Chronic kidney disease (CKD) is associated with high cardiovascular mortality that cannot be explained solely by traditional risk factors. A key CKD-specific pathway involves CKD–mineral and bone disorder (CKD–MBD), a syndrome encompassing abnormalities of calcium–phosphate balance, parathyroid hormone excess, vitamin D deficiency, and altered bone turnover. Beyond skeletal consequences, CKD–MBD is closely linked to extra-skeletal calcification, particularly vascular calcification (VC), which contributes to arterial stiffness, hypertension, left ventricular hypertrophy, and ischemic events.

Vascular calcification in CKD is increasingly recognized as an active, regulated process resembling bone formation. Elevated phosphate levels, oxidative stress, inflammation, and endocrine disturbances promote osteogenic differentiation of vascular smooth muscle cells, leading to deposition of calcium–phosphate mineral in the vascular wall. While imaging modalities such as plain radiography and CT quantify calcification, they are not always suitable for frequent monitoring. Biochemical markers provide indirect evidence of risk, yet they may not fully capture molecular mechanisms driving vascular calcification.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by modulating translation and mRNA stability. Circulating miRNAs are stable in blood due to packaging in exosomes and association with proteins, making them attractive biomarker candidates. Several miRNAs have been implicated in fibrosis, inflammation, and vascular remodeling—processes relevant to CKD and VC. miR-21, for instance, is associated with inflammatory signaling and tissue remodeling; miR-223 is linked with immune regulation; and miR-125b has been reported to exert protective effects against osteogenic transformation in vascular tissues. However, clinical evidence integrating miRNA patterns with CKD–MBD markers and calcification severity across CKD stages remains limited.

The present study sought to identify circulating miRNA signatures in CKD and explore their relationship with CKD–MBD parameters and vascular calcification, aiming to clarify their potential as accessible, non-invasive biomarkers for early detection and risk stratification.

This prospective, hospital-based, observational, analytical, cross-sectional study was conducted in the nephrology outpatient department and dialysis unit in collaboration with the Department of Biochemistry at an index medical hospital.

Study Duration

Total duration was 24 months:

• Recruitment: 18 months
• Laboratory analysis: 4 months
• Data analysis and interpretation: 2 months

Participants

A total of 400 participants were enrolled:

• CKD group (n=300): CKD stages 3–5 according to KDIGO criteria, including patients on dialysis
• Control group (n=100): apparently healthy age- and sex-matched individuals with eGFR ≥90 mL/min/1.73 m² and normal calcium, phosphate, and PTH

Inclusion Criteria

CKD group: adults ≥18 years, CKD stage 3–5, stable renal function for ≥3 months, consent provided.
Controls: age- and sex-matched, normal renal function, no known metabolic bone disease.

Exclusion Criteria

Acute kidney injury; active infection/inflammatory disease; malignancy; chronic liver disease; autoimmune/rheumatologic disorders; recent fractures (<6 months); medications affecting bone metabolism (bisphosphonates, denosumab, steroids); pregnancy/lactation.

Clinical and Laboratory Data Collection

Demographic and clinical information included CKD duration and etiology, comorbidities (diabetes, hypertension, cardiovascular disease), medication history (vitamin D analogs, phosphate binders, calcimimetics), and dialysis status.

Fasting venous blood (8–10 mL) was collected and separated into EDTA and plain vacutainers. Serum calcium, phosphate, ALP, urea, and creatinine were measured using automated analyzers. iPTH was measured by chemiluminescent immunoassay, and 25(OH) vitamin D by ELISA.

MicroRNA Quantification

Serum aliquots for miRNA analysis were stored at −80°C. Total RNA including small RNA was extracted using a commercial miRNA isolation kit. RNA concentration and purity were assessed spectrophotometrically. Reverse transcription was performed using miRNA-specific primers, followed by qRT-PCR using SYBR Green chemistry. Expression values were normalized to an endogenous control (U6), and relative expression was calculated using the 2⁻ΔΔCt method.

Vascular Calcification Assessment

Vascular calcification was assessed using lateral abdominal radiography with validated scoring systems or multislice CT for coronary calcium scoring where available. Calcification severity was categorized as none, mild, moderate, or severe.

Quality Control

All assays were performed in duplicate with equipment calibration, blinded miRNA analysis, and periodic internal quality audits.

Statistical Analysis

Data were entered in Microsoft Excel and analyzed using SPSS v26. Continuous variables were expressed as mean±SD or median (IQR), and categorical variables as percentages. Comparisons used t-test/Mann–Whitney and ANOVA/Kruskal–Wallis as appropriate. Correlations were assessed using Pearson/Spearman coefficients. Multivariate regression identified independent predictors of VC. ROC analysis evaluated diagnostic performance. p<0.05 was considered significant.

A total of 400 participants (300 CKD, 100 controls) completed the study. Groups were well matched for age, sex distribution, and BMI. Among CKD patients, diabetes and hypertension were highly prevalent, reflecting typical CKD epidemiology. CKD stages were nearly equally represented, supporting balanced subgroup analysis.

CKD–MBD Biochemical Profile

CKD patients demonstrated clear biochemical evidence of CKD–MBD, with significantly lower calcium and vitamin D and significantly higher phosphate, ALP, and iPTH compared with controls (all p<0.001). Stage-wise analysis revealed progressive worsening of phosphate and iPTH with advancing CKD, consistent with increasing mineral dysregulation as kidney function declines.

