Decoding myofibroblast origins in human kidney fibrosis.

Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist1-3. The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood1,2,4. Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis.

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.

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling.

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. The detailed characterization of organoid podocytes resulting from a hybrid culture protocol showed a podocyte population that resembles adult podocytes and was superior compared with 2D counterparts, based on single-cell RNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling, as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate, puromycin aminonucleoside treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

Human scattered tubular cells represent a heterogeneous population of glycolytic dedifferentiated proximal tubule cells.

Scattered tubular cells (STCs) are a phenotypically distinct cell population in the proximal tubule that increase in number after acute kidney injury. We aimed to characterize the human STC population. Three-dimensional human tissue analysis revealed that STCs are preferentially located within inner bends of the tubule and are barely present in young kidney tissue (<2 years), and their number increases with age. Increased STC numbers were associated with acute tubular injury (kidney injury molecule 1) and interstitial fibrosis (alpha smooth muscle actin). Isolated CD13+ CD24- CD133- proximal tubule epithelial cells (PTECs) and CD13+ CD24+ and CD13+ CD133+ STCs were analyzed using RNA sequencing. Transcriptome analysis revealed an upregulation of nuclear factor κB, tumor necrosis factor alpha, and inflammatory pathways in STCs, whereas metabolism, especially the tricarboxylic acid cycle and oxidative phosphorylation, was downregulated, without showing signs of cellular senescence. Using immunostaining and a publicly available single-cell sequencing database of human kidneys, we demonstrate that STCs represent a heterogeneous population in a transient state. In conclusion, STCs are dedifferentiated PTECs showing a metabolic shift toward glycolysis, which could facilitate cellular survival after kidney injury. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Regulating Chemokine-Receptor Interactions through the Site-Specific Bioorthogonal Conjugation of Photoresponsive DNA Strands.

Oligonucleotide conjugation has emerged as a versatile molecular tool for regulating protein activity. A state-of-the-art labeling strategy includes the site-specific conjugation of DNA, by employing bioorthogonal groups genetically incorporated in proteins through unnatural amino acids (UAAs). The incorporation of UAAs in chemokines has to date, however, remained underexplored, probably due to their sometimes poor stability following recombinant expression. In this work, we designed a fluorescent stromal-derived factor-1ß (SDF-1ß) chemokine fusion protein with a bioorthogonal functionality amenable for click reactions. Using amber stop codon suppression, p-azido-L-phenylalanine was site-specifically incorporated in the fluorescent N-terminal fusion partner, superfolder green fluorescent protein (sfGFP). Conjugation to single-stranded DNAs (ssDNA), modified with a photocleavable spacer and a reactive bicyclononyne moiety, was performed to create a DNA-caged species that blocked the receptor binding ability. This inhibition was completely reversible by means of photocleavage of the ssDNA strands. The results described herein provide a versatile new direction for spatiotemporally regulating chemokine-receptor interactions, which is promising for tissue engineering purposes.

The podocyte as a direct target of glucocorticoids in nephrotic syndrome.

Nephrotic syndrome (NS) is characterized by massive proteinuria; podocyte loss or altered function is a central event in its pathophysiology. Treatment with glucocorticoids is the mainstay of therapy, however, many patients experience one or multiple relapses and prolonged use may be associated with severe adverse effects. Recently the beneficial effects of glucocorticoids have been attributed to a direct effect on podocytes in addition to the well-known immunosuppressive effects. The molecular effects of glucocorticoid action have been studied using animal and cell models of NS. This review provides a comprehensive overview of different molecular mediators regulated by glucocorticoids, including an overview of the model systems that were used to study them. Glucocorticoids are described to stimulate podocyte recovery by restoring pro-survival signalling of slit diaphragm-related proteins and limiting inflammatory responses. Of special interest is the effect of glucocorticoids on stabilizing the cytoskeleton of podocytes, since these effects are also described for other therapeutic agents used in NS, such as cyclosporin. Current models provide much insight but do not fully recapitulate the human condition since the pathophysiology underlying NS is poorly understood. New and promising models include the glomerulus-on-a-chip and kidney organoids, which have the potential to be further developed into functional NS models in the future.

Establishment and characterization of a novel conditionally immortalized human parietal epithelial cell line.

