Organoids: Translation to clinical nephrology: Development of human kidney tubuloids and intestinal organoids to combat intrinsic and systemic causes of kidney disease

Abstract

In this thesis, we demonstrate that human kidney tubuloids and intestinal organoids provide powerful models that advance the field of nephrology. In chapter II we review the different nature of adult stem/progenitor cell (ASPC) kidney tubuloids and induced pluripotent stem cell (iPSC)-derived organoids. We identify these as complementary advanced in vitro models of the kidney with specific applications in studies of kidney development, regeneration, physiology, diseases and regenerative medicine. Chapter III summarizes available models for the distal convoluted tubule (DCT) and points out the need for accurate human in vitro models, emphasizing a promising role for tubuloids. After painting the background, chapter IV provides detailed methods for tubuloid culture establishment, incorporation in an organ-on-a-chip system and subsequent functional analyses. These protocols facilitate wide implementation in the field. In chapter V and VI we present new culture conditions that activate vasopressin, parathyroid hormone and aldosterone signaling to enable more mature differentiation towards all distal nephron segments and specifically collecting ducts (CDs) in human and mouse tubuloids respectively. This discovery is then leveraged to demonstrate diuretic-inhibitable electrolyte transport by thick ascending limb (TAL), DCT and principal cells (PCs). Given that tubuloids and iPSC organoids each have their complementary traits (chapters II, III), we merge these approaches to establish hybrid tubuloids with specific advantages to both (chapter VII). Hybrid tubuloid culture rekindles expansion, removes off-target and immature cells and enables easy functional evaluation compared with iPSC organoids. Hybrid tubuloids contain leak-tight epithelium capable of proximal tubule (PT) secretion and distal nephron electrolyte reabsorption. Chapters V and VIII present tubuloid-based human disease models for lithium tubulopathy and cystinosis that yield new mechanistic insights and support screening for therapeutic efficacy and toxicity. Findings include downregulation of TAL cell NKCC2 by lithium (which together with AQP2 loss further impairs urine concentration) and mitigation of pathologic α-ketoglutarate accumulation in cystinosis by cysteamine-bicalutamide combination treatment. Finally, we shift to intestinal organoids as a tool to interfere in systemic diseases that cause progressive renal injury. First, we explain how human intestinal organoids can be differentiated towards the different cell types of the absorptive and secretory lineages and genetically engineered to tailor them to specific research questions (chapter IX). These tools are then combined to identify bone morphogenetic protein (BMP) signaling as a key regulator of functional specialization of human enterocytes across the villus axis, where it drives enterocytes towards lipid absorption (chapter X). Finally, in chapter XI we show that nuclear receptors (NRs) further regulate enterocyte functions including nutrient and electrolyte absorption. In particular the liver X receptors (LXRs), farnesoid X receptor (FXR), pregnane X receptor (PXR) and peroxisome proliferator-activated receptor (PPAR) α stimulate lipid uptake. Therefore, BMP and NR signaling emerge as therapeutic targets for metabolic syndrome and diabetes mellitus, major causes of chronic kidney disease in the Western world.