Engineering Pluripotent Stem Cells to Uncover the Mechanisms of Human Kidney Diseases

Abstract

Chronic Kidney Disease (CKD) is a global health crisis for which successful targeted therapies remain elusive. In the United States, ~15% of the adult population suffers from CKD, costing ~$48 billion/year to treat <1% of the affected population. Clinical investigations have recently established that mild to moderate cardiovascular anomalies in humans enhance the risk for an abnormal Glomerular Filtration Rate. Although there is a growing recognition that Congenital Heart Disease (CHD) alters the predictive ability of risk factors involved in CKD, an in-depth understanding of the reasons behind this is still a mystery. From a developmental standpoint, the patterning of the human heart and kidneys is highly orchestrated and relies on shared molecular signaling pathways. Consequently, mutations in genes involved in organ patterning (e.g., SMAD2) can lead to a wide variety of congenital defects, notably within the heart and the kidneys. Here, I hypothesized that SMAD2 mutations may also cause abnormalities in kidney development and function. To address that, we introduced loss-of-function mutations in the SMAD2 gene in human induced pluripotent stem (iPS) cells, and by devising a method for directed differentiation towards major lineages, I illuminated mechanisms involved in cell-fate specifications at a defined developmental window. I discovered that loss of SMAD2 leads to biased lineage conversion and engages altered developmental gene regulatory networks to drive immature cell types through embryonic intermediates. Next, I modeled a kidney filtration barrier by developing a personalized kidney-chip, interfacing isogenic kidney podocytes and endothelial cells differentiated from the same iPS cells in an organ-on-a-chip device, and demonstrated that patients with SMAD2 mutations will develop early proteinuria. Building on my previous work, I realized that studying congenital kidney disease is crucial because if a child is born with impaired kidney function, their mortality rate is especially high. Recent GWAS studies reflect the challenge posed by the high aetiological and phenotypic heterogeneity of genetic Glomerulopathies, which makes it difficult to identify and treat disease subtypes on the basis of their molecular pathogenesis. Among those genes, patients with TRPC6 mutations are usually diagnosed with CKD in the first decade of their life. At this crossroad, I hypothesized that the pathogenicity of the TRPC6 might primarily occur in kidney podocytes – the specialized epithelium that encases kidney glomerular capillaries. By extending our podocyte differentiation strategy, I discovered that mutated TRPC6 disrupts protein trafficking of podocyte-lineage markers from the endoplasmic reticulum to the foot processes (FPs) due to defective endoplasmic reticulum (ER). Using high-resolution microscopy, I observed Podocin aggresomes near the nucleus. I also found that TRPC6-mutant podocytes fail to translocate Nephrin to the FPs. My work unraveled an unknown function of TRPC6, where its mutation stalls the proteostasis of Nephrin and Podocin from the ER to the FPs, further disturbing Nephrin localization to the FPs of the patients’ podocytes. I also found that TRPC6 mutations can disrupt autophagy in podocytes, forming protein aggresomes. Through a series of drug screenings and organ-on-a-chip experiments, I discovered that co-treatment with Sildenafil and Losartan can be used to increase proteostasis and repair ER while simultaneously reducing Podocin aggresomes, thereby favoring Nephrin-Podocin cuddling at the FPs. Using stem cell-based models and organ-on-a-chip technology, I illuminated the molecular mechanisms of genetic CKD. My work provides a viable platform for future therapeutic discovery.