OPTIMIZATION OF MICROFLUIDIC DEVICE FLOW PARAMETERS IN hiPSC-DERIVED ENDOTHELIAL CELLS TO INVESTIGATE VASCULAR DEFECTS IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited kidney disorder, caused mainly by mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Although ADPKD primarily affects the kidneys through progressive cyst formation, cardiovascular complications represent the major cause of morbidity and mortality in affected patients. In particular, early-onset hypertension and vascular abnormalities suggest a critical role of endothelial dysfunction in disease progression. Polycystins are expressed in endothelial primary cilia, where they contribute to mechanosensation and flow-dependent signaling. Alterations in these pathways may therefore impair endothelial homeostasis and vascular function. However, conventional static culture systems poorly reproduce physiological hemodynamic conditions, while animal models fail to fully recapitulate the genetic and phenotypic heterogeneity of human ADPKD. To overcome these limitations, we generated endothelial cells from human induced pluripotent stem cells (hiPSCs) obtained from two ADPKD patients carrying heterozygous PKD1 mutations, as well as from a CRISPR-Cas9-engineered Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited kidney disorder, caused mainly by mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Although ADPKD primarily affects the kidneys through progressive cyst formation, cardiovascular complications represent the major cause of morbidity and mortality in affected patients. In particular, early-onset hypertension and vascular abnormalities suggest a critical role of endothelial dysfunction in disease progression. Polycystins are expressed in endothelial primary cilia, where they contribute to mechanosensation and flow-dependent signalling. Alterations in these pathways may therefore impair endothelial homeostasis and vascular function. While animal models fail to fully recapitulate the genetic and phenotypic heterogeneity of human ADPKD, we generated endothelial cells from human induced pluripotent stem cells (hiPSCs) obtained from two ADPKD patients carrying heterozygous PKD1 mutations, as well as from a CRISPR-Cas9-engineered PKD1 complete knockout line and its corresponding isogenic control. All hiPSC lines were differentiated into endothelial cells and subsequently characterized by immunofluorescence (IF) and Real-Time PCR for endothelial markers. Functional characterization through tube formation assays revealed impaired angiogenic capacity in both ADPKD patient-derived and PKD1 knockout endothelial cells compared to healthy controls. We subsequently implemented a microfluidic flow system to expose endothelial cells to controlled shear stress under more physiological conditions. Experimental parameters, including shear stress magnitude, incubation time, and cell density, were optimized to establish a reproducible flow-based model. Downstream molecular analyses aimed at identifying signalling pathways involved in ADPKD-associated endothelial dysfunction, including Western blot assays, are currently ongoing.