Circulating miRNA Dysregulation

Circulating miRNA profiling showed a distinct pattern in CKD:

• miR-21 and miR-223 were markedly upregulated compared to controls.
• miR-125b was significantly downregulated. Moreover, miR-21 increased progressively across CKD stages, while miR-125b declined with CKD progression, suggesting that worsening renal function is accompanied by escalating pro-calcific signaling and diminishing protective regulation.

Vascular Calcification Burden

Vascular calcification was common: approximately half of CKD patients exhibited moderate to severe calcification. The prevalence of moderate–severe VC increased significantly with CKD stage (p<0.001), underscoring the contribution of advancing CKD to calcification burden.

Associations Between miRNAs and CKD–MBD Markers

Correlation analysis demonstrated that miR-21 had strong positive relationships with phosphate and iPTH, and a negative relationship with vitamin D (p<0.001). miR-125b demonstrated inverse correlations, supporting the concept that it may be linked with anti-calcific or protective pathways.

Independent Prediction and Diagnostic Performance

In multivariate regression, miR-21 remained an independent predictor of vascular calcification after adjustment for phosphate, iPTH, and diabetes mellitus. ROC curve analysis further showed that miR-21 provided the best discrimination for severe calcification (AUC 0.84), indicating clinically meaningful diagnostic accuracy.

Table 1. Baseline Demographic Characteristics

No significant differences in age, sex distribution, or BMI indicate appropriate matching.

Table 2. Clinical Characteristics of CKD Patients

Diabetes and hypertension were the predominant comorbidities.

Table 3. Distribution of CKD Stages

*Included in Stage 5 analysis.

Nearly equal distribution across stages supported balanced subgroup comparisons.

Table 4. Biochemical Parameters: CKD vs Controls

CKD patients had significant CKD–MBD abnormalities (hyperphosphatemia, elevated iPTH, and vitamin D deficiency).

Table 5. Biochemical Parameters Across CKD Stages

Mineral abnormalities worsened progressively with advancing CKD stage.

Table 6. Relative Expression of Circulating miRNAs

Pro-calcific miRNAs (miR-21, miR-223) were upregulated while miR-125b was downregulated in CKD.

Table 7. miRNA Expression Across CKD Stages

miRNA dysregulation increased with CKD severity (miR-21 increased; miR-125b decreased).

Table 8. Prevalence of Vascular Calcification in CKD Patients

Approximately half the CKD cohort showed moderate–severe calcification.

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

VC burden increased strongly with advancing CKD stage.

Table 10. Correlation Between miRNAs and Mineral Parameters

*p < 0.001

miR-21 aligned with a pro-calcific biochemical profile; miR-125b showed inverse (potentially protective) associations.

Table 11. Multivariate Regression Analysis for Predictors of Vascular Calcification

miR-21 remained an independent predictor of vascular calcification.

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

miR-21 demonstrated the best diagnostic accuracy for severe vascular calcification.

This study demonstrates three interlinked phenomena in CKD patients: (1) significant biochemical CKD–MBD abnormalities, (2) high prevalence of vascular calcification that increases with CKD stage, and (3) distinct circulating miRNA dysregulation associated with both mineral markers and calcification severity.

The observed mineral profile—hyperphosphatemia, elevated iPTH, and vitamin D deficiency—reflects expected CKD–MBD physiology. As renal function declines, phosphate excretion is impaired and vitamin D activation decreases, promoting hypocalcemia and secondary hyperparathyroidism. These endocrine and mineral disturbances do not remain confined to the skeleton; rather, they directly influence vascular tissue through calcification-promoting pathways.

A notable finding was the robust performance of miR-21. Its progressive increase across CKD stages, strong correlation with phosphate and iPTH, and independent predictive value for vascular calcification suggest that miR-21 may capture molecular processes beyond traditional biochemical measurements. Mechanistically, miR-21 has been implicated in inflammatory and fibrotic signaling and vascular remodeling—processes relevant to calcification progression. While the present study was not designed to confirm mechanistic causality, the clinical associations support the hypothesis that miR-21 reflects a pro-calcific milieu in CKD.

In contrast, miR-125b was consistently reduced and inversely associated with mineral disturbances. This pattern aligns with a potential protective role for miR-125b in vascular smooth muscle biology, where reduced expression may permit osteogenic transformation and calcification progression.

miR-223 was also upregulated and demonstrated moderate predictive accuracy for severe calcification. Given its link with immune regulation, miR-223 may reflect inflammatory activation contributing to vascular pathology, though its predictive value appeared lower than miR-21 in this dataset.

ROC analysis showed miR-21 as the strongest discriminator for severe calcification (AUC 0.84). This suggests a possible future role for miR-21 as a screening or risk stratification marker to identify CKD patients who may benefit from closer vascular monitoring or early interventions aimed at mineral correction and cardiovascular risk reduction.

Circulating microRNAs show significant dysregulation in CKD patients and are closely associated with CKD–MBD biochemical abnormalities and vascular calcification severity. miR-21, in particular, demonstrates strong correlations with phosphate and iPTH, independently predicts vascular calcification, and provides good diagnostic accuracy for severe calcification. These findings support the potential of miRNA profiling—especially miR-21—as a non-invasive biomarker approach for early detection and risk stratification of vascular calcification in CKD.

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