a-standardized-porcine-kidney-auto-transplant-protocol-for-studying

A Standardized Porcine Kidney Auto-transplant Protocol for Studying Delayed Graft Function

Delayed graft function (DGF) is commonly defined as the need for dialysis within 7 days after kidney transplantation; however, current definitions often ...

University of North Carolina

In an attempt to address the significant morbidity, mortality, and economic cost associated with tunneled dialysis catheter (TDC) dysfunction, we report the development of nitric oxide-releasing dialysis catheter lock solutions. Catheter lock solutions with a range of NO payloads and release kinetics were prepared using low-molecular-weight -diazeniumdiolate nitric oxide donors. Nitric oxide released through the catheter surface as a dissolved gas was maintained at therapeutically relevant levels for at least 72 h, supporting clinical translatability (interdialytic period). Slow, sustained NO release from the catheter surface prevented bacterial adhesion in vitro by 88.9 and 99.7% for  and , respectively, outperforming a burst NO-release profile. Furthermore, bacteria adhered to the catheter surface in vitro prior to lock solution use was reduced by 98.7 and 99.2% for  and , respectively, when using a slow releasing NO donor, demonstrating both preventative and treatment potential. The adhesion of proteins to the catheter surface, a process often preceding biofilm formation and thrombosis, was also lessened by 60-65% by sustained NO release. In vitro cytotoxicity of catheter extract solutions to mammalian cells was minimal, supporting the non-toxic nature of the NO-releasing lock solutions. The use of the NO-releasing lock solution in an in vivo TDC porcine model demonstrated decreased infection and thrombosis, enhanced catheter functionality, and improved outcome (i.e., likelihood of survival) as a result of catheter use.

Nitric Oxide-Releasing Hemodialysis Catheter Lock Solutions.

In this study, we investigated the hemodynamic characteristics of arteriovenous fistulae (AVF) in murine models using micro-CT based computational fluid dynamics (CFD). By combining high-resolution micro-CT imaging with ultrasound flow measurements, our methodology offers a cost-effective and efficient alternative to traditional MRI-based approaches. CFD simulations performed at 7 and 21 days post-surgery revealed significant temporal changes in both geometry and hemodynamics. Geometric analysis showed that: the proximal artery diameter increased from 0.29 mm to 0.38 mm, while the initial 2 mm fistula segment showed a 21.6% decrease (0.74 mm to 0.58 mm). Blood flow through the AVF nearly doubled from 1.33 mL/min to 2.57 mL/min. Time-averaged wall shear stress (TAWSS) peak values increased from 142 Pa (day 7) within the proximal artery to 200 Pa (day 21), in the stenotic region. The oscillatory shear index (OSI) showed marked elevation at the anastomosis (increasing from 0.22 to 0.48), indicating disturbed flow development. An inverse relationship between TAWSS and OSI was identified consistent with previous studies. Our methodology demonstrates the capability to analyze relationships between early hemodynamics and subsequent geometric changes. This approach could enable identification of regions susceptible to stenosis development and monitoring of AVF maturation, which could ultimately lead to quantitative metrics to evaluate surgical outcomes and early therapeutic interventions.

Temporal evolution of hemodynamics in murine arteriovenous fistula: a micro-CT based computational fluid dynamics study.

Arteriovenous fistulae (AVF) are the preferred mode of dialysis vascular access but have a maturation failure rate of over 50%. Venous segment stenosis is one of the main reasons. However, the reasons for the prevalence of stenosis in the venous segment, as opposed to the arterial segment, remain unknown. We hypothesize that differences in the protein expression profiles and in the activation of key signaling pathways of the vascular smooth muscle cells (VSMCs) in these two segments may contribute to this difference. In this study, arterial porcine VSMCs (ApSMCs) and venous porcine VSMCs (VpSMCs) were isolated to examine relevant protein expression and cell signaling pathway activation via Western blots and immunofluorescent staining. Our results revealed that growth-medium-stimulated cell proliferation was significantly higher in VpSMCs compared to ApSMCs, but no difference was detected in PDGF-BB-stimulated proliferation. VpSMCs migration was significantly greater than ApSMCs under both the serum-free condition and in the growth medium condition. In addition, VpSMCs appeared more susceptible to PDGF-BB-stimulated cell dedifferentiation. Furthermore, P53, vitronectin, collagen 1A1 and integrin β3 had lower expression in VpSMCs, whereas fibronectin, FAK, VCAM-1, MMP-9, TIMP-2 and TIMP-3 had higher expression as compared to ApSMCs. Meanwhile, PDGF-BB stimulated P38, JNKs and AKT activation, which were more potent in VpSMCs. Our results identify significant differences between VpSMCs and ApSMCs in the expression of functional proteins and the activation of signaling pathways, which may be responsible for more aggressive venous-as compared to arterial-stenosis in the clinical setting of arteriovenous access or other similar procedures.

