Tubuloid differentiation to model the human distal nephron and collecting duct in health and disease.

Organoid technology is rapidly gaining ground for studies on organ (patho)physiology. Tubuloids are long-term expanding organoids grown from adult kidney tissue or urine. The progenitor state of expanding tubuloids comes at the expense of differentiation. Here, we differentiate tubuloids to model the distal nephron and collecting ducts, essential functional parts of the kidney. Differentiation suppresses progenitor traits and upregulates genes required for function. A single-cell atlas reveals that differentiation predominantly generates thick ascending limb and principal cells. Differentiated human tubuloids express luminal NKCC2 and ENaC capable of diuretic-inhibitable electrolyte uptake and enable disease modeling as demonstrated by a lithium-induced tubulopathy model. Lithium causes hallmark AQP2 loss, induces proliferation, and upregulates inflammatory mediators, as seen in vivo. Lithium also suppresses electrolyte transport in multiple segments. In conclusion, this tubuloid model enables modeling of the human distal nephron and collecting duct in health and disease and provides opportunities to develop improved therapies.

Certain heterozygous variants in the kinase domain of the serine/threonine kinase NEK8 can cause an autosomal dominant form of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) resulting from pathogenic variants in PKD1 and PKD2 is the most common form of PKD, but other genetic causes tied to primary cilia function have been identified. Biallelic pathogenic variants in the serine/threonine kinase NEK8 cause a syndromic ciliopathy with extra-kidney manifestations. Here we identify NEK8 as a disease gene for ADPKD in 12 families. Clinical evaluation was combined with functional studies using fibroblasts and tubuloids from affected individuals. Nek8 knockout mouse kidney epithelial (IMCD3) cells transfected with wild type or variant NEK8 were further used to study ciliogenesis, ciliary trafficking, kinase function, and DNA damage responses. Twenty-one affected monoallelic individuals uniformly exhibited cystic kidney disease (mostly neonatal) without consistent extra-kidney manifestations. Recurrent de novo mutations of the NEK8 missense variant p.Arg45Trp, including mosaicism, were seen in ten families. Missense variants elsewhere within the kinase domain (p.Ile150Met and p.Lys157Gln) were also identified. Functional studies demonstrated normal localization of the NEK8 protein to the proximal cilium and no consistent cilia formation defects in patient-derived cells. NEK8-wild type protein and all variant forms of the protein expressed in Nek8 knockout IMCD3 cells were localized to cilia and supported ciliogenesis. However, Nek8 knockout IMCD3 cells expressing NEK8-p.Arg45Trp and NEK8-p.Lys157Gln showed significantly decreased polycystin-2 but normal ANKS6 localization in cilia. Moreover, p.Arg45Trp NEK8 exhibited reduced kinase activity in vitro. In patient derived tubuloids and IMCD3 cells expressing NEK8-p.Arg45Trp, DNA damage signaling was increased compared to healthy passage-matched controls. Thus, we propose a dominant-negative effect for specific heterozygous missense variants in the NEK8 kinase domain as a new cause of PKD.

Tubuloid culture enables long-term expansion of functional human kidney tubule epithelium from iPSC-derived organoids.

Kidney organoids generated from induced pluripotent stem cells (iPSC) have proven valuable for studies of kidney development, disease, and therapeutic screening. However, specific applications have been hampered by limited expansion capacity, immaturity, off-target cells, and inability to access the apical side. Here, we apply recently developed tubuloid protocols to purify and propagate kidney epithelium from d7+18 (post nephrogenesis) iPSC-derived organoids. The resulting 'iPSC organoid-derived (iPSCod)' tubuloids can be exponentially expanded for at least 2.5 mo, while retaining expression of important tubular transporters and segment-specific markers. This approach allows for selective propagation of the mature tubular epithelium, as immature cells, stroma, and undesirable off-target cells rapidly disappeared. iPSCod tubuloids provide easy apical access, which enabled functional evaluation and demonstration of essential secretion and electrolyte reabsorption processes. In conclusion, iPSCod tubuloids provide a different, complementary human kidney model that unlocks opportunities for functional characterization, disease modeling, and regenerative nephrology.

T cell interaction with activated endothelial cells primes for tissue-residency.

