CORRELATION OF HISTOMORPHOMETRIC PARAMETERS OF VENOUS WALL WITH AVF NON-MATURATION IN NON-DIABETIC CHRONIC KIDNEY DISEASE

The structural integrity of the venous wall plays a pivotal role in the maturation of arteriovenous fistulas (AVFs), which remains the Achilles heel. Despite appropriate preoperative vascular mapping and advancements in surgical techniques, arteriovenous fistula (AVF) non-maturation occurs in 20–60% of patients. Pre-AVF creation ultrasound assessments primarily focus on arterial and venous diameters, but often overlook the role of venous and arterial wall morphology in fistula outcomes. Ankle–brachial index (ABI) can act as a prognostic marker and influence AVF outcome by identifying underlying peripheral arterial disease. Further, shear stress experienced by the venous wall also plays an important role in AVF maturation. Adequate wall shear stress (WSS) promotes endothelial nitric oxide–mediated remodeling and venous maturation, while low or disturbed WSS drives neointimal proliferation and hyperplasia leading to stenosis and non-maturation.

Pre-existing abnormalities in the venous wall, particularly intimal hyperplasia and medial fibrosis, significantly influence the vascular remodeling process after AVF creation, predisposing to its primary or secondary failure assessment of these features provides objective insights into the vein’s adaptive potential. Quantitative morphometry with digital software like ImageJ enables precise, reproducible measurements of venous wall layers and extracellular matrix, providing quantified data that can be correlated with outcomes. Previous studies have emphasized descriptive findings of venous wall histology, but did not quantify detailed venous wall morphometry to subsequent AVF maturation. Further, no one has systematically quantified the preoperative venous wall (e.g., intima/media area ratio, fibrosis burden) and tested its independent association with AVF non-maturation, particularly in a non-diabetic CKD cohort.

The present study examined the correlation of pre-operative venous wall morphometry with AVF non-maturation in non-diabetic CKD. In parallel, it also evaluated the utility of parameters such as luminal stenosis and intima-media ratio (IMR), and their correlation with systemic and local hemodynamic variables, including ankle-brachial index (ABI) and wall shear stress (WSS), which helped in developing a unified predictive model for AVF non-maturation.

Study Design and Setting

This was a prospective, single-center observational study conducted from October 2023 to December 2024 at the Department of Nephrology of a tertiary teaching institution after obtaining approval from the institutional ethics committee.

Sample Size Calculation

The sample size was calculated based on the assumption of a moderate correlation (r = 0.3) between histological variables and AVF maturation outcomes according to a previous study using G-Power (3.1.9.4). Using a two-tailed α of 0.05 and a power (1–β) of 80%, the minimum required sample size was calculated as 84. Accounting for potential dropouts, loss to follow-up with a 25% attrition rate, a sample size of 104 was considered for enrollment.

Study population.

Adults aged 18 to 60 years with non-diabetic CKD stage 4 and 5, who were planned for AVF creation (radio cephalic or brachiocephalic), were included. Diabetes mellitus (type 1 or type 2), dialysis vintage more than 3 months, presence of peripheral vascular disease, and anticipated need for brachio-basilic or two-stage AVF creation were excluded.

Data collection

Pre-operative assessments

All consenting eligible patients underwent detailed demographic and clinical evaluation to obtain data on age, sex, etiology of CKD, body mass index, blood pressure, and medication history. Laboratory investigations included hemoglobin, serum creatinine, calcium, phosphate, and intact parathyroid hormone (iPTH)were also performed thereafter.

On the day of surgery, all patients underwent preoperative, ultrasound-guided venous mapping of both forearms using a linear array probe (5–10 MHz). The radial and brachial arteries and the cephalic or basilic veins were evaluated for diameter, depth, patency, and the presence of thrombus. In addition, the ABI was measured with a Sonosite ultrasound Doppler probe (5-10 MHz) after a 10-minute rest by obtaining systolic blood pressures at both brachial arteries and at ankle arteries (posterior tibial or dorsalis pedis) in the supine position with the heart and leg at heart level with an appropriately sized cuff. The ABI was calculated as the mean of three ankle systolic pressures divided by the mean of three brachial systolic pressures.

