ADAR regulates APOL1 via A-to-I RNA editing by inhibition of MDA5 activation in a paradoxical biological circuit.

APOL1 risk variants are associated with increased risk of kidney disease in patients of African ancestry, but not all individuals with the APOL1 high-risk genotype develop kidney disease. As APOL1 gene expression correlates closely with the degree of kidney cell injury in both cell and animal models, the mechanisms regulating APOL1 expression may be critical determinants of risk allele penetrance. The APOL1 messenger RNA includes Alu elements at the 3' untranslated region that can form a double-stranded RNA structure (Alu-dsRNA) susceptible to posttranscriptional adenosine deaminase acting on RNA (ADAR)-mediated adenosine-to-inosine (A-to-I) editing, potentially impacting gene expression. We studied the effects of ADAR expression and A-to-I editing on APOL1 levels in podocytes, human kidney tissue, and a transgenic APOL1 mouse model. In interferon-γ (IFN-γ)-stimulated human podocytes, ADAR down-regulates APOL1 by preventing melanoma differentiation-associated protein 5 (MDA5) recognition of dsRNA and the subsequent type I interferon (IFN-I) response. Knockdown experiments showed that recognition of APOL1 messenger RNA itself is an important contributor to the MDA5-driven IFN-I response. Mathematical modeling suggests that the IFN-ADAR-APOL1 network functions as an incoherent feed-forward loop, a biological circuit capable of generating fast, transient responses to stimuli. Glomeruli from human kidney biopsies exhibited widespread editing of APOL1 Alu-dsRNA, while the transgenic mouse model closely replicated the edited sites in humans. APOL1 expression in mice was inversely correlated with Adar1 expression under IFN-γ stimuli, supporting the idea that ADAR regulates APOL1 levels in vivo. ADAR-mediated A-to-I editing is an important regulator of APOL1 expression that could impact both penetrance and severity of APOL1-associated kidney disease.

Characterization of Hippo Pathway Components by Gene Inactivation.

The Hippo pathway is important for regulating tissue homeostasis, and its dysregulation has been implicated in human cancer. However, it is not well understood how the Hippo pathway becomes dysregulated because few mutations in core Hippo pathway components have been identified. Therefore, much work in the Hippo field has focused on identifying upstream regulators, and a complex Hippo interactome has been identified. Nevertheless, it is not always clear which components are the most physiologically relevant in regulating YAP/TAZ. To provide an overview of important Hippo pathway components, we created knockout cell lines for many of these components and compared their relative contributions to YAP/TAZ regulation in response to a wide range of physiological signals. By this approach, we provide an overview of the functional importance of many Hippo pathway components and demonstrate NF2 and RHOA as important regulators of YAP/TAZ and TAOK1/3 as direct kinases for LATS1/2.

AIM2 Suppresses Inflammation and Epithelial Cell Proliferation during Glomerulonephritis.

Absent in melanoma-2 (AIM2) is an inflammasome-forming innate immune sensor for dsDNA but also exhibits inflammasome-independent functions such as restricting cellular proliferation. AIM2 is expressed in the kidney, but its localization and function are not fully characterized. In normal human glomeruli, AIM2 localized to podocytes. In patients with glomerulonephritis, AIM2 expression increased in CD44+-activated parietal epithelial cells within glomerular crescents. To explore AIM2 effects in glomerular disease, studies in Aim2 -/- mice were performed. Aim2-/- glomeruli showed reduced expression of Wilm tumor gene-1 (WT1), WT1-driven podocyte genes, and increased proliferation in outgrowth assays. In a nephrotoxic serum (NTS)-induced glomerulonephritis model, Aim2-/- (B6) mice exhibited more severe glomerular crescent formation, tubular injury, inflammation, and proteinuria compared with wild-type controls. Inflammasome activation markers were absent in both Aim2 -/- and wild-type kidneys, despite an increased inflammatory transcriptomic signature in Aim2 -/- mice. Aim2 -/- mice also demonstrated dysregulated cellular proliferation and an increase in CD44+ parietal epithelial cells during glomerulonephritis. The augmented inflammation and epithelial cell proliferation in Aim2 -/- (B6) mice was not due to genetic background, as Aim2 -/- (B6.129) mice demonstrated a similar phenotype during NTS glomerulonephritis. The AIM2-like receptor (ALR) locus was necessary for the inflammatory glomerulonephritis phenotype observed in Aim2 -/- mice, as NTS-treated ALR -/- mice displayed equal levels of injury as wild-type controls. Podocyte outgrowth from ALR -/- glomeruli was still increased, however, confirming that the ALR locus is dispensable for AIM2 effects on epithelial cell proliferation. These results identify a noncanonical role for AIM2 in suppressing inflammation and epithelial cell proliferation during glomerulonephritis.

