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.

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).

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.

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.

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.

The nucleic acid binding protein YB-1-controlled expression of CXCL-1 modulates kidney damage in liver fibrosis.

Acute kidney injury is a common complication of advanced liver disease and increased mortality of these patients. Here, we analyzed the role of Y-box protein-1 (YB-1), a nucleic acid binding protein, in the bile duct ligation model of liver fibrosis and monitored liver and subsequent kidney damage. Following bile duct ligation, both serum levels of liver enzymes and expression of hepatic extracellular matrix components such as type I collagen were significantly reduced in mice with half-maximal YB-1 expression (Yb1+/-) as compared to their wild-type littermates. By contrast, expression of the chemokine CXCL1 was significantly augmented in these Yb1+/- mice. YB-1 was identified as a potent transcriptional repressor of the Cxcl1 gene. Precision-cut kidney slices from Yb1+/- mice revealed higher expression of the CXCL1 receptor CXCR2 as well as enhanced responsivity to CXCL1 compared to those from wild-type mice. Increased CXCL1 content in Yb1+/- mice led to pronounced bile duct ligation-induced damage of the kidneys monitored as parameters of tubular epithelial injury and immune cell infiltration. Pharmacological blockade of CXCR2 as well as application of an inhibitory anti-CXCL1 antibody significantly mitigated early systemic effects on the kidneys following bile duct ligation whereas it had only a modest impact on hepatic inflammation and function. Thus, our analyses provide direct evidence that YB-1 crucially contributes to hepatic fibrosis and modulates liver-kidney crosstalk by maintaining tight control over chemokine CXCL1 expression.

New drugs for NAFLD: lessons from basic models to the clinic.

The term nonalcoholic fatty liver disease (NAFLD) comprises a spectrum of increasingly harmful conditions ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) to liver fibrosis and end-stage cirrhosis. NAFLD is the currently most common form of chronic liver disease in both adults and children worldwide. As NAFLD evolves as a global pandemic alongside the still growing prevalence of metabolic syndrome, obesity, and diabetes, it is inevitable to develop effective counterstrategies. Over the last decades, great effort has been dedicated to the understanding of the pathogenesis of NAFLD. This includes the development of an array of models for NAFLD, ranging from advanced in vitro (primary cells, 3D cultures, biochip, spheroids, organoids) to in vivo rodent models (particularly in mice). Based on these approaches novel therapies have been proposed and subsequently evaluated for patients with advanced forms of NAFLD, in particular those with NASH and liver fibrosis or cirrhosis. In this review, we delineate the current understanding of disease pathophysiology and depict how novel therapeutic strategies aim to exploit these different mechanisms to ameliorate, treat, or stop progression of NASH. We also discuss obstacles and chances along the way from basic models to promising clinical treatment options.

DC subset-specific induction of T cell responses upon antigen uptake via Fcγ receptors in vivo.

Dendritic cells (DCs) are efficient antigen-presenting cells equipped with various cell surface receptors for the direct or indirect recognition of pathogenic microorganisms. Interestingly, not much is known about the specific expression pattern and function of the individual activating and inhibitory Fcγ receptors (FcγRs) on splenic DC subsets in vivo and how they contribute to the initiation of T cell responses. By targeting antigens to select activating and the inhibitory FcγR in vivo, we show that antigen uptake under steady-state conditions results in a short-term expansion of antigen-specific T cells, whereas under inflammatory conditions especially, the activating FcγRIV is able to induce superior CD4+ and CD8+ T cell responses. Of note, this effect was independent of FcγR intrinsic activating signaling pathways. Moreover, despite the expression of FcγRIV on both conventional splenic DC subsets, the induction of CD8+ T cell responses was largely dependent on CD11c+CD8+ DCs, whereas CD11c+CD8- DCs were critical for priming CD4+ T cell responses.

Human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment.

In mice, conventional and plasmacytoid dendritic cells (DCs) derive from separate hematopoietic precursors before they migrate to peripheral tissues. Moreover, two classes of conventional DCs (cDC1 and cDC2 DCs) and one class of plasmacytoid DCs (pDCs) have been shown to be transcriptionally and functionally distinct entities. In humans, these three DC subtypes can be identified using the cell surface markers CD1c (cDC2), CD141 (cDC1), and CD303 (pDCs), albeit it remains elusive whether DC functionality is mainly determined by ontogeny or the tissue microenvironment. By phenotypic and transcriptional profiling of these three DC subtypes in different human tissues derived from a large number of human individuals, we demonstrate that DC subpopulations in organs of the lymphohematopoietic system (spleen, thymus, and blood) are strongly defined by ontogeny rather than by signals from the microenvironment. In contrast, DC subsets derived from human lung or skin differed substantially, strongly arguing that DCs react toward modulatory signals from tissue microenvironments. Collectively, the data obtained in this study may serve as a major resource to guide further studies into human DC biology during homeostasis and inflammation.