Characterization of Protein Expression and Signaling Pathway Activation That May Contribute to Differential Biological Functions in Porcine Arterial and Venous Smooth Muscle Cells.

In this study, we investigated the hemodynamic characteristics of arteriovenous fistulas (AVFs) in murine models using micro-CT based computational fluid dynamics (CFD). By combining high-resolution micro-CT imaging with ultrasound flow measurements, our methodology offers a cost-effective and efficient alternative to traditional MRI-based approaches. CFD simulations performed at 7 and 21 days post-surgery revealed substantial temporal changes in both geometry and hemodynamics. Geometric analysis showed that the proximal artery diameter increased from 0.29 mm to 0.38 mm, whereas the initial 2 mm fistula segment showed a 21.6% decrease (0.74 mm to 0.58 mm). Blood flow through the AVF nearly doubled from 1.33 mL/min to 2.57 mL/min. Time-averaged wall shear stress (TAWSS) peak values and locations changed from 142 Pa (day 7) within the proximal artery to 200 Pa (day 21) in the stenotic region. The oscillatory shear index (OSI) showed marked elevation at the anastomosis (increasing from 0.22 to 0.48), indicating disturbed flow development. An inverse relationship between TAWSS and OSI was identified, consistent with previous studies. Our methodology demonstrates the capability to analyze relationships between early hemodynamics and subsequent geometric changes. This approach can enable identification of regions susceptible to stenosis development and monitoring of AVF maturation, which can ultimately lead to quantitative metrics to evaluate surgical outcomes and early therapeutic interventions.

Temporal evolution of hemodynamics in murine arteriovenous fistula: A micro-CT based CFD study.

Many patients with end-stage kidney disease (ESKD) frequently suffer from both aggressive vascular access stenosis in the venous segment of arteriovenous fistula or arteriovenous graft, and widespread cardiovascular disease (CVD) or peripheral arterial disease (PAD). Despite the magnitude of these clinical problems, the pathogenic role of the uremic state in both of these conditions remains unclear. To investigate the underlying mechanisms, we used porcine-derived arterial smooth muscle cells (ApSMCs) and venous smooth muscle cells (VpSMCs) to examine several key aspects of cell behavior in response to uremic serum. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated that 30% of uremic serum was able to stimulate the proliferation of both subtypes of cells equally. Cell migration, measured by the scratch assay, showed that uremic serum increased migration of both cells, but was more robust in VpSMCs. Importantly, uremic serum induced phenotypic switching (e.g., dedifferentiation) in both subtypes of cells, as indicated by increased proliferating cell nuclear antigen expression and reduced calponin expression. Intriguingly, we found that several key aspects of this uremia-induced phenotypic switch were stronger in ApSMCs as compared with VpSMCs, including the production of extracellular matrix (ECM) proteins, such as fibronectin, cellular calcification [high expression of RUNX family transcription factor 2 (Runx2), alkaline phosphatase (ALP), and Krüppel-like factor 4 (KLF-4)], and a proinflammatory state [high expression of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6)]. Our findings suggest that uremia plays an important role in both the aggressive arteriovenous stenosis and CVD/PAD that affect many patients undergoing hemodialysis. This information could contribute to the development of novel uremia-specific therapies for both vascular access dysfunction and CVD/PAD in patients with ESKD. This study was the first one to directly explore the differential response of arterial VSMCs and venous VSMCs to uremic serum exposure side by side. Both similarities and differences were detected in this in vitro study, which provides insight into the clinical manifestations we observed in patients with ESKD. Furthermore, these results may also be valuable information for uremia-specific therapies for both vascular access dysfunction and CVD/PAD in patients with ESKD.

Uremic serum exposure leads to differential phenotypic switch in porcine arterial and venous smooth muscle cells.