DGAT2 Inhibition Potentiates Lipid Droplet Formation To Reduce Cytotoxicity in APOL1 Kidney Risk Variants.

BACKGROUND: Two variants in the gene encoding apolipoprotein L1 (APOL1) that are highly associated with African ancestry are major contributors to the large racial disparity in rates of human kidney disease. We previously demonstrated that recruitment of APOL1 risk variants G1 and G2 from the endoplasmic reticulum to lipid droplets leads to reduced APOL1-mediated cytotoxicity in human podocytes. METHODS: We used CRISPR-Cas9 gene editing of induced pluripotent stem cells to develop human-derived APOL1G0/G0 and APOL1G2/G2 kidney organoids on an isogenic background, and performed bulk RNA sequencing of organoids before and after treatment with IFN-γ. We examined the number and distribution of lipid droplets in response to treatment with inhibitors of diacylglycerol O-acyltransferases 1 and 2 (DGAT1 and DGAT2) in kidney cells and organoids. RESULTS: APOL1 was highly upregulated in response to IFN-γ in human kidney organoids, with greater increases in organoids of high-risk G1 and G2 genotypes compared with wild-type (G0) organoids. RNA sequencing of organoids revealed that high-risk APOL1G2/G2 organoids exhibited downregulation of a number of genes involved in lipogenesis and lipid droplet biogenesis, as well as upregulation of genes involved in fatty acid oxidation. There were fewer lipid droplets in unstimulated high-risk APOL1G2/G2 kidney organoids than in wild-type APOL1G0/G0 organoids. Whereas DGAT1 inhibition reduced kidney organoid lipid droplet number, DGAT2 inhibition unexpectedly increased organoid lipid droplet number. DGAT2 inhibition promoted the recruitment of APOL1 to lipid droplets, with associated reduction in cytotoxicity. CONCLUSIONS: Lipogenesis and lipid droplet formation are important modulators of APOL1-associated cytotoxicity. Inhibition of DGAT2 may offer a potential therapeutic strategy to attenuate cytotoxic effects of APOL1 risk variants.

Recruitment of APOL1 kidney disease risk variants to lipid droplets attenuates cell toxicity.

Two coding variants in the apolipoprotein L1 (APOL1) gene (termed G1 and G2) are strongly associated with increased risk of nondiabetic kidney disease in people of recent African ancestry. The mechanisms by which the risk variants cause kidney damage, although not well-understood, are believed to involve injury to glomerular podocytes. The intracellular localization and function of APOL1 in podocytes remain unclear, with recent studies suggesting possible roles in the endoplasmic reticulum (ER), mitochondria, endosomes, lysosomes, and autophagosomes. Here, we demonstrate that APOL1 also localizes to intracellular lipid droplets (LDs). While a large fraction of risk variant APOL1 (G1 and G2) localizes to the ER, a significant proportion of wild-type APOL1 (G0) localizes to LDs. APOL1 transiently interacts with numerous organelles, including the ER, mitochondria, and endosomes. Treatment of cells that promote LD formation with oleic acid shifted the localization of G1 and G2 from the ER to LDs, with accompanying reduction of autophagic flux and cytotoxicity. Coexpression of G0 APOL1 with risk variant APOL1 enabled recruitment of G1 and G2 from the ER to LDs, accompanied by reduced cell death. The ability of G0 APOL1 to recruit risk variant APOL1 to LDs may help explain the recessive pattern of kidney disease inheritance. These studies establish APOL1 as a bona fide LD-associated protein, and reveal that recruitment of risk variant APOL1 to LDs reduces cell toxicity, autophagic flux, and cell death. Thus, interventions that divert APOL1 risk variants to LDs may serve as a novel therapeutic strategy to alleviate their cytotoxic effects.

