Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Lipid metabolism in sickness and in health: Emerging regulators of lipotoxicity.

Lipids play crucial roles in signal transduction, contribute to the structural integrity of cellular membranes, and regulate energy metabolism. Questions remain as to which lipid species maintain metabolic homeostasis and which disrupt essential cellular functions, leading to metabolic disorders. Here, we discuss recent advances in understanding lipid metabolism with a focus on catabolism, synthesis, and signaling. Technical advances, including functional genomics, metabolomics, lipidomics, lipid-protein interaction maps, and advances in mass spectrometry, have uncovered new ways to prioritize molecular mechanisms mediating lipid function. By reviewing what is known about the distinct effects of specific lipid species in physiological pathways, we provide a framework for understanding newly identified targets regulating lipid homeostasis with implications for ameliorating metabolic diseases.

Gasdermin D pore structure reveals preferential release of mature interleukin-1.

As organelles of the innate immune system, inflammasomes activate caspase-1 and other inflammatory caspases that cleave gasdermin D (GSDMD). Caspase-1 also cleaves inactive precursors of the interleukin (IL)-1 family to generate mature cytokines such as IL-1ß and IL-18. Cleaved GSDMD forms transmembrane pores to enable the release of IL-1 and to drive cell lysis through pyroptosis1-9. Here we report cryo-electron microscopy structures of the pore and the prepore of GSDMD. These structures reveal the different conformations of the two states, as well as extensive membrane-binding elements including a hydrophobic anchor and three positively charged patches. The GSDMD pore conduit is predominantly negatively charged. By contrast, IL-1 precursors have an acidic domain that is proteolytically removed by caspase-110. When permeabilized by GSDMD pores, unlysed liposomes release positively charged and neutral cargoes faster than negatively charged cargoes of similar sizes, and the pores favour the passage of IL-1ß and IL-18 over that of their precursors. Consistent with these findings, living-but not pyroptotic-macrophages preferentially release mature IL-1ß upon perforation by GSDMD. Mutation of the acidic residues of GSDMD compromises this preference, hindering intracellular retention of the precursor and secretion of the mature cytokine. The GSDMD pore therefore mediates IL-1 release by electrostatic filtering, which suggests the importance of charge in addition to size in the transport of cargoes across this large channel.

Mechanisms of Protein Trafficking and Quality Control in the Kidney and Beyond.

Numerous trafficking and quality control pathways evolved to handle the diversity of proteins made by eukaryotic cells. However, at every step along the biosynthetic pathway, there is the potential for quality control system failure. This review focuses on the mechanisms of disrupted proteostasis. Inspired by diseases caused by misfolded proteins in the kidney (mucin 1 and uromodulin), we outline the general principles of protein biosynthesis, delineate the recognition and degradation pathways targeting misfolded proteins, and discuss the role of cargo receptors in protein trafficking and lipid homeostasis. We also discuss technical approaches including live-cell fluorescent microscopy, chemical screens to elucidate trafficking mechanisms, multiplexed single-cell CRISPR screening platforms to systematically delineate mechanisms of proteostasis, and the advancement of novel tools to degrade secretory and membrane-associated proteins. By focusing on components of trafficking that go awry, we highlight ongoing efforts to understand fundamental mechanisms of disrupted proteostasis and implications for the treatment of human proteinopathies.

Single-Cell Transcriptomics Reveal Disrupted Kidney Filter Cell-Cell Interactions after Early and Selective Podocyte Injury.

The health of the kidney filtration barrier requires communication among podocytes, endothelial cells, and mesangial cells. Disruption of these cell-cell interactions is thought to contribute to disease progression in chronic kidney diseases (CKDs). Podocyte ablation via doxycycline-inducible deletion of an essential endogenous molecule, CTCF [inducible podocyte-specific CTCF deletion (iCTCFpod-/-)], is sufficient to drive progressive CKD. However, the earliest events connecting podocyte injury to disrupted intercellular communication within the kidney filter remain unclear. Single-cell RNA sequencing of kidney tissue from iCTCFpod-/- mice after 1 week of doxycycline induction was performed to generate a map of the earliest transcriptional effects of podocyte injury on cell-cell interactions at single-cell resolution. A subset of podocytes had the earliest signs of injury due to disrupted gene programs for cytoskeletal regulation and mitochondrial function. Surviving podocytes up-regulated collagen type IV ɑ5, causing reactive changes in integrin expression in endothelial populations and mesangial cells. Intercellular interaction analysis revealed several receptor-ligand-target gene programs as drivers of endothelial cell injury and abnormal matrix deposition. This analysis reveals the earliest disruptive changes within the kidney filter, pointing to new, actionable targets within a therapeutic window that may allow us to maximize the success of much needed therapeutic interventions for CKDs.

HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes.

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of hundreds of chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell-type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome single-cell RNA-sequencing, making HyPR-seq a powerful method for targeted RNA profiling in single cells.

Discovery of Autoantibodies Targeting Nephrin in Minimal Change Disease Supports a Novel Autoimmune Etiology.

BACKGROUND: Failure of the glomerular filtration barrier, primarily by loss of slit diaphragm architecture, underlies nephrotic syndrome in minimal change disease. The etiology remains unknown. The efficacy of B cell-targeted therapies in some patients, together with the known proteinuric effect of anti-nephrin antibodies in rodent models, prompted us to hypothesize that nephrin autoantibodies may be present in patients with minimal change disease. METHODS: We evaluated sera from patients with minimal change disease, enrolled in the Nephrotic Syndrome Study Network (NEPTUNE) cohort and from our own institutions, for circulating nephrin autoantibodies by indirect ELISA and by immunoprecipitation of full-length nephrin from human glomerular extract or a recombinant purified extracellular domain of human nephrin. We also evaluated renal biopsies from our institutions for podocyte-associated punctate IgG colocalizing with nephrin by immunofluorescence. RESULTS: In two independent patient cohorts, we identified circulating nephrin autoantibodies during active disease that were significantly reduced or absent during treatment response in a subset of patients with minimal change disease. We correlated the presence of these autoantibodies with podocyte-associated punctate IgG in renal biopsies from our institutions. We also identified a patient with steroid-dependent childhood minimal change disease that progressed to end stage kidney disease; she developed a massive post-transplant recurrence of proteinuria that was associated with high pretransplant circulating nephrin autoantibodies. CONCLUSIONS: Our discovery of nephrin autoantibodies in a subset of adults and children with minimal change disease aligns with published animal studies and provides further support for an autoimmune etiology. We propose a new molecular classification of nephrin autoantibody minimal change disease to serve as a framework for instigation of precision therapeutics for these patients.

PIP2 regulation of TRPC5 channel activation and desensitization.

Transient receptor potential canonical type 5 (TRPC5) ion channels are expressed in the brain and kidney and have been identified as promising therapeutic targets whose selective inhibition can protect against diseases driven by a leaky kidney filter, such as focal segmental glomerular sclerosis. TRPC5 channels are activated not only by elevated levels of extracellular Ca2+or lanthanide ions but also by G protein (Gq/11) stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C enzymes leads to PKC-mediated phosphorylation of TRPC5 channels and their subsequent desensitization. However, the roles of PIP2 in activation and maintenance of TRPC5 channel activity via its hydrolysis product diacyl glycerol (DAG), as well as the mechanism of desensitization of TRPC5 activity by DAG-stimulated PKC activity, remain unclear. Here, we designed experiments to distinguish between the processes underlying channel activation and inhibition. Employing whole-cell patch-clamp, we used an optogenetic tool to dephosphorylate PIP2 and assess channel-PIP2 interactions influenced by activators, such as DAG, or inhibitors, such as PKC phosphorylation. Using total internal reflection microscopy, we assessed channel cell surface density. We show that PIP2 controls both the PKC-mediated inhibition and the DAG- and lanthanide-mediated activation of TRPC5 currents via control of gating rather than channel cell surface density. These mechanistic insights promise to aid in the development of more selective and precise inhibitors to block TRPC5 channel activity and illuminate new opportunities for targeted therapies for a group of chronic kidney diseases for which there is currently a great unmet need.

Multigram Preparation of BRD4780 Enantiomers and Assignment of Absolute Stereochemistry.

The development of a multigram synthesis of 3-exo-isopropylbicyclo[2.2.1]heptan-2-endo-amine hydrochloride (1) (also known as BRD4780 and AGN-192403) is described. The process involves protection of the amine as 4-nitrobenzyl carbamate, pNZ, which enables chiral SFC chromatography. The absolute configuration (AC) of the individual enantiomers has been determined by Mosher's amide method, VCD spectroscopy, and X-ray crystallography. We highlight the VCD approach as a rapid and effective means of AC determination that can be deployed directly on the target compounds.

Clinical and genetic spectra of autosomal dominant tubulointerstitial kidney disease due to mutations in UMOD and MUC1.

