Chronic kidney disease (CKD) is a major global health burden with rising prevalence due to increasing rates of diabetes mellitus, hypertension, and aging populations. CKD is associated with a range of complications affecting nearly every organ system, but cardiovascular disease remains the leading cause of mortality in this population. The relationship between CKD and cardiovascular disease is multifactorial and includes traditional risk factors such as hypertension, dyslipidemia, and diabetes, as well as non-traditional risk factors unique to CKD.

One of the most important non-traditional risk factors is CKD–mineral and bone disorder (CKD–MBD). CKD–MBD is characterized by abnormalities in mineral metabolism (calcium, phosphate), hormonal disturbances (parathyroid hormone and vitamin D), and alterations in bone turnover. Importantly, CKD–MBD is strongly linked to extra-skeletal calcification, particularly vascular calcification (VC). Vascular calcification is not a passive process but rather an actively regulated phenomenon resembling bone formation. It involves phenotypic switching of vascular smooth muscle cells into osteoblast-like cells, deposition of calcium-phosphate crystals in the vascular wall, and inflammatory and oxidative mechanisms.

The clinical significance of vascular calcification in CKD cannot be overstated. It contributes to arterial stiffness, increased pulse pressure, left ventricular hypertrophy, ischemic heart disease, heart failure, and increased mortality. Conventional imaging techniques such as radiography and CT can identify calcification, but these may not be feasible for frequent monitoring. Moreover, biochemical markers like phosphate, calcium, and iPTH provide indirect insights but may not fully reflect ongoing molecular changes driving vascular calcification.

In recent years, microRNAs (miRNAs) have gained attention as potential regulators of CKD progression and cardiovascular complications. miRNAs are short non-coding RNA molecules (~22 nucleotides) that regulate gene expression post-transcriptionally by binding to messenger RNA and inhibiting translation or promoting degradation. Circulating miRNAs are stable in blood, can be quantified reliably, and are increasingly studied as biomarkers in cardiovascular disease, malignancy, and metabolic disorders.

Among miRNAs, miR-21 has been associated with fibrosis, inflammation, and vascular remodeling. miR-223 has been linked to immune modulation and inflammatory pathways. miR-125b has been suggested to have protective roles against vascular smooth muscle osteogenic transformation. However, their specific relationship with CKD–MBD biochemical abnormalities and vascular calcification severity across CKD stages requires further evaluation, especially in clinical settings.

Therefore, this study was designed to evaluate circulating miRNA expression patterns in CKD patients and determine their association with mineral bone disorder markers and vascular calcification burden. The findings may contribute to the development of non-invasive biomarkers for early detection and monitoring of vascular calcification and improved cardiovascular risk stratification in CKD.

Study Design

This was a prospective, hospital-based, observational, analytical, cross-sectional study conducted in the nephrology outpatient department and dialysis unit in collaboration with the Biochemistry Department of an index medical hospital. The study was designed to evaluate the association between circulating microRNAs, mineral bone disorder parameters, and vascular calcification in patients with chronic kidney disease.

Study Duration

The total study duration was 24 months, including:

• 18 months for patient recruitment
• 4 months for laboratory analysis
• 2 months for data analysis and interpretation

Source of Data

Data were obtained from:

• Adult patients diagnosed with CKD attending the nephrology outpatient clinic and dialysis unit
• Age- and sex-matched healthy volunteers recruited from hospital staff and patient attendants

Clinical data, biochemical parameters, imaging findings, and molecular analysis results were prospectively collected using a structured case record form.

Sample Size Calculation

Based on previous studies demonstrating a moderate correlation (r = 0.35–0.45) between circulating microRNAs and vascular calcification scores, the sample size was calculated using the formula for correlation studies. Assuming:

• Confidence level (α) = 95%
• Power (1−β) = 90%
• Expected correlation coefficient (r) = 0.35

The minimum sample size required was calculated as 230 CKD patients. To compensate for dropouts and inadequate samples, the sample size was increased by 15%.

Final sample size:

• CKD patients: 300
• Healthy controls: 100
• Total participants: 400

Study Population

CKD Group (n = 300)

Patients were stratified according to KDIGO CKD stages:

• Stage 3: ~100 patients
• Stage 4: ~100 patients
• Stage 5 (including dialysis): ~100 patients

Control Group (n = 100)

Apparently healthy individuals with normal renal function and no known metabolic bone disease.