Surgical Procedure and Tissue Collection

AVF creation was performed under 2% lignocaine using a standard side-to-side anastomosis technique at either the radio-cephalic or brachiocephalic site, depending on vessel suitability. All procedures were performed by an experienced Nephrologist with a consistent technique to minimize surgical variability. After obtaining consent from the patient, a 3–5 mm segment of the vein distal to the site of AVF anastomosis was excised.  Sample was transported to lab in 10% neutral buffered formalin and submitted for histological processing. Post-surgery AVF were examined for the presence of thrill and bruit. In the absence of post-operative thrill, venous dilatation with a dilator is followed by heparinized saline flush from the distal end of the vein used for AVF anastomosis. In the absence of thrill or bruit thereafter, defined as surgical failure. All were advised routine post-operative care, including prevention of hypotension.

Postoperative Follow-up and Maturation Assessment

All were followed up clinically and radiologically for a period of 12 weeks i.e.  2nd, 6th, and 12thweeks. At 2 weeks, sutures were removed, and the AVF was inspected for complications such as infection, hematoma, and gangrene. Thrill and bruit were checked. At the 6th week, a thorough physical exam was followed by a Doppler ultrasound to assess AVF maturation clinically and radiologically. The patient lay supine with the arm externally rotated ~45°, and internal diameter and depth were measured in B‑mode at the AVF body ~5 cm proximal to the anastomosis; peak systolic velocity (Vmax) was obtained at a 60° Doppler angle, and access flow (mL/min) was auto-calculated from mean velocity and cross-sectional area, with three measurements averaged per parameter. Wall shear stress (WSS) is a temporal change in shear forces acting on the venous wall. This was estimated using Doppler-derived parameters and recorded. The formula used for calculation of  WSS was 2μVmax/R, where μ represents the dynamic blood viscosity (assumed to be 2.7 centipoise), Vₘₐₓ is the peak systolic velocity in the vein, and R is the vessel radius.

Radiologic maturation was established during that period if AVF blood flow ≥500 mL/min and diameter ≥5 mm, and clinical maturation was defined as successful two-needle cannulation for three consecutive dialysis sessions. At week 12, physical and Doppler assessments were repeated, applying the same radiologic thresholds and clinical criteria as above. Further, AVFs not meeting these criteria were classified as primary AVF failure.

Histological Processing and Morphometric Analysis

Venous tissue samples transported from the OT were embedded in paraffin, sectioned at 4 μm thickness, and stained using hematoxylin and eosin (H&E) and Masson’s trichrome stains. H&E provided structural details of intimal and medial layers, while Masson’s trichrome highlighted collagen deposition, useful for assessing fibrosis.

Quantitative morphometric analysis was performed using NIH(National Institute of Health) ImageJ software. Images were captured at 4x and 10x magnifications under standardized lighting.  Parameters measured were 1. Medial fibrosis: Percentage of the medial area occupied by collagen (blue-stained regions in trichrome-stained sections). 2. Intimal hyperplasia: Calculated as the intimal area divided by the total vessel wall area. 3. Luminal stenosis: Ratio of intimal thickness to combined intima + media thickness, and 4. Intima-media ratio (IMR): Ratio of intimal to medial thickness at the point of maximal intimal proliferation.

Morphometric analysis was performed on venous sections using ImageJ (NIH, Bethesda, MD, USA) to quantify the Intima–Media Ratio (IMR), percentage medial fibrosis, percentage intimal hyperplasia, luminal area, and percentage luminal stenosis. Stained images captured at 4× and 10× magnification were opened and calibrated to the 100 µm scale bar by drawing a reference line and setting the pixel-to-micron conversion to ensure accurate measurements. To facilitate layer differentiation, images were converted to 8‑bit grayscale and segmented by threshold using ImageJ’s Threshold tool, creating masks for the lumen, intima, and media; regions were outlined with polygon tools, measured via the analysis functions, and managed using the ROI (region of interest) Manager. IMR was obtained by measuring intimal and medial thicknesses with the line tool and computing Intima Thickness/Media Thickness; medial fibrosis was quantified by isolating fibrotic regions within the media using color thresholding (blue color) and expressing the fibrotic area as a percentage of total medial area. Intimal hyperplasia was calculated as the percentage change of measured intimal area relative to a reference intimal area and was also represented as luminal stenosis categories by degree of luminal narrowing. Percentage luminal stenosis was calculated as (Total Vessel Area − Luminal Area)/Total Vessel Area × 100 after outlining both areas. All measurements were recorded (with repeated measures averaged where applicable), exported as CSV files for statistical analysis, and annotated images were saved in TIFF format for documentation.

Independent observers performed the measurements, and mean values were used. Inter-observer agreement was confirmed using intra-class correlation coefficients (ICCs).