Distance education for anatomy and surgical training - A systematic review.

Rapid development of COVID-19 has resulted in a massive shift from traditional to online teaching. This review aims to evaluate the effectiveness of distance learning on anatomy and surgical training. This systematic review was conducted in line with the PRISMA statement and current methodological literature. The databases CINAHL, Cochrane, EMBASE and Pubmed were searched using the search terms "Distant learning" OR "Distance learning" AND "Anatomy OR Surgery". 182 non-duplicate studies were identified. 20 studies were included for qualitative analysis. 10 studies evaluated students' performance with distance learning. 3 studies suggested that students' learning motivation improved with distance learning pedagogy. 5 studies found improved student performance with distance learning (performance or task completion time) when compared to conventional physical method. While 2 other studies found non-inferior student performance. 10 studies evaluated students' feedback on distance learning. Most feedbacks were positive, with flexibility, efficiency, increased motivation and better viewing angles as the most-liked features of distance teaching. 4 studies pointed out some limitations of distance learning, including the lack of personal contact with tutor, poor network and reduced student concentration. 7 studies evaluated tutors' feedback on distance learning. Tutors generally liked online platforms for the ease of tracking silent students, monitoring performance and updating fast-changing knowledge. Yet the lack of hands-on experience for students, technical issues and high costs are the main concerns for tutors. In conclusion, distance learning is a feasible alternative for anatomy and surgical teaching.

Post-mortem molecular investigations of SARS-CoV-2 in an unexpected death of a recent kidney transplant recipient.

Solid organ transplant recipients are vulnerable to severe infection during induction therapy. We report a case of a 67-year-old male who died unexpectedly 10 days after receiving a kidney transplant on February 10, 2020. There was no clear cause of death, but COVID-19 was considered retrospectively, as the death occurred shortly after the first confirmed case of COVID-19 in Canada. We confirmed the presence of SARS-CoV-2 components in the renal allograft and native lung tissue using immunohistochemistry for SARS-CoV-2 spike protein and RNA scope in situ hybridization for SARS-CoV-2 RNA. Results were reaffirmed with the Food and Drug Administration Emergency Use Authorization approved Bio-Rad SARS-CoV-2 digital droplet PCR for the kidney specimen. Our case highlights the importance of patient autopsies in an unfolding global pandemic and demonstrates the utility of molecular assays to diagnose SARS-CoV-2 post-mortem. SARS-CoV-2 infection during induction therapy may portend a fatal clinical outcome. We also suggest COVID-19 may be transmittable via renal transplant.

FSGS-Causing INF2 Mutation Impairs Cleaved INF2 N-Fragment Functions in Podocytes.

BACKGROUND: Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly. Why the disease-causing mutations are restricted to the N terminus and how they cause human disease has been unclear. METHODS: We examined INF2 isoforms present in podocytes and evaluated INF2 cleavage as an explanation for immunoblot findings. We evaluated the expression of INF2 N- and C-terminal fragments in human kidney disease conditions. We also investigated the localization and functions of the DID-containing N-terminal fragment in podocytes and assessed whether the FSGS-associated R218Q mutation impairs INF2 cleavage or the function of the N-fragment. RESULTS: The INF2-CAAX isoform is the predominant isoform in podocytes. INF2 is proteolytically cleaved, a process mediated by cathepsin proteases, liberating the N-terminal DID to function independently. Although the N-terminal region normally localizes to podocyte foot processes, it does not do so in the presence of FSGS-associated INF2 mutations. The C-terminal fragment localizes to the cell body irrespective of INF2 mutations. In podocytes, the N-fragment localizes to the plasma membrane, binds mDIA1, and promotes cell spreading in a cleavage-dependent way. The disease-associated R218Q mutation impairs these N-fragment functions but not INF2 cleavage. CONCLUSIONS: INF2 is cleaved into an N-terminal DID-containing fragment and a C-terminal DAD-containing fragment. Cleavage allows the N-terminal fragment to function independently and helps explain the clustering of FSGS-associated mutations.

