, potentially improving outcomes in this high-risk population.[20] [21] [22]

This study has several limitations. First, it relies on a rat model of CRF induced by glycerol, which may not fully replicate the multifactorial etiology of human CKD. Translational gaps exist, as rodent lungs differ anatomically and physiologically from human lungs, potentially overestimating or underestimating proliferative responses. Second, the sample size per subgroup (n = 25) and the focus on only three age groups limit generalizability, and long-term outcomes were not assessed. Third, although QuPath provided quantitative analysis, manual oversight introduces subjectivity, and molecular assays to confirm mechanisms, such as specific cytokine levels, were lacking. Future studies should incorporate human cohorts, advanced imaging, and broader therapeutic evaluations to address these constraints.

Building on the insights from this study, several avenues for future research emerge to deepen our understanding of kidney–lung interactions in CRF and to translate these findings into clinical applications. First, extending the investigation to human cohorts would be invaluable. While rat models provide a controlled environment to mimic CRF-induced pulmonary changes, prospective studies involving lung biopsies or bronchoalveolar lavage from CKD patients could validate the observed reductions in K_i-67 expression. Non-invasive techniques, such as high-resolution computed tomography combined with circulating biomarkers of proliferation (e.g., serum K_i-67 levels or exosomal miRNAs regulating cell cycle), could assess proliferative deficits in real-time, correlating them with clinical outcomes, such as forced vital capacity or diffusion capacity for carbon monoxide.

Second, delving into molecular mechanisms underlying the suppressed K_i-67 expression warrants further exploration. Advanced omics approaches, including RNA sequencing, proteomics, or single-cell RNA-seq on lung tissues from CRF models, could identify key pathways—such as hypoxia-inducible factor signaling, NF-κB-mediated inflammation, or TGF-β-driven fibrosis—that mediate proliferative arrest. Integrating these with CRISPR-based gene editing to knock out or overexpress candidate genes (e.g., those involved in erythropoietin deficiency or uremic toxin accumulation) would clarify causal relationships and pinpoint therapeutic targets.

Third, expanding the scope to include additional proliferative and fibrotic markers, such as PCNA, p53, or α-SMA, alongside K_i-67, could offer a more comprehensive profile of lung remodeling in CRF. Comparative studies across different CRF induction methods (e.g., adenine diet versus unilateral ureteral obstruction) and species (e.g., mice or non-human primates) would enhance model robustness and reveal species-specific responses. Additionally, investigating sex differences, as our study focused on male rats, could uncover gender-specific vulnerabilities in pulmonary proliferation under renal stress.

Finally, longitudinal studies tracking long-term pulmonary outcomes in CRF, including survival analysis and functional assessments (e.g., spirometry in larger animal models), would elucidate the prognostic value of K_i-67 as a biomarker. Incorporating machine learning to analyze IHC data from QuPath or similar tools could predict disease progression based on proliferative indices. Collaborative, multicenter trials bridging preclinical and clinical research will be essential to develop targeted therapies that address the kidney–lung axis, ultimately reducing the burden of respiratory complications in CKD patients worldwide.

Conclusion

In conclusion, this study provides compelling evidence of the profound impact of CRF on pulmonary cellular proliferation in a rat model, as assessed through K_i-67 IHC expression in alveolar lung tissue. Our findings reveal a clear age-dependent pattern in healthy control rats, where proliferative activity naturally diminishes over time—from a robust 55.86% in 6-month-old animals to 52.32% at 9 months and a marked 23.44% at 12 months—reflecting physiological aging processes that involve reduced cellular renewal and regenerative capacity in the lung parenchyma. This baseline decline underscores the vulnerability of older organisms to external stressors, aligning with established notions of senescence in respiratory tissues.

Under induced CRF conditions, however, K_i-67 expression was dramatically suppressed across all age groups, plummeting to 21.75% in 6-month-olds, 11.06% in 9-month-olds, and a near-negligible 2.32% in 12-month-olds. These results highlight the detrimental effects of kidney–lung cross-talk, in which systemic factors such as uremic toxins, chronic inflammation, oxidative stress, and hypoxia converge to inhibit cell cycle progression and promote degenerative changes, including fibrosis, in the lungs. The near-abolition of proliferation in older CRF rats suggests accelerated involution, potentially exacerbating pulmonary complications and mirroring the high morbidity seen in advanced CKD stages.

Clinically, these observations reinforce the need for integrated management of renal and respiratory health in CKD patients, where reduced lung proliferation may contribute to common complications, such as pulmonary fibrosis, edema, hypertension, and impaired gas exchange. By elucidating mechanisms of proliferative impairment, our work supports targeted interventions to preserve lung function, ultimately improving quality of life and prognosis in this population.

Future research should expand on these results by exploring molecular pathways (e.g., via RNA sequencing or cytokine profiling) to pinpoint the exact mediators of K_i-67 suppression, testing regimens in larger cohorts or other CKD models, and translating findings to human studies through biopsy analyses or non-invasive imaging. Addressing these gaps could refine therapeutic strategies and deepen understanding of multiorgan interactions in chronic diseases. Overall, this investigation advances the field by quantifying CRF's pulmonary repercussions at the cellular level, emphasizing proliferation as a key biomarker for organ crosstalk and recovery potential.

Conflict of Interest

None declared.

Acknowledgments

The authors would like to extend their gratitude to the laboratory members and professors of Bukhara State Medical Institute for their valuable insights and constructive feedback on this article.

Authors' Contributions

T.T.B.U. collected the data. A.D.B. prepared the manuscript draft. K.D.A. reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript.

Artificial intelligence (AI) tools, including language models, have been used solely for linguistic purposes, such as translation and grammatical correction. No part of the scientific content, data analysis, interpretation, or conclusions was generated using AI.

Declaration of GenAI Use

Artificial intelligence (AI) tools, including language models, have been used solely for linguistic purposes, such as translation and grammatical correction. No part of the scientific content, data analysis, interpretation, or conclusions was generated using AI.

Compliance with Ethical Principles

Ethical approval was obtained from the Local Ethics Committee of Bukhara State Medical Institute (Protocol No. 5, November 27, 2024; Extract No. 12028 dated December 02, 2024).

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Publication History

Article published online:
12 January 2026