Human stem cell models to uncover the roles of extracellular matrix homeostasis and YAP signaling in the podocyte injury response

Abstract

Early diagnoses and therapeutic intervention are critical to patients at risk for chronic kidney disease (CKD). Yet, available detection agents are not sensitive enough to detect pathogenesis and current treatment options are limited to symptom management since they cannot address the direct causes of injury. Thus, kidney function progressively degenerates through 5 stages, eventually to renal failure, or ESKD. To combat CKD’s global health epidemic, it is imperative to unveil novel indicators of injury and develop targeted therapies. The glomerular filtration barrier is highly susceptible to injury since it is the primary site for blood filtration. The glomerular filtration barrier is composed of two specialized cell types, vascular endothelium and podocytes, separated by a unique fusion of their extracellular matrices called the glomerular basement membrane. Injury to this site is deleterious to kidney function, especially because the podocytes are post-mitotic and have little to no known regenerative capacity. Thus, drivers of podocytopathies may be prime early disease biomarkers and catalysts for phenotypic or functional rescue. Specifically, as the barrier selectively filters molecules based on size and charge, it must withstand a large demand of molecular signals and mechanical forces. Therefore, the work presented in three (3) original research chapters of this dissertation investigates the mechanobiological properties of podocyte homeostasis and injury response. In particular, this work focuses on the effects of outside-in and inside-out signaling via the podocyte-extracellular matrix interface and yes-associate protein (YAP) activity, respectively. To carry out this objective, we leverage the proliferative and pluripotent prowess of human induced pluripotent stem (iPS) cells and a method to derive mature podocytes and vascular endothelium, as well as tissue engineering techniques with organ-on-a-chip devices to recapitulate the glomerular filtration barrier and build models of disease, in vitro. First (Chapter 2), we recognized that pathologic remodeling at the podocyte-extracellular matrix interface is a hallmark of diabetic nephropathy, the leading cause of ESKD. Thus, we generated a human iPS cell-derived model of diabetic podocytopathy to investigate disease pathogenesis and progression. The model recapitulated hallmarks of podocytopathy that precede proteinuria including retraction of foot processes and podocytopenia (detachment from the extracellular matrix (ECM)). Moreover, hyperglycemia-induced injury to podocytes exacerbated remodeling of the ECM. Specifically, mature podocytes aberrantly increased expression and excessively deposited collagen (IV)α1α1α2 that is normally abundant in the embryonic glomerulus. This collagen (IV) imbalance coincided with dysregulation of lineage-specific proteins, structural abnormalities of the ECM, and podocytopenia – a mechanism not shared with endothelium and is distinct from drug-induced injury. Intriguingly, repopulation of hyperglycemia-injured podocytes on decellularized ECM scaffolds isolated from healthy podocytes attenuated the loss of synaptopodin (a mechanosensitive protein associated with podocyte health). These results demonstrate that human iPS cell-derived podocytes can facilitate in vitro studies to uncover the mechanisms of chronic hyperglycemia and ECM remodeling and guide disease target identification. Next (Chapter 3), we engineered a personalized glomerulus chip system reconstituted from human induced pluripotent stem (iPS) cell-derived vascular endothelial cells (ECs) and podocytes from a single patient. Our stem cell-derived kidney glomerulus chip successfully mimics the structure and some essential functions of the glomerular filtration barrier. We further modeled glomerular injury in our tissue chips by administering a clinically relevant dose of the chemotherapy drug Adriamycin. The drug disrupts the structural integrity of the endothelium and the podocyte tissue layers, leading to significant albuminuria as observed in patients with glomerulopathies. Finally (Chapter 4), we hypothesized that YAP and its target genes, CTGF and Cyr61, could serve as viable therapeutic targets. To examine the role of YAP signaling in podocyte homeostasis and injury response, we developed a series of lentiviral-mediated short hairpin RNA- and TetOn-constructs to conditionally knockdown and overexpress the gene targets, respectively. We found that loss of either CTGF, Cyr61, or YAP significantly perturbed podocyte morphology by reducing cell spread. Interestingly, CTGF/Cyr61 double knockdown, along with YAP knockdown, increased podocyte sensitivity to Adriamycin. While we expect that monitored CTGF/Cyr61 secretion could serve as a predictive biomarker for early podocyte injury in vivo, their cytoprotective effects from overexpression were negligible when exposed to Adriamycin (ADR). YAP overexpression did, however, desensitize podocytes to insult from ADR but could not rescue their metabolic viability when supplied after injury. Instead, we unveil a critical interaction that supports the re-extension of primary processes following injury: actin remodeling and enhanced actomyosin activity. Finally, we translate the structural repair into functional repair in a higher-order microphysiological device, the Glomerulus Chip, where we found an improvement in albuminuria when YAP was overexpressed following ADR injury. We believe these results will expand effective targeted treatment for kidney disease and serve as a blueprint for ex vivo gene editing for other chronic diseases.