Contributions of Rare Gene Variants to Familial and Sporadic FSGS.

BACKGROUND: Over the past two decades, the importance of genetic factors in the development of FSGS has become increasingly clear. However, despite many known monogenic causes of FSGS, single gene defects explain only 30% of cases. METHODS: To investigate mutations underlying FSGS, we sequenced 662 whole exomes from individuals with sporadic or familial FSGS. After quality control, we analyzed the exome data from 363 unrelated family units with sporadic or familial FSGS and compared this to data from 363 ancestry-matched controls. We used rare variant burden tests to evaluate known disease-associated genes and potential new genes. RESULTS: We validated several FSGS-associated genes that show a marked enrichment of deleterious rare variants among the cases. However, for some genes previously reported as FSGS related, we identified rare variants at similar or higher frequencies in controls. After excluding such genes, 122 of 363 cases (33.6%) had rare variants in known disease-associated genes, but 30 of 363 controls (8.3%) also harbored rare variants that would be classified as "causal" if detected in cases; applying American College of Medical Genetics filtering guidelines (to reduce the rate of false-positive claims that a variant is disease related) yielded rates of 24.2% in cases and 5.5% in controls. Highly ranked new genes include SCAF1, SETD2, and LY9. Network analysis showed that top-ranked new genes were located closer than a random set of genes to known FSGS genes. CONCLUSIONS: Although our analysis validated many known FSGS-causing genes, we detected a nontrivial number of purported "disease-causing" variants in controls, implying that filtering is inadequate to allow clinical diagnosis and decision making. Genetic diagnosis in patients with FSGS is complicated by the nontrivial rate of variants in known FSGS genes among people without kidney disease.

Mitochondrial NLRP3 protein induces reactive oxygen species to promote Smad protein signaling and fibrosis independent from the inflammasome.

Nucleotide-binding domain and leucine-rich repeat containing PYD-3 (NLRP3) is a pattern recognition receptor that is implicated in the pathogenesis of inflammation and chronic diseases. Although much is known regarding the NLRP3 inflammasome that regulates proinflammatory cytokine production in innate immune cells, the role of NLRP3 in non-professional immune cells is unclear. Here we report that NLRP3 is expressed in cardiac fibroblasts and increased during TGFß stimulation. NLRP3-deficient cardiac fibroblasts displayed impaired differentiation and R-Smad activation in response to TGFß. Only the central nucleotide binding domain of NLRP3 was required to augment R-Smad signaling because the N-terminal Pyrin or C-terminal leucine-rich repeat domains were dispensable. Interestingly, NLRP3 regulation of myofibroblast differentiation proceeded independently from the inflammasome, IL-1ß/IL-18, or caspase 1. Instead, mitochondrially localized NLRP3 potentiated reactive oxygen species to augment R-Smad activation. In vivo, NLRP3-deficient mice were protected against angiotensin II-induced cardiac fibrosis with preserved cardiac architecture and reduced collagen 1. Together, these results support a distinct role for NLRP3 in non-professional immune cells independent from the inflammasome to regulate differential aspects of wound healing and chronic disease.

Inflammasome-independent NLRP3 augments TGF-ß signaling in kidney epithelium.