Inclusion Criteria

CKD Patients

• Age ≥18 years
• Diagnosed CKD stages 3–5 as per KDIGO guidelines
• Stable renal function for at least 3 months
• Willing to provide informed consent

Controls

• Age- and sex-matched healthy individuals
• eGFR ≥90 mL/min/1.73 m²
• Normal serum calcium, phosphate, and PTH

Exclusion Criteria

• Acute kidney injury
• Active infection or inflammatory disease
• Known malignancy
• Chronic liver disease
• Autoimmune or rheumatologic disorders
• Recent fractures (<6 months)
• Use of medications affecting bone metabolism (bisphosphonates, denosumab, steroids)
• Pregnant or lactating women

METHODOLOGY

Clinical Assessment

Detailed clinical evaluation included:

• Demographic details
• Duration and etiology of CKD
• Comorbidities (diabetes mellitus, hypertension, cardiovascular disease)
• Medication history (vitamin D analogs, phosphate binders, calcimimetics)
• Dialysis status and duration (if applicable)

Sample Collection and Processing

Blood Collection

• 8–10 mL of fasting venous blood was collected under aseptic conditions.
• Samples were divided into EDTA and plain vacutainers.

Biochemical Analysis

• Serum calcium, phosphate, ALP, urea, creatinine were measured using automated analyzers.
• Intact PTH was measured by chemiluminescent immunoassay.
• 25-hydroxyvitamin D was measured by ELISA.

Sample Storage

Serum samples for miRNA analysis were aliquoted and stored at −80°C until analysis.

MicroRNA Extraction and Quantification

• Total RNA, including small RNA, was extracted using a commercial miRNA isolation kit.
• RNA concentration and purity were assessed using spectrophotometry.
• Reverse transcription was performed using miRNA-specific primers.
• Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green chemistry.
• Expression levels were normalized to endogenous control miRNA (U6).
• Relative expression was calculated using the 2⁻ΔΔCt method.

Assessment of Vascular Calcification

Vascular calcification was assessed using:

• Plain radiographs (lateral abdominal X-ray) using validated scoring systems OR
• Multislice CT (where available) for coronary artery calcium scoring

Patients were categorized into:

• No calcification
• Mild calcification
• Moderate calcification
• Severe calcification

Statistical Analysis

Data entered into Microsoft Excel and analyzed using SPSS version 26.0. Continuous variables expressed as mean ± SD or median (IQR). Categorical variables expressed as percentages. Comparison between groups using Student’s t-test / Mann–Whitney U test and One-way ANOVA / Kruskal–Wallis test. Correlation assessed using Pearson or Spearman correlation coefficients. Multivariate regression analysis performed to identify independent predictors. ROC curve analysis used to assess diagnostic performance of miRNAs. p < 0.05 considered statistically significant

A total of 400 participants were included: 300 CKD patients (Stages 3–5) and 100 healthy controls. All samples were analyzed successfully with no major protocol deviations.

Table 1. Baseline Demographic Characteristics of Study Population

There was no statistically significant difference in age, sex distribution, or BMI between CKD patients and controls, indicating appropriate matching.

Table 2. Clinical Characteristics of CKD Patients

Hypertension and diabetes were the most common comorbidities, reflecting typical CKD epidemiology.

Table 3. Distribution of CKD Stages

                                                                *Included in Stage 5 analysis.

                                    Nearly equal representation across CKD stages ensured balanced subgroup analysis.

Table 4. Biochemical Parameters: CKD vs Controls

CKD patients showed significant mineral bone disorder with hyperphosphatemia, secondary hyperparathyroidism, and vitamin D deficiency.

Table 5. Biochemical Parameters Across CKD Stages

                           There was a progressive worsening of mineral abnormalities with advancing CKD stage.

Table 6. Relative Expression of Circulating miRNAs

Pro-calcific miRNAs were significantly upregulated, while protective miRNAs were downregulated in CKD.

Table 7. miRNA Expression Across CKD Stages

miRNA dysregulation increased progressively with CKD severity.

Table 8. Prevalence of Vascular Calcification in CKD Patients

Approximately 50% of CKD patients had moderate to severe vascular calcification.

Table 9. Association Between CKD Stage and Moderate–Severe Vascular Calcification

Advanced CKD stages were strongly associated with higher vascular calcification burden.

Table 10. Correlation Between miRNAs and Mineral Parameters

                                                               *p < 0.001

miRNAs showed strong correlations with key CKD–MBD markers.

Table 11. Multivariate Regression Analysis for Predictors of Vascular Calcification

miR-21 emerged as an independent predictor of vascular calcification.