Parietal epithelial cells (PECs) are epithelial cells in the kidney, surrounding Bowman's space. When activated, PECs increase in cell volume, proliferate, migrate to the glomerular tuft and excrete extracellular matrix. Activated PECs are crucially involved in the formation of sclerotic lesions, seen in focal segmental glomerulosclerosis (FSGS). In FSGS, a number of glomeruli show segmental sclerotic lesions. Further disease progression will lead to increasing number of involved glomeruli and gradual destruction of the affected glomeruli. Although the involvement of PECs in FSGS has been acknowledged, little is known about the molecular processes driving PEC activation. To get more insights in this process, accurate in vivo and in vitro models are needed. Here, we describe the development and characterization of a novel conditionally immortalized human PEC (ciPEC) line. We demonstrated that ciPECs are differentiated when grown under growth-restrictive conditions and express important PEC-specific markers, while lacking podocyte and endothelial markers. In addition, ciPECs showed PEC-like morphology and responded to IL-1ß treatment. We therefore conclude that we have successfully generated a novel PEC line, which can be used for future studies on the role of PECs in FSGS.

Remote sensing and signaling in kidney proximal tubules stimulates gut microbiome-derived organic anion secretion.

Membrane transporters and receptors are responsible for balancing nutrient and metabolite levels to aid body homeostasis. Here, we report that proximal tubule cells in kidneys sense elevated endogenous, gut microbiome-derived, metabolite levels through EGF receptors and downstream signaling to induce their secretion by up-regulating the organic anion transporter-1 (OAT1). Remote metabolite sensing and signaling was observed in kidneys from healthy volunteers and rats in vivo, leading to induced OAT1 expression and increased removal of indoxyl sulfate, a prototypical microbiome-derived metabolite and uremic toxin. Using 2D and 3D human proximal tubule cell models, we show that indoxyl sulfate induces OAT1 via AhR and EGFR signaling, controlled by miR-223. Concomitantly produced reactive oxygen species (ROS) control OAT1 activity and are balanced by the glutathione pathway, as confirmed by cellular metabolomic profiling. Collectively, we demonstrate remote metabolite sensing and signaling as an effective OAT1 regulation mechanism to maintain plasma metabolite levels by controlling their secretion.

Nephrotic syndrome in a dish: recent developments in modeling in vitro.

Nephrotic syndrome is a heterogeneous disease, and one of the most frequent glomerular disorders among children. Depending on the etiology, it may result in end-stage renal disease and the need for renal replacement therapy. A dysfunctional glomerular filtration barrier, comprising of endothelial cells, the glomerular basement membrane and podocytes, characterizes nephrotic syndrome. Podocytes are often the primary target cells in the pathogenesis, in which not only the podocyte function but also their crosstalk with other glomerular cell types can be disturbed due to a myriad of factors. The pathophysiology of nephrotic syndrome is highly complex and studying molecular mechanisms in vitro requires state-of-the-art cell-based models resembling the in vivo situation and preferably a fully functional glomerular filtration barrier. Current advances in stem cell biology and microfluidic platforms have heralded a new era of three-dimensional (3D) cultures that might have the potential to recapitulate the glomerular filtration barrier in vitro. Here, we highlight the molecular basis of nephrotic syndrome and discuss requirements to accurately study nephrotic syndrome in vitro, including an overview of specific podocyte markers, cutting-edge stem cell organoids, and the implementation of microfluidic platforms. The development of (patho) physiologically relevant glomerular models will accelerate the identification of molecular targets involved in nephrotic syndrome and may be the harbinger of a new era of therapeutic avenues.

The importance of breast cancer resistance protein to the kidneys excretory function and chemotherapeutic resistance.

The relevance of membrane transporters gained momentum in recent years and it is now widely recognized that transporters are key players in drug disposition and chemoresistance. As such, the kidneys harbor a variety of drug transporters and are one of the main routes for xenobiotic excretion. The breast cancer resistance protein (BCRP/ABCG2) is widely accepted as a key mediator of anticancer drug resistance and is a prominent renal drug transporter. Here, we review the role of BCRP in both processes and present a multitude of variables that can influence its activity. An increasing number of renally cleared chemotherapeutics, including tyrosine kinase inhibitors, described as BCRP substrates can modulate its activity via transcription factors and cellular signaling pathways, such as the phosphoinositide 3-kinase (PI3K) pathway. In addition to pharmacological actions, genetic variations, as well as differences between species and gender can affect BCRP function, which are also discussed. Furthermore, the role of BCRP in light of cancer treatments and the implications for novel therapeutic interventions that take into account renal function are discussed.