Tubulointerstitial inflammation and fibrosis are strongly associated with the outcome of chronic kidney disease. We recently demonstrated that the NOD-like receptor, pyrin domain containing-3 (NLRP3) contributes to renal inflammation, injury, and fibrosis following unilateral ureteric obstruction in mice. NLRP3 expression in renal tubular epithelial cells (TECs) was found to be an important component of experimental disease pathogenesis, although the biology of NLRP3 in epithelial cells is unknown. In human and mouse primary renal TECs, NLRP3 expression was increased in response to TGF-ß1 stimulation and associated with epithelial-mesenchymal transition (EMT) and the expression of α-smooth muscle actin (αSMA) and matrix metalloproteinase (MMP) 9. TGF-ß1-induced EMT and the induction of MMP-9 and αSMA were significantly decreased in mouse Nlrp3(-/-) renal TECs, suggesting a role for Nlrp3 in TGF-ß-dependent signaling. Although apoptosis-associated speck-like protein containing a CARD domain(-/-) TECs demonstrated a phenotype similar to that of Nlrp3(-/-) cells in response to TGF-ß1, the effect of Nlrp3 on MMP-9 and αSMA expression was inflammasome independent, as IL-1ß, IL-18, MyD88, and caspase-1 were dispensable. Smad2 and Smad3 phosphorylation in response to TGF-ß1 was attenuated in Nlrp3(-/-) and apoptosis-associated speck-like protein containing a CARD domain(-/-) cells, accounting for the dampened EMT and TGF-ß1 responsiveness in these cells. Consistent with these findings, overexpression of NLRP3 in 293T cells resulted in increased Smad3 phosphorylation and activity. Taken together, these data support a novel and direct role for NLRP3 in promoting TGF-ß signaling and R-Smad activation in epithelial cells independent of the inflammasome.

Comparative effectiveness of lenalidomide/dexamethasone-based triplet regimens for treatment of relapsed and/or refractory multiple myeloma in the United States: An analysis of real-world electronic health records data.

BACKGROUND: This retrospective longitudinal study compared the effectiveness of dexamethasone+lenalidomide (Rd)-based triplet regimens containing proteasome inhibitors (PIs) ixazomib (IRd), carfilzomib (KRd), and bortezomib (VRd) or monoclonal antibodies (MABs) elotuzumab (ERd) and daratumumab (DRd) in patients with relapsed/refractory multiple myeloma (RRMM)-including those with high cytogenetic risk-primarily treated at community oncology clinics in the United States. METHODS: Electronic health records of adult RRMM patients in a deidentified real-world database (01/01/2014-09/30/2020) who initiated IRd, KRd, VRd, ERd, or DRd in the second or later line of therapy (LOT) were analyzed. The index date was the date of initiation of each LOT and baseline was the 6-month pre-index period. Duration of therapy (DOT), time to next therapy (TTNT), progression-free survival (PFS), and overall survival (OS) were compared across regimens with multivariable Cox proportional hazards models. RESULTS: Of the 1,185 patients contributing 1,332 LOTs, 985 had standard cytogenetic risk (median age, 71 years) and 180 had high risk (median age, 69 years). Compared with other regimens, DRd was associated with longer DOT overall (adjusted hazard ratio [95 % confidence interval]: 1.84 [1.42, 2.38] vs. KRd, 1.65 [1.20, 2.28] vs. ERd, 1.58 [1.23, 2.04] vs. IRd, and 1.54 [1.18, 2.00] vs. VRd), and longer TTNT and PFS. KRd was associated with shorter OS compared with DRd (1.45 [1.01, 2.08]) and VRd (1.32 [1.01, 1.73]). High-risk patients had similar outcomes with all triplet regimens. CONCLUSION: Although DRd improved clinical outcomes overall, Rd-based triplet regimens containing a PI or MAB are similarly effective in high-risk RRMM.

Understanding the Current Landscape of Kidney Disease in Canada to Advance Precision Medicine Guided Personalized Care.

