Podocytes are a major component of the glomerular blood filtration barrier, and alterations to the morphology of their unique actin-based foot processes (FP) are a common feature of kidney disease. Adjacent FP are connected by a specialized intercellular junction known as the slit diaphragm (SD), which serves as the ultimate barrier to regulate passage of macromolecules from the blood. While the link between SD dysfunction and reduced filtration selectivity has been recognized for nearly 50 years, our understanding of the underlying molecular circuitry began only 20 years ago, sparked by the identification of NPHS1, encoding the transmembrane protein nephrin. Nephrin not only functions as the core component of the extracellular SD filtration network but also as a signaling scaffold via interactions at its short intracellular region. Phospho-regulation of several conserved tyrosine residues in this region influences signal transduction pathways which control podocyte cell adhesion, shape, and survival, and emerging studies highlight roles for nephrin phospho-dynamics in mechanotransduction and endocytosis. The following review aims to summarize the last 5 years of advancement in our knowledge of how signaling centered at nephrin directs SD barrier formation and function. We further provide insight on promising frontiers in podocyte biology, which have implications for SD signaling in the healthy and diseased kidney.

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin Y3F/Y3F mice). Homozygous nephrin Y3F/Y3F mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin Y3F/Y3F mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome.Compartmentalization of the eukaryotic cell into membrane-bound and membrane-less organelles and other subcellular niches allows biological processes to occur synchronously 1 . Proteins often localize to specific subcellular niches to fulfil their function and dynamic movement of proteins between compartments is essential for cellular processes including signalling, growth, proliferation, motility and programmed cell death; indeed, cells employ dedicated mechanisms to ensure the correct trafficking of proteins and mislocalization of proteins has been implicated in various different pathological states 2,3 . Mutations causing aberrant protein localization underpin some forms of obesity 4 , cancers 5 , laminopathies 6 and lung and liver disease 7 , and translation at inappropriate subcellular locations has been linked to cancer 8 and dementia 9 .Determining the subcellular location of a protein and how it changes upon perturbation or varies between different cell types is essential for understanding the protein's biochemical function. This is complicated in the case of multi-localized proteins (MLPs), which reside in multiple subcellular locations because trafficking between locations is part of their cellular function or enables them to adopt different functions in the cell in a context specific manner 10,11 . Up to 50% of the proteome is estimated to be composed of MLPs 11 . Recently, community-led spatial proteomics approaches and the refinement of experimental techniques have made substantial progress in determining and understanding the subcellular localization of proteins and assembling subcellular protein atlases [11][12][13][14][15][16][17][18] . These experimental metho...

Purpose of review: Uremic pruritus is a highly prevalent and debilitating symptom in patients with chronic kidney disease (CKD) and end-stage kidney disease (ESKD). The purpose of this review is to examine current evidence on the mechanisms and treatments of pruritus in CKD and highlight promising areas for future research. Sources of information: Published literature, including randomized controlled trials, cohort studies, case reports, and review articles, was searched for evidence pertaining to the pathophysiology and treatment of uremic pruritus. Methods: A comprehensive narrative review was conducted to explore the molecular mechanisms underlying uremic pruritus, as well as the evidence (or lack thereof) supporting pharmacological and nonpharmacological treatments for uremic pruritus. The potential role of patient sex in the pathophysiology and management of uremic pruritus is also discussed. Key findings: The pathophysiology of uremic pruritus involves a complex interplay of uremic toxins, systemic inflammation, mast cell activation, and imbalance of opioid receptors. Classic treatment strategies for uremic pruritus include optimization of dialysis parameters, amelioration of CKD-related mineral and bone disease, topical emollients and analgesics, antihistamines, the anticonvulsant medications gabapentin and pregabalin, and ultraviolet light B (UV-B) phototherapy. Strong data to support many of these classical treatments for uremic pruritus are limited. Newly evolving treatment approaches for uremic pruritus include opioid receptor modulators, neurokinin-1 inhibitors, and cannabinoids. Further studies regarding their efficacy, pharmacodynamics, and safety in the CKD and ESKD population are needed before these agents are accepted into widespread use. Additional nonpharmacological strategies aimed at treating uremic pruritus include psychotherapy, acupuncture, omega-3 fatty acids, and exercise. Finally, sex differences may exist regarding uremic pruritus, but studies directly addressing sex-specific mechanisms of uremic pruritus remain absent. Limitations: High-quality evidence in the management of uremic pruritus remains lacking. Most recommendations are based on expert opinion or studies involving small numbers of patients. In addition, our understanding of the pathophysiological mechanisms behind uremic pruritus is incomplete and continues to evolve over time. Implications: Uremic pruritus is a common symptom which reduces quality of life in CKD and ESKD. The identification of novel targeted treatment approaches may ease the burden of uremic pruritus in the future.