Tissue-resident memory T cells (TRM) are suspected drivers of chronic inflammation, but their induction remains unclear. Since endothelial cells (EC) are obligate interaction partners for T cells trafficking into inflamed tissues, they may play a role in TRM development. Here, we used an in vitro co-culture system of human cytokine-activated EC and FACS-sorted T cells to study the effect of EC on T(RM) cell differentiation. T cell phenotypes were assessed by flow cytometry, including proliferation measured by CellTrace Violet dilution assay. Soluble mediators were analyzed by multiplex immunoassay. Co-culture of T cells with cytokine-activated, but not resting EC induced CD69 expression without activation (CD25, Ki67) or proliferation. The dynamic of CD69 expression induced by EC was distinct from that induced by TCR triggering, with rapid induction and stable expression over 7 days. CD69 induction by activated EC was higher in memory than naive T cells, and most pronounced in CD8+ effector memory T cells. Early CD69 induction was mostly mediated by IL-15, whereas later effects were also mediated by interactions with ICAM-1 and/or VCAM-1. CD69+ T cells displayed a phenotype associated with tissue-residency, with increased CD49a, CD103, CXCR6, PD-1 and CD57 expression, and decreased CD62L and S1PR1. EC-induced CD69+ T cells were poised for high production of pro-inflammatory cytokines and showed increased expression of T-helper 1 transcription factor T-bet. Our findings demonstrate that activated EC can induce functional specialization in T cells with sustained CD69 expression, increased cytokine response and a phenotypic profile reminiscent of TRM. Interaction with activated EC during transmigration into (inflamed) tissues thus contributes to TRM-residency priming.

Differentiation and CRISPR-Cas9-mediated genetic engineering of human intestinal organoids.

Intestinal organoids are three-dimensional cultures that resemble key aspects of the epithelium of origin. Here, we describe how to differentiate human small intestinal organoids by combining growth media variations and genetic engineering. We detail the differentiation of human intestinal organoids in the presence and absence of BMP agonists to recapitulate a broader scope of functional cell states found in vivo. Using transient overexpression of the transcription factor Neurogenin-3, we describe the enhancement of differentiation toward rare enteroendocrine cells. For complete details on the use and execution of this protocol, please refer to Beumer et al. (2022).

Differentiated kidney tubular cell-derived extracellular vesicles enhance maturation of tubuloids.

The prevalence of end-stage kidney disease (ESKD) is rapidly increasing with the need for regenerative therapies. Adult stem cell derived kidney tubuloids have the potential to functionally mimic the adult kidney tubule, but still lack the expression of important transport proteins needed for waste removal. Here, we investigated the potential of extracellular vesicles (EVs) obtained from matured kidney tubular epithelial cells to modulate in vitro tubuloids functional maturation. We focused on organic anion transporter 1 (OAT1), one of the most important proteins involved in endogenous waste excretion. First, we show that EVs from engineered proximal tubule cells increased the expression of several transcription factors and epithelial transporters, resulting in improved OAT1 transport capacity. Next, a more in-depth proteomic data analysis showed that EVs can trigger various biological pathways, including mesenchymal-to-epithelial transition, which is crucial in the tubular epithelial maturation. Moreover, we demonstrated that the combination of EVs and tubuloid-derived cells can be used as part of a bioartificial kidney to generate a tight polarized epithelial monolayer with formation of dense cilia structures. In conclusion, EVs from kidney tubular epithelial cells can phenotypically improve in vitro tubuloid maturation, thereby enhancing their potential as functional units in regenerative or renal replacement therapies.

Substrate Stiffness Determines the Establishment of Apical-Basal Polarization in Renal Epithelial Cells but Not in Tubuloid-Derived Cells.