Purpose of Review: To understand the impact of kidney disease in Canada and the priority areas of kidney research that can benefit from patient-oriented, precision medicine research using novel technologies. Sources of Information: Information was collected through discussions between health care professionals, researchers, and patient partners. Literature was compiled using search engines (PubMed, PubMed central, Medline, and Google) and data from the Canadian Organ Replacement Register. Methods: We reviewed the impact, prevalence, economic burden, causes of kidney disease, and priority research areas in Canada. After reviewing the priority areas for kidney research, potential avenues for future research that can integrate precision medicine initiatives for patient-oriented research were outlined. Key Findings: Chronic kidney disease (CKD) remains among the top causes of morbidity and mortality in the world and exerts a large financial strain on the health care system. Despite the increasing number of people with CKD, funding for basic kidney research continues to trail behind other diseases. Current funding strategies favor existing clinical treatment and patient educational strategies. The identification of genetic factors for various forms of kidney disease in the adult and pediatric populations provides mechanistic insight into disease pathogenesis. Allocation of resources and funding toward existing high-yield personalized research initiatives have the potential to significantly affect patient-oriented research outcomes but will be difficult due to a constant decline of funding for kidney research. Limitations: This is an overview primarily focused on Canadian-specific literature rather than a comprehensive systematic review of the literature. The scope of our findings and conclusions may not be applicable to health care systems in other countries.

Justification: Cette étude visait à mieux comprendre l'impact de l'insuffisance rénale au Canada et à déterminer les domaines de recherche prioritaires en santé rénale qui pourraient tirer profit des recherches axées sur le patient qui sont menées en médecine de précision avec de nouvelles technologies. Sources: L'information provient de discussions entre les professionnels de la santé, les chercheurs et les patients partenaires. La littérature a été compilée à l'aide de moteurs de recherche (PubMed, PubMed central, Medline et Google) et de données provenant du Registre canadien des insuffisances et des transplantations d'organes. Méthodologie: Nous avons examiné les répercussions, la prévalence, le fardeau économique et les causes des maladies rénales, ainsi que les domaines prioritaires de la recherche au Canada. Après avoir examiné les domaines de recherche prioritaires en santé rénale, nous avons décrit les possibles orientations de recherche pouvant intégrer des initiatives de la recherche axée sur le patient menée en médecine de précision. Principaux résultats: L'insuffisance rénale chronique (IRC) demeure l'une des principales causes de morbidité et de mortalité dans le monde et elle exerce une forte pression financière sur le système de santé. Malgré le nombre croissant de personnes atteintes d'IRC, le financement de la recherche fondamentale en santé rénale reste à la traîne des autres maladies; et le financement actuel favorise les stratégies existantes pour le traitement clinique et l'éducation des patients. L'identification de facteurs génétiques liés aux diverses formes de néphropathies dans les populations adultes et pédiatriques fournit une compréhension mécanistique de la pathogenèse de la maladie. L'attribution des ressources et du financement à des initiatives de recherche personnalisées existantes et connues pour leur rendement élevé pourrait avoir une incidence considérable sur les résultats de la recherche axée sur le patient, mais elle sera difficile compte tenu de la diminution constante du financement de la recherche en santé rénale. Limites: Il s'agit d'un portrait axé principalement sur la littérature canadienne et non d'un examen systématique complet de la littérature. La portée des résultats et conclusions pourrait ne pas s'appliquer aux systèmes de santé d'autres pays.

Economic burden of early-stage non-small-cell lung cancer: an assessment of healthcare resource utilization and medical costs.

Aim: To quantify the economic burden of early-stage non-small-cell lung cancer (NSCLC) among patients with and without adjuvant therapy. Methods: All-cause and NSCLC-related healthcare resource utilization and medical costs were assessed among patients with resected stage IB-IIIA NSCLC in the SEER-Medicare database (1 January 2011-31 December 2019), from NSCLC diagnosis to death, end of continuous enrollment, or end of data availability (whichever occurred first). Results: Patients receiving adjuvant therapy had the lowest mean NSCLC-related medical costs (adjuvant [n = 1776]: $3738; neoadjuvant [n = 56]: $5793; both [n = 47]: $4818; surgery alone [n = 3478]: $4892, per-person-per-month), driven by lower NSCLC-related hospitalization rates. Conclusion: Post-surgical management of early-stage NSCLC was associated with high economic burden. Adjuvant therapy was associated with numerically lower medical costs over surgical resection alone.