Table 12. ROC Curve Analysis of miRNAs for Predicting Severe Calcification

miR-21 demonstrated good diagnostic accuracy for identifying severe vascular calcification

This study investigated circulating microRNAs in CKD patients and their association with mineral bone disorder parameters and vascular calcification. The key findings include: (1) CKD patients exhibited significant CKD–MBD biochemical derangements compared to controls, (2) circulating miRNAs showed distinct dysregulation patterns in CKD, (3) vascular calcification was highly prevalent and increased with CKD stage, and (4) miR-21 emerged as a strong independent predictor of vascular calcification with good diagnostic accuracy.

In this study, CKD patients demonstrated significantly lower serum calcium and vitamin D levels, along with elevated phosphate, ALP, and iPTH. These findings are consistent with the pathophysiology of CKD–MBD, where declining renal function results in phosphate retention and reduced calcitriol synthesis. Phosphate retention stimulates fibroblast growth factor-23 and contributes to hypocalcemia, triggering secondary hyperparathyroidism. Over time, this hormonal imbalance contributes to high bone turnover, vascular calcification, and cardiovascular risk.

Stage-wise analysis showed progressive worsening of phosphate and iPTH levels across CKD stages. This confirms that mineral metabolism abnormalities intensify with CKD progression and supports the importance of early monitoring and intervention.

Circulating miRNAs have emerged as promising biomarkers due to their stability in blood and association with disease pathways. In the present study, miR-21 and miR-223 were significantly upregulated in CKD patients, while miR-125b was downregulated. These patterns suggest that CKD is associated with activation of pro-inflammatory and pro-calcific molecular pathways, along with suppression of protective regulatory mechanisms.

The stage-wise trend in miRNA expression further supports their relationship with CKD severity. miR-21 expression increased progressively from stage 3 to stage 5, suggesting that worsening renal dysfunction and mineral abnormalities may contribute to increased miR-21 release or expression. Conversely, miR-125b decreased progressively, suggesting loss of protective anti-calcific signaling.

Vascular calcification was prevalent in the CKD cohort, with nearly half of patients exhibiting moderate to severe calcification. This finding is clinically important because vascular calcification strongly predicts cardiovascular events and mortality in CKD patients. The association between CKD stage and vascular calcification severity was statistically significant, with stage 5 patients showing the highest burden. This aligns with the known progression of calcification as kidney function declines and CKD–MBD becomes more pronounced.

A major strength of this study is the correlation analysis demonstrating that miR-21 showed strong positive correlations with serum phosphate and iPTH, and a negative correlation with vitamin D. These results suggest that miR-21 may reflect the metabolic environment promoting calcification. Elevated phosphate is a direct promoter of vascular smooth muscle osteogenic differentiation, while elevated iPTH reflects secondary hyperparathyroidism and altered bone turnover. Vitamin D deficiency contributes to inflammation and endothelial dysfunction and may further worsen calcification risk.

miR-125b showed inverse correlations with phosphate and iPTH and positive correlation with vitamin D, supporting the possibility that miR-125b plays a protective role against vascular calcification pathways.

Multivariate regression analysis identified miR-21 as an independent predictor of vascular calcification even after adjusting for phosphate, iPTH, and diabetes mellitus. This suggests that miR-21 may provide additional information beyond traditional biochemical markers. Diabetes mellitus also emerged as a significant predictor, consistent with its role in vascular damage and accelerated calcification.

ROC curve analysis demonstrated that miR-21 had the highest AUC (0.84), indicating good diagnostic accuracy for predicting severe vascular calcification. This supports the potential clinical utility of miR-21 as a biomarker for identifying high-risk CKD patients. miR-223 and miR-125b also demonstrated predictive ability, though to a lesser extent.

The findings of this study suggest that miRNA profiling may complement biochemical and imaging assessments in CKD patients. A non-invasive blood-based biomarker such as miR-21 could help in early identification of patients at high risk for vascular calcification, enabling timely intervention. Additionally, miRNAs may serve as targets for future therapies aimed at preventing vascular calcification progression.

This prospective cross-sectional study demonstrates significant dysregulation of circulating microRNAs in CKD patients, with strong associations between miRNA expression, CKD–MBD biochemical abnormalities, and vascular calcification severity. miR-21 was significantly upregulated, correlated strongly with phosphate and iPTH, independently predicted vascular calcification, and demonstrated good diagnostic performance for severe calcification. These findings support the potential role of circulating miRNAs, particularly miR-21, as non-invasive biomarkers for cardiovascular risk stratification and monitoring in CKD patients.

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