We recently identified significant differences between porcine arterial and venous smooth muscle cells (ApSMCs and VpSMCs) in the expression of numerous genes and activity of several important signaling pathways. To understand the mechanisms that are responsible for these differences, we performed a genome-wide comparison of VpSMCs and ApSMCs using bulk RNA sequencing. A principal component analysis (PCA) plot and heatmaps revealed a clear separation of the two groups of samples. Using a standard cutoff (≥2-fold change, false discovery rate (FDR) ≤ 0.05), 466 genes were highly expressed in ApSMCs, and 358 genes were highly expressed in VpSMCs. Functional pathway analyses were conducted using the Gene Set Enrichment Analysis (GSEA) tool. The top 15 enriched pathways of the GSEA and Overrepresentation Analysis (ORA) results were detected by comparing the dataset against the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO) biological process, GO cellular component, GO molecular function, and WikiPathways databases. Both the GSEA and ORA results revealed that the top enriched pathways are mostly linked to cell cycle, cell structure, and cell differentiation. Further analysis of differentially expressed genes (DEGs) in a specific pathway identified that different sets of genes were utilized to regulate the same pathway between ApSMCs and VpSMCs. For example, in the cell cycle pathway, TGFB1, GADD45A, and TP53 were expressed highly in ApSMCs, while SKP2, PCK1, CDK1, and PPP2CA were expressed highly in VpSMCs. This study identified key differences in the gene expression patterns of two subsets of VSMCs and found that different sets of genes are utilized in specific signaling pathways within the different subtypes of cells, which provides crucial information for developing vein- or artery-specific strategies to prevent corresponding vascular diseases.

Bulk RNA Sequencing Reveals Signature Differences in Key Cell Signaling Pathways Between Porcine Venous and Arterial Smooth Muscle Cells.

Reliable vascular access remains a major clinical challenge for hemodialysis patients, as expanded polytetrafluoroethylene (PTFE) grafts exhibit poor patency and frequent complications driven by thrombosis and neointimal hyperplasia. Tissue-engineered vascular grafts offer a regenerative alternative but often lack the mechanical resilience required for high-flow arteriovenous (AV) environments. Here, we developed a reinforced, biofunctionalized coaxial electrospun graft comprising a poly(ε-caprolactone) mechanical core and a norbornene-functionalized poly(ethylene glycol) sheath incorporating pro-endothelialization cues. Circumferential PTFE rings were added to improve kink resistance. Grafts were implanted in a porcine AV configuration that recapitulates clinical hemodynamic conditions. Mechanical characterization included compliance, burst pressure, and kink resistance; host remodeling was assessed using histology, immunofluorescence, and multiphoton imaging at 4 weeks. Ring-reinforced electrospun grafts demonstrated a kink radius of 0.187 cm, compliance of 1.04 ± 0.29%/100 mmHg, and burst pressure of 1505 ± 565 mmHg, values all comparable to Gore-Tex PTFE and within industrial performance standards. In vivo, the electrospun grafts showed extensive host cell infiltration, collagen deposition, and formation of smooth muscle-like tissue, whereas PTFE controls remained largely acellular. Immunofluorescence confirmed intramural α-SMA and CD31 cell populations, and multiphoton microscopy revealed significantly greater collagen and elastin content compared with PTFE ( < 0.05). Collectively, these findings demonstrate that the reinforced electrospun graft maintains mechanical integrity under physiological AV loading while supporting in situ endothelialization and extracellular matrix remodeling in a clinically relevant, large animal model. This work provides one of the first demonstrations of functional tissue regeneration within a fully synthetic, acellular scaffold in a porcine hemodialysis model and advances the translational development of durable, regenerative vascular access grafts that couple mechanical resilience with bioactive healing capacity.

Article	Year
Nitric Oxide-Releasing Hemodialysis Catheter Lock Solutions. ACS applied materials & interfaces\| PubMed ID: 37285144	2023
Temporal evolution of hemodynamics in murine arteriovenous fistula: a micro-CT based computational fluid dynamics study. bioRxiv : the preprint server for biology\| PubMed ID: 40196505	2025
Characterization of Protein Expression and Signaling Pathway Activation That May Contribute to Differential Biological Functions in Porcine Arterial and Venous Smooth Muscle Cells. International journal of molecular sciences\| PubMed ID: 40243809	2025
Temporal evolution of hemodynamics in murine arteriovenous fistula: A micro-CT based CFD study. PLoS computational biology\| PubMed ID: 40825001	2025
Uremic serum exposure leads to differential phenotypic switch in porcine arterial and venous smooth muscle cells. American journal of physiology. Renal physiology\| PubMed ID: 41138212	2025
Bulk RNA Sequencing Reveals Signature Differences in Key Cell Signaling Pathways Between Porcine Venous and Arterial Smooth Muscle Cells. International journal of molecular sciences\| PubMed ID: 41465376	2025
Biofunctionalized Vascular Access Graft Improves Patency and Endothelialization in a Porcine Arteriovenous Model. Journal of functional biomaterials\| PubMed ID: 41745527	2026

Unimunkh Uriyanghai

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