Mechanical guidance of tissue morphogenesis is an emerging method of regenerative medicine that can be employed to steer functional kidney architecture for the purpose of bioartificial kidney design or renal tissue engineering strategies. In kidney morphogenesis, apical-basal polarization of renal epithelial cells is paramount for tubule formation and subsequent tissue functions like excretion and resorption. In kidney epithelium, polarization is initiated by integrin-mediated cell-matrix adhesion at the cell membrane. Cellular mechanobiology research has indicated that this integrin-mediated adhesion is responsive to matrix stiffness, raising the possibility to use matrix stiffness as a handle to steer cell polarization. Herein, we evaluate apical-basal polarization in response to 2D substates of different stiffness (1, 10, 50 kPa and glass) in Madin Darby Canine Kidney cells (MDCKs), a classic canine-derived cell model of epithelial polarization, and in tubuloid-derived cells, established from human primary cells derived from adult kidney tissue. Our results show that sub-physiological (1 kPa) substrate stiffness with low integrin-based adhesion induces polarization in MDCKs, while MDCKs on supraphysiological (>10 kPa) stiffness remain unpolarized. Inhibition of integrin, indeed, allows for polarization on the supraphysiological substrates, suggesting that increased cellular adhesion on stiff substrates opposes polarization. In contrast, tubuloid-derived cells do not establish apical-basal polarization on 2D substrates, irrespective of substrate stiffness, despite their ability to polarize in 3D environments. Further analysis implies that the 2D cultured tubuloid-derived cells have a diminished mechanosensitive capacity when presented with different substrate stiffnesses due to immature focal adhesions and the absence of a connection between focal adhesions and the cytoskeleton. Overall, this study demonstrates that apical-basal polarization is a complex process, where cell type, the extracellular environment, and both the mechanical and chemical aspects in cell-matrix interactions performed by integrins play a role.

Cysteamine-bicalutamide combination therapy corrects proximal tubule phenotype in cystinosis.

Nephropathic cystinosis is a severe monogenic kidney disorder caused by mutations in CTNS, encoding the lysosomal transporter cystinosin, resulting in lysosomal cystine accumulation. The sole treatment, cysteamine, slows down the disease progression, but does not correct the established renal proximal tubulopathy. Here, we developed a new therapeutic strategy by applying omics to expand our knowledge on the complexity of the disease and prioritize drug targets in cystinosis. We identified alpha-ketoglutarate as a potential metabolite to bridge cystinosin loss to autophagy, apoptosis and kidney proximal tubule impairment in cystinosis. This insight combined with a drug screen revealed a bicalutamide-cysteamine combination treatment as a novel dual-target pharmacological approach for the phenotypical correction of cystinotic kidney proximal tubule cells, patient-derived kidney tubuloids and cystinotic zebrafish.

Culture and analysis of kidney tubuloids and perfused tubuloid cells-on-a-chip.

Advanced in vitro kidney models are of great importance to the study of renal physiology and disease. Kidney tubuloids can be established from primary cells derived from adult kidney tissue or urine. Tubuloids are three-dimensional multicellular structures that recapitulate tubular function and have been used to study infectious, malignant, metabolic, and genetic diseases. For tubuloids to more closely represent the in vivo kidney, they can be integrated into an organ-on-a-chip system that has a more physiological tubular architecture and allows flow and interaction with vasculature or epithelial and mesenchymal cells from other organs. Here, we describe a detailed protocol for establishing tubuloid cultures from tissue and urine (1-3 weeks), as well as for generating and characterizing tubuloid cell-derived three-dimensional tubular structures in a perfused microfluidic multi-chip platform (7 d). The combination of the two systems yields a powerful in vitro tool that better recapitulates the complexity of the kidney tubule with donor-specific properties.

Modeling Distal Convoluted Tubule (Patho)Physiology: An Overview of Past Developments and an Outlook Toward the Future.

The kidneys are essential for maintaining electrolyte homeostasis. Blood electrolyte composition is controlled by active reabsorption and secretion processes in dedicated segments of the kidney tubule. Specifically, the distal convoluted tubule (DCT) and connecting tubule are important for regulating the final excretion of sodium, magnesium, and calcium. Studies unravelling the specific function of these segments have greatly improved our understanding of DCT (patho)physiology. Over the years, experimental models used to study the DCT have changed and the field has advanced from early dissection studies with rats and rabbits to the use of various transgenic mouse models. Developments in dissection techniques and cell culture methods have resulted in immortalized mouse DCT cell lines and made it possible to specifically obtain DCT fragments for ex vivo studies. However, we still do not fully understand the complex (patho)physiology of this segment and there is need for advanced human DCT models. Recently, kidney organoids and tubuloids have emerged as new complex cell models that provide excellent opportunities for physiological studies, disease modeling, drug discovery, and even personalized medicine in the future. This review presents an overview of cell models used to study the DCT and provides an outlook on kidney organoids and tubuloids as model for DCT (patho)physiology. Impact statement This study provides a detailed overview of past and future developments on cell models used to study kidney (patho)physiology and specifically the distal convoluted tubule (DCT) segment. Hereby, we highlight the need for an advanced human cell model of this segment and summarize recent advances in the field of kidney organoids and tubuloids with a focus on DCT properties. The findings reported in this review are significant for future developments toward an advanced human model of the DCT that will help to increase our understanding of DCT (patho)physiology.