Ocular involvement in TEMPI syndrome.

Purpose: We report the first case of ocular involvement in TEMPI syndrome, a rare disease characterized by telangiectasias, elevated erythropoietin with erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intra-pulmonary shunting. Observations: A 64-year-old Caucasian man with history of TEMPI syndrome presented with subacute bilateral painless vision loss. Ocular examination showed chronic retinal ischemia with microvascular damage, which was likely associated with the chronic systemic hypoxemia, and spontaneous wax and wane of cystoid macular edema, presumedly related to the systemic bortezomib treatment. Conclusions and importance: Our case demonstrates that pathologic retinal vascular changes could be seen in association with TEMPI syndrome and suggests that a comprehensive ophthalmological examination may be beneficial for these patients.

Infecting kidney organoids with a cDNA reporter clone of SARS-CoV-2.

Induced pluripotent stem cell (iPSC)-derived kidney organoids can be used for disease modeling and drug testing. Here, we describe a protocol to prepare stocks of an infectious clone of SARS-CoV-2 expressing a stable mNeonGreen reporter (icSARS-CoV-2-mNG). We demonstrate the infection of kidney organoids, primarily at the proximal tubular cells, with icSARS-CoV-2-mNG. Using a TCID50 (tissue culture infectious dose 50) assay and confocal microscopy, we show the quantification of SARS-CoV-2-mNG signal in proximal tubular cells of the kidney organoids. For complete details on the use and execution of this protocol, please refer to Rahmani et al. (2022).

Attenuation of SARS-CoV-2 infection by losartan in human kidney organoids.

COVID-19-associated acute kidney injury (COVID-AKI) is a common complication of SARS-CoV-2 infection in hospitalized patients. The susceptibility of human kidneys to direct SARS-CoV-2 infection and modulation of the renin-angiotensin II signaling (RAS) pathway by viral infection remain poorly characterized. Using induced pluripotent stem cell-derived kidney organoids, SARS-CoV-1, SARS-CoV-2, and MERS-CoV tropism, defined by the paired expression of a host receptor (ACE2, NRP1 or DPP4) and protease (TMPRSS2, TMPRSS4, FURIN, CTSB or CTSL), was identified primarily among proximal tubule cells. Losartan, an angiotensin II receptor blocker being tested in patients with COVID-19, inhibited angiotensin II-mediated internalization of ACE2, upregulated interferon-stimulated genes (IFITM1 and BST2) known to restrict viral entry, and attenuated the infection of proximal tubule cells by SARS-CoV-2. Our work highlights the susceptibility of proximal tubule cells to SARS-CoV-2 and reveals a putative protective role for RAS inhibitors during SARS-CoV-2 infection.

Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury.

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1-/- mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

The NLRP3 inflammasome promotes renal inflammation and contributes to CKD.

Inflammation significantly contributes to the progression of chronic kidney disease (CKD). Inflammasome-dependent cytokines, such as IL-1ß and IL-18, play a role in CKD, but their regulation during renal injury is unknown. Here, we analyzed the processing of caspase-1, IL-1ß, and IL-18 after unilateral ureteral obstruction (UUO) in mice, which suggested activation of the Nlrp3 inflammasome during renal injury. Compared with wild-type mice, Nlrp3(-/-) mice had less tubular injury, inflammation, and fibrosis after UUO, associated with a reduction in caspase-1 activation and maturation of IL-1ß and IL-18; these data confirm that the Nlrp3 inflammasome upregulates these cytokines in the kidney during injury. Bone marrow chimeras revealed that Nlrp3 mediates the injurious/inflammatory processes in both hematopoietic and nonhematopoietic cellular compartments. In tissue from human renal biopsies, a wide variety of nondiabetic kidney diseases exhibited increased expression of NLRP3 mRNA, which correlated with renal function. Taken together, these results strongly support a role for NLRP3 in renal injury and identify the inflammasome as a possible therapeutic target in the treatment of patients with progressive CKD.