Statistical Analysis

All data were entered and analyzed using IBM SPSS Statistics for Windows, Version 26.0. Normality of continuous variables was assessed using the Kolmogorov-Smirnov test. Normally distributed data were expressed as mean ± standard deviation, and non-parametric data as median (interquartile range).Comparisons between groups (matured vs. non-matured) were made using the independent samples t-test or Mann-Whitney U test, as appropriate. Categorical variables were analyzed using the Chi-square or Fisher’s exact test. Correlations between continuous variables were assessed using Pearson’s or Spearman’s correlation coefficients. Multivariate logistic regression analysis was performed to identify independent predictors of non-maturation. Variables with p < 0.1 in univariate analysis were entered into the regression model. Operating Characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of morphometric variables and to determine optimal cutoff values for predicting AVF non-maturation.

A total of 259 CKD patients were screened for native arteriovenous fistula (AVF) creation. After standardized vascular mapping and ankle–brachial index (ABI) assessment, 124 patients met the eligibility criteria and were enrolled. Venous tissue biopsy specimens were obtained intraoperatively from 104 enrolled patients. Postoperative clinical assessments were conducted at 2, 6, and 12 weeks (Figure 1).

Demographics and Baseline Characteristics.

Demographic variables such as age, gender, body mass index, blood pressure, and comorbidities were similar between the maturation and non-maturation group. There were no significant differences in biochemical parameters at baseline. iPTH levels were significantly higher in the non-matured group. Additionally, ABI values were notably lower in the non-matured group as depicted in Table 1.

Preoperative screening, intra-op vascular characteristic, and post-operative dilatation.

Preoperative Doppler evaluation showed comparable arterial and venous diameters across both groups. However, intraoperative arterial wall calcification was significantly higher in non-matured patients (42.8%) compared to matured AVFs (8.7%; p = 0.04). Post-operative dilatation was performed in 44 (42.3%) patients.

Postoperative Hemodynamics and Functional Maturation

On follow up, AVF maturation occurred in 84 (80.8%), matured AVFs showed significant increase in flow, diameter and higher WSS value between 6th and 12th week when compared with matured AVFs. It shows adequate venous remodeling in matured AVF in contrast to non-matured AVF (Table 2). There was non-significant difference of maturation rates in AVFs with (84.1%) or without (78.3%) post-operative dilatation (χ²=0.235, p=0.628).

Histo-morphometric Analysis of Venous Tissue

On histopathological examination of the venous tissue, complete circumferential architecture of the vein was present in 81(77.0%) specimens. The remaining segments exhibited partial circumferential representation, ranging from approximately 10% to 90% of the vessel wall. On histo-morphometric evaluation by ImageJ, pre-existing intimal hyperplasia and medial fibrosis was observed in 36%and 67% of total venous tissue respectively. There were significantly higher percentage of intimal hyperplasia (90%) and medial fibrosis (80%) in non-matured AVFs when compared to matured AVF where it was 21% and 61% respectively. Further, there was significant higher prevalence of intimal hyperplasia in non-matured AVF (90%vs 21%, p=0.000) and significant correlation was seen between prevalence of IH and MF with non-maturation of AVF (Figure 2a and Figure 2b).

In further morphometric analysis of each venous tissue, it was found that correlation of percentage of medial fibrosis, percentage of luminal narrowing, and IMR with AVF non-maturation were r= 0.236, r= 0.325 and r=0.431 respectively. (Figure 3a and 3b), (Table 3).

To find out the association of AVF non-maturation, fifteen biochemical, histological, and radiological factors were evaluated through univariate analysis initially. Of them six factors such as intimal hyperplasia, IMR, medial fibrosis, luminal narrowing and flow & diameter at 12 weeks were significantly associated with AVF non-maturation. Pre-existing intimal hyperplasia was the strongest predictor of AVF non-maturation. Multivariate analysis showed pre-existing intimal hyperplasia (OR 27.08; p < 0.0001) and a high IMR were independent predictors of non-maturation (IMR OR 88.6; p = 0.003 Table 4).

To determine the diagnostic value of IMR towards AVF non-maturation, ROC curve analysis was performed. It showed the range of IMR was 0.2 to 0.6 which had strong predictive ability for AVF non-maturation (AUC- 0.879). IMR was further stratified in to four categories- (a). less than 0.2, (b). 0.2-0.3, (c). 0.3- 0.4 and (d). 0.4-0.6. Analysis of this revealed AVFs with venous IMR of less than 0.3matured successfully. The IMR of 0.36 has shown the highest sensitivity (80.5%) and moderate specificity (73.9%), towards AVF non-maturation. The curves demonstrated high sensitivity at low false-positive rates and remained well-separated from the reference line, confirming its strong, non-random association with AVF failure (Figure 4a, 4b, 4c and 4d); (Table 5).