Kidney Organoids and Tubuloids.

In the past five years, pluripotent stem cell (PSC)-derived kidney organoids and adult stem or progenitor cell (ASC)-based kidney tubuloids have emerged as advanced in vitro models of kidney development, physiology, and disease. PSC-derived organoids mimic nephrogenesis. After differentiation towards the kidney precursor tissues ureteric bud and metanephric mesenchyme, their reciprocal interaction causes self-organization and patterning in vitro to generate nephron structures that resemble the fetal kidney. ASC tubuloids on the other hand recapitulate renewal and repair in the adult kidney tubule and give rise to long-term expandable and genetically stable cultures that consist of adult proximal tubule, loop of Henle, distal tubule, and collecting duct epithelium. Both organoid types hold great potential for: (1) studies of kidney physiology, (2) disease modeling, (3) high-throughput screening for drug efficacy and toxicity, and (4) regenerative medicine. Currently, organoids and tubuloids are successfully used to model hereditary, infectious, toxic, metabolic, and malignant kidney diseases and to screen for effective therapies. Furthermore, a tumor tubuloid biobank was established, which allows studies of pathogenic mutations and novel drug targets in a large group of patients. In this review, we discuss the nature of kidney organoids and tubuloids and their current and future applications in science and medicine.

Bioengineering Organs for Blood Detoxification.

For patients with severe kidney or liver failure the best solution is currently organ transplantation. However, not all patients are eligible for transplantation and due to limited organ availability, most patients are currently treated with therapies using artificial kidney and artificial liver devices. These therapies, despite their relative success in preserving the patients' life, have important limitations since they can only replace part of the natural kidney or liver functions. As blood detoxification (and other functions) in these highly perfused organs is achieved by specialized cells, it seems relevant to review the approaches leading to bioengineered organs fulfilling most of the native organ functions. There, the culture of cells of specific phenotypes on adapted scaffolds that can be perfused takes place. In this review paper, first the functions of kidney and liver organs are briefly described. Then artificial kidney/liver devices, bioartificial kidney devices, and bioartificial liver devices are focused on, as well as biohybrid constructs obtained by decellularization and recellularization of animal organs. For all organs, a thorough overview of the literature is given and the perspectives for their application in the clinic are discussed.

The development of offspring from mothers with systemic lupus erythematosus. A systematic review.

OBJECTIVE: To analyze published data on the influence of maternal systemic lupus erythematosus (SLE) on different aspects of child development. METHODS: A systematic review was conducted using PubMed and Embase searches for SLE or SLE-related antibodies and physical, neurocognitive, psychiatric or motor development outcomes in children. RESULTS: In total 24 cohort and 4 case-control studies were included after initial screening of 1853 hits. Learning disorders (LD) were reported in 21.4-26% of SLE offspring, exceeding the prevalence in the general population. Four studies reported that dyslexia and reading problems were present in 14.3-21.6% of lupus offspring with a clear male predominance. Furthermore, a twofold increased rate of autism spectrum disorders (ASD) (n=1 study) and a two- to threefold increased risk for speech disorders (n=3 studies) were reported in lupus offspring compared to controls, although the latter was not statistically significant. More divergent results were found for attention deficit (n=5 studies) and behavior disorders (n=3 studies). In two large controlled studies attention disorders were more prevalent and a trend towards more behavior disorders was reported in 2 of 3 studies analyzing this subject. Finally, IQ and motor skills were not affected in respectively 7 and 5 studies. Cardiopulmonary functioning and mood disorders were scarcely investigated (both n=1). Maternal anti-SSA antibodies were associated with LD in offspring in one study. Other SLE-related antibodies were rarely studied. CONCLUSION: This systematic review suggests that maternal SLE is associated with LD (specifically dyslexia), ASD, attention deficit and probably speech problems in offspring. However, over half of the studies were assigned a low or moderate evidence level. Therefore, further research is necessary to substantiate the found evidence and expand the scope to lesser researched areas such as cardiopulmonary functioning.