Disposition and clinical implications of protein-bound uremic toxins.

In patients with chronic kidney disease (CKD), adequate renal clearance is compromised, resulting in the accumulation of a plethora of uremic solutes. These uremic retention solutes, also named uremic toxins, are a heterogeneous group of organic compounds with intrinsic biological activities, many of which are too large to be filtered and/or are protein bound. The renal excretion of protein-bound toxins depends largely on active tubular secretion, which shifts the binding and allows for active secretion of the free fraction. To facilitate this process, renal proximal tubule cells are equipped with a range of transporters that co-operate in basolateral uptake and luminal excretion. Many of these transporters have been characterized as mediators of drug disposition, but have recently been recognized for their importance in the proximal renal tubular transport of uremic toxins as well. This also indicates that during uremia, drug disposition may be severely affected as a result of drug-uremic toxin interaction. In addition, CKD patients receive various drugs to treat their complications potentially resulting in drug-drug interactions (DDIs), also for drugs that are non-renally excreted. This review discusses the current knowledge on formation, disposition and removal of protein-bound uremic toxins. Furthermore, implications associated with drug treatment in kidney failure, as well as innovative renal replacement therapies targetting the protein-bound uremic toxins are being discussed. It will become clear that the complex problems associated with uremia warrant a transdisciplinary approach that unites research experts in the area of fundamental biomedical research with their colleagues in clinical nephrology.

Cetuximab Prevents Methotrexate-Induced Cytotoxicity in Vitro through Epidermal Growth Factor Dependent Regulation of Renal Drug Transporters.

The combination of methotrexate with epidermal growth factor receptor (EGFR) recombinant antibody, cetuximab, is currently being investigated in treatment of head and neck carcinoma. As methotrexate is cleared by renal excretion, we studied the effect of cetuximab on renal methotrexate handling. We used human conditionally immortalized proximal tubule epithelial cells overexpressing either organic anion transporter 1 or 3 (ciPTEC-OAT1/ciPTEC-OAT3) to examine OAT1 and OAT3, and the efflux pumps breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp) in methotrexate handling upon EGF or cetuximab treatment. Protein kinase microarrays and knowledge-based pathway analysis were used to predict EGFR-mediated transporter regulation. Cytotoxic effects of methotrexate were evaluated using the dimethylthiazol bromide (MTT) viability assay. Methotrexate inhibited OAT-mediated fluorescein uptake and decreased efflux of Hoechst33342 and glutathione-methylfluorescein (GS-MF), which suggested involvement of OAT1/3, BCRP, and MRP4 in transepithelial transport, respectively. Cetuximab reversed the EGF-increased expression of OAT1 and BCRP as well as their membrane expressions and transport activities, while MRP4 and P-gp were increased. Pathway analysis predicted cetuximab-induced modulation of PKC and PI3K pathways downstream EGFR/ERBB2/PLCg. Pharmacological inhibition of ERK decreased expression of OAT1 and BCRP, while P-gp and MRP4 were increased. AKT inhibition reduced all transporters. Exposure to methotrexate for 24 h led to a decreased viability, an effect that was reversed by cetuximab. In conclusion, cetuximab downregulates OAT1 and BCRP while upregulating P-gp and MRP4 through an EGFR-mediated regulation of PI3K-AKT and MAPKK-ERK pathways. Consequently, cetuximab attenuates methotrexate-induced cytotoxicity, which opens possibilities for further research into nephroprotective comedication therapies.

Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter.

Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP(+)). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP(+) uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP(+) uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 µM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 µM ASP(+), which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 µM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP(+) uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.

Protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell-derived kidney organoids using Seahorse XF96.

Metabolic derangement is a key culprit in kidney pathophysiology. Organoids have emerged as a promising in vitro tool for kidney research. Here, we present a fine-tuned protocol to analyze bioenergetics in single human induced-pluripotent-stem-cell (iPSC)-derived kidney organoids using Seahorse XF96. We describe the generation of self-organized three-dimensional kidney organoids, followed by preparation of organoids for Seahorse XF96 analysis. We then detail how to carry out stress tests to determine mitochondrial and glycolytic rates in single kidney organoids.

Enhanced fatty acid oxidation through metformin and baicalin as therapy for COVID-19 and associated inflammatory states in lung and kidney.