This study is the first to demonstrate a strong association between AVF non-maturation and venous remodeling, quantitatively assessed through histo-morphometry in non-diabetic CKD patients. Pre-existing intimal hyperplasia, medial fibrosis, and elevated intima–media ratio (IMR) were identified as the main histological predictors of failure. Complementary Doppler findings showed that altered wall shear stress (WSS) and a low ankle–brachial index (ABI < 0.9) were associated with increased non-maturation risk, highlighting the interplay between vessel structure and hemodynamics.

Most participants were middle-aged; age had little effect due to the exclusion of elderly patients. Fewer females were included because of suboptimal vessel quality, but outcomes did not differ by sex, aligning with previous reports (3,5,14). Hypertension offered no advantage for maturation and may instead impair it by promoting vascular stiffness and endothelial dysfunction (5,6,15,16). Biochemical variables such as hemoglobin, calcium, and creatinine were not associated with outcomes, reaffirming that local vascular and hemodynamic factors play a greater role than systemic parameters. Serum phosphorus also showed no correlation with non-maturation, consistent with the HFM study (14,17). Elevated iPTH, though not statistically significant, may contribute to stiffness and calcification, indirectly affecting remodeling (9,17).

Preoperative arterial and venous diameters did not predict maturation, indicating that vascular function depends more on compliance and distensibility than size. Patients with non-matured AVFs had lower ABI values, suggesting underlying arterial disease. Though not significant due to sample size, ABI may serve as a simple noninvasive screening marker (18–20). Postoperative Doppler parameters—blood flow and venous diameter—were strongly associated with maturation by 12 weeks, reflecting time-dependent remodeling (4,14).

Laminar flow with adequate WSS supports favorable remodeling, while low or turbulent WSS promotes intimal hyperplasia and stenosis (21,22). In this study, WSS values were slightly higher in non-matured AVFs, consistent with prior findings that both low and excessively high WSS can impair adaptation (14,21–23).

Venous medial fibrosis correlated with non-maturation, supporting Martinez et al., who reported a 55% increased failure risk for each 10% rise in fibrosis (OR 1.55). Other studies, including Allon et al. and Shiu et al., found no such link, suggesting that lesion severity, not presence alone, determines outcome. Likewise, pre-existing intimal hyperplasia showed strong correlation with failure in our study. While Allon et al. and the HFM study observed limited effect of mild lesions, Martinez et al. emphasized that marked postoperative intimal proliferation predicted failure, underscoring the importance of both baseline and adaptive changes.

A key contribution of this work is the diagnostic use of IMR. An IMR > 0.36 demonstrated excellent predictive value (AUC = 0.879, sensitivity 80.5%, specificity 73.9%), outperforming individual histological indices. Comparable ultrasound studies have shown venous IMT ≥ 0.26 mm predicts maturation, while ≤ 0.20 mm predicts failure (24–26). IMR thus provides a dynamic measure of wall imbalance and may serve as a histologic correlate of ultrasound-based predictors.

Previous immunohistochemical studies show intimal hyperplasia comprises SMA-positive, vimentin-positive, desmin-negative myofibroblasts, key mediators of vascular remodeling (27,28). The non-diabetic composition of our cohort helped isolate intrinsic structural factors from metabolic confounders. On multivariate regression, only intimal hyperplasia and high IMR independently predicted AVF non-maturation, confirming their dominant role in adverse venous remodeling.

Strengths include the prospective design, exclusive non-diabetic cohort, standardized morphometry, and integration of clinical, Doppler, and histological data. Limitations are its single-center design, modest sample size, lack of long-term patency follow-up, and single-site biopsy sampling. Despite these, the findings provide compelling evidence that venous histo-morphometry is a reliable predictor of AVF outcomes.

In conclusion, pre-existing venous intimal hyperplasia, medial fibrosis, and elevated IMR are the strongest independent predictors of AVF non-maturation in non-diabetic CKD patients. Non-invasive indices such as ABI and WSS may complement histological risk assessment. Integrating venous morphometry with hemodynamic evaluation could enable individualized AVF planning and early risk stratification. Future work should explore rapid tissue evaluation techniques and targeted therapies against intimal hyperplasia and fibrosis to improve AVF success rates.