Autosomal dominant tubulointerstitial kidney disease (ADTKD) is an increasingly recognized cause of end-stage kidney disease, primarily due to mutations in UMOD and MUC1. The lack of clinical recognition and the small size of cohorts have slowed the understanding of disease ontology and development of diagnostic algorithms. We analyzed two registries from Europe and the United States to define genetic and clinical characteristics of ADTKD-UMOD and ADTKD-MUC1 and develop a practical score to guide genetic testing. Our study encompassed 726 patients from 585 families with a presumptive diagnosis of ADTKD along with clinical, biochemical, genetic and radiologic data. Collectively, 106 different UMOD mutations were detected in 216/562 (38.4%) of families with ADTKD (303 patients), and 4 different MUC1 mutations in 72/205 (35.1%) of the families that are UMOD-negative (83 patients). The median kidney survival was significantly shorter in patients with ADTKD-MUC1 compared to ADTKD-UMOD (46 vs. 54 years, respectively), whereas the median gout-free survival was dramatically reduced in patients with ADTKD-UMOD compared to ADTKD-MUC1 (30 vs. 67 years, respectively). In contrast to patients with ADTKD-UMOD, patients with ADTKD-MUC1 had normal urinary excretion of uromodulin and distribution of uromodulin in tubular cells. A diagnostic algorithm based on a simple score coupled with urinary uromodulin measurements separated patients with ADTKD-UMOD from those with ADTKD-MUC1 with a sensitivity of 94.1%, a specificity of 74.3% and a positive predictive value of 84.2% for a UMOD mutation. Thus, ADTKD-UMOD is more frequently diagnosed than ADTKD-MUC1, ADTKD subtypes present with distinct clinical features, and a simple score coupled with urine uromodulin measurements may help prioritizing genetic testing.

Outcomes of patient self-referral for the diagnosis of several rare inherited kidney diseases.

PURPOSE: To evaluate self-referral from the Internet for genetic diagnosis of several rare inherited kidney diseases. METHODS: Retrospective study from 1996 to 2017 analyzing data from an academic referral center specializing in autosomal dominant tubulointerstitial kidney disease (ADTKD). Individuals were referred by academic health-care providers (HCPs) nonacademic HCPs, or directly by patients/families. RESULTS: Over 21 years, there were 665 referrals, with 176 (27%) directly from families, 269 (40%) from academic HCPs, and 220 (33%) from nonacademic HCPs. Forty-two (24%) direct family referrals had positive genetic testing versus 73 (27%) families from academic HCPs and 55 (25%) from nonacademic HCPs (P = 0.72). Ninety-nine percent of direct family contacts were white and resided in zip code locations with a mean median income of $77,316 ± 34,014 versus US median income $49,445. CONCLUSION: Undiagnosed families with Internet access bypassed their physicians and established direct contact with an academic center specializing in inherited kidney disease to achieve a diagnosis. Twenty-five percent of all families diagnosed with ADTKD were the result of direct family referral and would otherwise have been undiagnosed. If patients suspect a rare disorder that is undiagnosed by their physicians, actively pursuing self-diagnosis using the Internet can be successful. Centers interested in rare disorders should consider improving direct access to families.

Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound, AC1903.

The transient receptor potential cation channel 5 (TRPC5) has been previously shown to affect podocyte survival in the kidney. As such, inhibitors of TRPC5 are interesting candidates for the treatment of chronic kidney disease (CKD). Herein, we report the synthesis and biological characterization of a series of N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors. Work reported here evaluates the benzimidazole scaffold and substituents resulting in the discovery of AC1903, a TRPC5 inhibitor that is active in multiple animal models of CKD.

Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease.

BACKGROUND: Autosomal dominant tubulointerstitial kidney disease caused by mucin-1 gene (MUC1) mutations (ADTKD-MUC1) is characterized by progressive kidney failure. Genetic evaluation for ADTKD-MUC1 specifically tests for a cytosine duplication that creates a unique frameshift protein (MUC1fs). Our goal was to develop immunohistochemical methods to detect the MUC1fs created by the cytosine duplication and, possibly, by other similar frameshift mutations and to identify novel MUC1 mutations in individuals with positive immunohistochemical staining for the MUC1fs protein. METHODS: We performed MUC1fs immunostaining on urinary cell smears and various tissues from ADTKD-MUC1-positive and -negative controls as well as in individuals from 37 ADTKD families that were negative for mutations in known ADTKD genes. We used novel analytic methods to identify MUC1 frameshift mutations. RESULTS: After technique refinement, the sensitivity and specificity for MUC1fs immunostaining of urinary cell smears were 94.2% and 88.6%, respectively. Further genetic testing on 17 families with positive MUC1fs immunostaining revealed six families with five novel MUC1 frameshift mutations that all predict production of the identical MUC1fs protein. CONCLUSIONS: We developed a noninvasive immunohistochemical method to detect MUC1fs that, after further validation, may be useful in the future for diagnostic testing. Production of the MUC1fs protein may be central to the pathogenesis of ADTKD-MUC1.