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response, metabolic derangement and ultimate tissue scarring. A positive balance of cellular energy may result crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in cellular energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID-19 patients that had been previously treated with metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-ß-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two indole derivatives, IND6 and IND8 with AMPK activating capacity. Consistently, a reduced time of hospitalization and need of intensive care was observed in COVID-19 patients previously exposed to metformin. Baicalin also mitigated the activation of pro-inflammatory bone marrow-derived macrophages (BMDMs) and reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19. In human epithelial lung and kidney cells, both drugs improved mitochondrial function and prevented TGF-ß-induced renal epithelial cell dedifferentiation. Our results support that favoring cellular energy production through enhanced FAO may prove useful in the prevention of COVID-19-induced lung and renal damage.

Transformative Materials to Create 3D Functional Human Tissue Models In Vitro in a Reproducible Manner.

Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state-of-the-art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large-scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life-like human tissue models that provide a platform to answer health-based scientific questions.

Motile Cilia on Kidney Proximal Tubular Epithelial Cells Are Associated With Tubular Injury and Interstitial Fibrosis.

It is well established that mammalian kidney epithelial cells contain a single non-motile primary cilium (9 + 0 pattern). However, we noted the presence of multiple motile cilia with a central microtubular pair (9 + 2 pattern) in kidney biopsies of 11 patients with various kidney diseases, using transmission electron microscopy. Immunofluorescence staining revealed the expression of the motile cilia-specific markers Radial Spoke Head Protein 4 homolog A, Forkhead-box-protein J1 and Regulatory factor X3. Multiciliated cells were exclusively observed in proximal tubuli and a relative frequent observation in human kidney tissue: in 16.7% of biopsies with tubular injury and atrophy (3 of 18 tissues), in 17.6% of biopsies from patients with membranous nephropathy (3 of 17 tissues) and in 10% of the human kidney tissues derived from the unaffected pole after tumour nephrectomy (3 of 30 tissues). However, these particular tissues showed marked tubular injury and fibrosis. Further analysis showed a significant relation between the presence of multiciliated cells and an increased expression of alpha-smooth-muscle-actin (p-value < 0.01) and presence of Kidney-injury-molecule-1 (p-value < 0.01). Interestingly, multiciliated cells co-showed staining for the scattered tubular cell markers annexin A2, annexin A3, vimentin and phosphofructokinase platelet but not with cell senescence associated markers, like (p16) and degradation of lamin B. In conclusion, multiciliated proximal tubular cells with motile cilia were frequently observed in kidney biopsies and associated with tubular injury and interstitial fibrosis. These data suggest that proximal tubular cells are able to transdifferentiate into multiciliated cells.

Using human iPSC-derived kidney organoids to decipher SARS-CoV-2 pathology on single cell level.

We describe a protocol for single-cell RNA sequencing of SARS-CoV-2-infected human induced pluripotent stem cell (iPSC)-derived kidney organoids. After inoculation of kidney organoids with virus, we use mechanical and enzymatic disruption to obtain single cell suspensions. Next, we process the organoid-derived cells into sequencing-ready SARS-CoV-2-targeted libraries. Subsequent sequencing analysis reveals changes in kidney cells after virus infection. The protocol was designed for kidney organoids cultured in a 6-well transwell format but can be adapted to organoids with different organ backgrounds. For complete details on the use and execution of this protocol, please refer to Jansen et al. (2022).

Parietal epithelial cells maintain the epithelial cell continuum forming Bowman's space in focal segmental glomerulosclerosis.

In the glomerulus, Bowman's space is formed by a continuum of glomerular epithelial cells. In focal segmental glomerulosclerosis (FSGS), glomeruli show segmental scarring, a result of activated parietal epithelial cells (PECs) invading the glomerular tuft. The segmental scars interrupt the epithelial continuum. However, non-sclerotic segments seem to be preserved even in glomeruli with advanced lesions. We studied the histology of the segmental pattern in Munich Wistar Frömter rats, a model for secondary FSGS. Our results showed that matrix layers lined with PECs cover the sclerotic lesions. These PECs formed contacts with podocytes of the uninvolved tuft segments, restoring the epithelial continuum. Formed Bowman's spaces were still connected to the tubular system. In biopsies of patients with secondary FSGS, we also detected matrix layers formed by PECs, separating the uninvolved from the sclerotic glomerular segments. PECs have a major role in the formation of glomerulosclerosis; we show here that in FSGS they also restore the glomerular epithelial cell continuum that surrounds Bowman's space. This process may be beneficial and indispensable for glomerular filtration in the uninvolved segments of sclerotic glomeruli.

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.