Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2–/– organoids and blocked via treatment with de novo–designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

The lipid kinase VPS34 orchestrates autophagy, endocytosis, and metabolism and is implicated in cancer and metabolic disease. The proximal tubule in the kidney is a key metabolic organ that controls reabsorption of nutrients such as fatty acids, amino acids, sugars, and proteins. Here, by combining metabolomics, proteomics, and phosphoproteomics analyses with functional and superresolution imaging assays of mice with an inducible deficiency in proximal tubular cells, we revealed that VPS34 controlled the metabolome of the proximal tubule. In addition to inhibiting pinocytosis and autophagy, VPS34 depletion induced membrane exocytosis and reduced the abundance of the retromer complex necessary for proper membrane recycling and lipid retention, leading to a loss of fuel and biomass. Integration of omics data into a kidney cell metabolomic model demonstrated that VPS34 deficiency increased β-oxidation, reduced gluconeogenesis, and enhanced the use of glutamine for energy consumption. Furthermore, the omics datasets revealed that VPS34 depletion triggered an antiviral response that included a decrease in the abundance of apically localized virus receptors such as ACE2. VPS34 inhibition abrogated SARS-CoV-2 infection in human kidney organoids and cultured proximal tubule cells in a glutamine-dependent manner. Thus, our results demonstrate that VPS34 adjusts endocytosis, nutrient transport, autophagy, and antiviral responses in proximal tubule cells in the kidney.

Diabetic retinal disease (DRD) remains the most common cause of vision loss in adults of working age. Progress on the development of new therapies for DRD has been limited by the complexity of the human eye, which constrains the utility of traditional research techniques, including animal and tissue culture models—a problem shared by those in the field of kidney disease research. By contrast, significant progress in the study of diabetic kidney disease (DKD) has resulted from the successful employment of systems biology approaches. Systems biology is widely used to comprehensively understand complex human diseases through the unbiased integration of genetic, environmental, and phenotypic aspects of the disease with the functional and structural manifestations of the disease. The application of a systems biology approach to DRD may help to clarify the molecular basis of the disease and its progression. Acquiring this type of information might enable the development of personalized treatment approaches, with the goal of discovering new therapies targeted to an individual’s specific DRD pathophysiology and phenotype. Furthermore, recent efforts have revealed shared and distinct pathways and molecular targets of DRD and DKD, highlighting the complex pathophysiology of these diseases and raising the possibility of therapeutics beneficial to both organs. The objective of this review is to survey the current understanding of DRD pathophysiology and to demonstrate the investigative approaches currently applied to DKD that could promote a more thorough understanding of the structure, function, and progression of DRD.

Hypoxia drives diabetic kidney disease (DKD) progression through Hypoxia Inducible Factor (HIF) signaling. The kidney’s cellular heterogeneity and complex architecture pose challenges for directly assessing the pharmacologic effects on kidney oxygenation and hypoxia-responsive pathways in vivo, such as treatment with SGLT2 inhibitors (SGLT2i), presumed to impact kidney oxygenation. Using single-cell transcriptional profiling of kidney tissue from youth with type 2 diabetes (T2D) who showed minimal clinical evidence of DKD, we identified cell type enrichment of HIF-regulated genes, findings that replicated in people with later-stage DKD in the Kidney Precision Medicine Project (KPMP). Using conserved transcription factor (TF) binding motifs, higher-order promoter regulatory structures identified potential cooperating TFs that explained the cell type enrichment pattern. From these promoter elements, 7 interconnected regulatory pathways were identified, comprising a network of 237 genes. Analysis of multiome data from reference tissue in KPMP demonstrated that 80% of the network genes resided in accessible chromatin. Expression of network genes increased significantly in the late compared to the early stage DKD and was validated in a hypoxic human organoid model system. Kidney tissue from individuals with T2D treated with SGLT2i demonstrated reversal of the accumulated changes in the HIF network compared to those not treated with SGLT2i. Most high-confidence genes showed concordant differential expression in spatial transcriptomics from individuals with T2D. Hypoxic kidney organoids treated with SGLT2i confirmed these protective effects. Our promoter-anchored HIF regulatory network provides a multi-component read-out that captures disease progression and quantifies therapeutic response to SGLT2i.

BACKGROUND AND AIMS: Kidney organoids are a valuable and innovative model to understand genetic diseases, kidney development and transcriptomic dynamics. However, their proteome has not been analyzed so far. It is unclear how their proteome changes during differentiation, and if more complex disease processes such as inflammatory tissue responses could be modelled with this approach. METHOD: Here, we used proteomics to compare organoids with existing model systems such as native glomeruli and cultured cells. We characterize the trajectory of organoid differentiation and delineate innate immune responses in organoids to expand its scope as a model system in nephrology. We also compared our proteomics with bulk and single cell transcriptomic data. RESULTS: Genes involved in podocytopathies and cystic kidney diseases were abundantly expressed on protein level, distinguishing organoids from almost every available cell culture model. On their pathway to terminal differentiation, organoids developed increased deposition of extracellular matrix. Single cell transcriptomic analysis suggests that most changes locate to podocytes and early podocyte progenitors. This matrix deposition is different from commonly used animal models of glomerular disease. A novel signaling system discovered was the TNFα system, a system also available in podocytes. Incubation of organoids with high concentrations of TNFα led to an activation of NF-kB signaling, and secretion of cytokines and complement components, alongside with extracellular matrix components. CONCLUSION: Interestingly, this signaling system directly links inflammatory signaling, production of cytokines and complement, and production of extracellular matrix. Thus, we provide a repository of human kidney organoid proteins that revealed the potential to model pathophysiological pathways beyond genetic diseases.