Lysine trimethylation regulates 78-kDa glucose-regulated protein proteostasis during endoplasmic reticulum stress.

The up-regulation of chaperones such as the 78-kDa glucose-regulated protein (GRP78, also referred to as BiP or HSPA5) is part of the adaptive cellular response to endoplasmic reticulum (ER) stress. GRP78 is widely used as a marker of the unfolded protein response, associated with sustained ER stress. Here we report the discovery of a proteostatic mechanism involving GRP78 trimethylation in the context of ER stress. Using mass spectrometry-based proteomics, we identified two GRP78 fractions, one homeostatic and one induced by ER stress. ER stress leads to de novo biosynthesis of non-trimethylated GRP78, whereas homeostatic, METTL21A-dependent lysine 585-trimethylated GRP78 is reduced. This proteostatic mechanism, dependent on the posttranslational modification of GRP78, allows cells to differentially regulate specific protein abundance during cellular stress.

Synaptopodin Is a Coincidence Detector of Tyrosine versus Serine/Threonine Phosphorylation for the Modulation of Rho Protein Crosstalk in Podocytes.

Tyrosine and serine/threonine signal-transduction pathways influence many aspects of cell behavior, including the spatial and temporal regulation of the actin cytoskeleton. However, little is known about how input from diverse tyrosine and serine/threonine kinases is integrated to control Rho protein crosstalk and actin remodeling, which are critically important in podocyte health and disease. Here we unveil the proteolytically-regulated, actin organizing protein synaptopodin as a coincidence detector of tyrosine versus serine/threonine phosphorylation. We show that serine/threonine and tyrosine kinases duel for synaptopodin stability versus degradation. EGFR/Src-mediated tyrosine phosphorylation of synaptopodin in podocytes promotes binding to the serine/threonine phosphatase calcineurin. This leads to the loss of 14-3-3 binding, resulting in synaptopodin degradation, Vav2 activation, enhanced Rac1 signaling, and ultimate loss of stress fibers. Our studies reveal how synaptopodin, a single proteolytically-controlled protein, integrates antagonistic tyrosine versus serine/threonine phosphorylation events for the dynamic control of the actin cytoskeleton in podocytes.

Calcium-permeable ion channels in the kidney.

Calcium ions (Ca(2+)) are crucial for a variety of cellular functions. The extracellular and intracellular Ca(2+) concentrations are thus tightly regulated to maintain Ca(2+) homeostasis. The kidney, one of the major organs of the excretory system, regulates Ca(2+) homeostasis by filtration and reabsorption. Approximately 60% of the Ca(2+) in plasma is filtered, and 99% of that is reabsorbed by the kidney tubules. Ca(2+) is also a critical signaling molecule in kidney development, in all kidney cellular functions, and in the emergence of kidney diseases. Recently, studies using genetic and molecular biological approaches have identified several Ca(2+)-permeable ion channel families as important regulators of Ca(2+) homeostasis in kidney. These ion channel families include transient receptor potential channels (TRP), voltage-gated calcium channels, and others. In this review, we provide a brief and systematic summary of the expression, function, and pathological contribution for each of these Ca(2+)-permeable ion channels. Moreover, we discuss their potential as future therapeutic targets.

Abatacept in B7-1-positive proteinuric kidney disease.

Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize ß1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.

Human genetics of nephrotic syndrome and the quest for precision medicine.

PURPOSE OF REVIEW: In this review, we take a combined membrane biologist's and geneticist's view of the podocyte, to examine how genetics have informed our understanding of membrane receptors, channels, and other signaling molecules affecting podocyte health and disease. RECENT FINDINGS: An integral part of the kidney, the glomerulus, is responsible for the kidney's filter function. Within the glomerulus, the podocyte is a unique cell serving a critically important role: it is exposed to signals from the urinary space in Bowman's capsule, it receives and transmits signals to/from the basement membrane upon which it elaborates, and it receives signals from the vascular space with which it also communicates, thus exposed to toxins, viruses, chemicals, proteins, and cellular components or debris that flow in the blood stream. Our understanding of how podocytes perform their important role has been largely informed by human genetics, and the recent revolution afforded by exome sequencing has brought a tremendous wealth of new genetic data to light. SUMMARY: Genetically defined, rare/orphan podocytopathies, as reviewed here, are critically important to study as they may reveal the next generation targets for precision medicine in nephrology.