A Tight Control of Non-Canonical TGF-β Pathways and MicroRNAs Downregulates Nephronectin in Podocytes

Nephronectin (NPNT) is an extracellular matrix protein in the glomerular basement membrane that is produced by podocytes and is important for the integrity of the glomerular filtration barrier. Upregulated transforming growth factor β (TGF-β) and altered NPNT are seen in different glomerular diseases. TGF-β downregulates NPNT and upregulates NPNT-targeting microRNAs (miRs). However, the pathways involved were previously unknown. By using selective inhibitors of the canonical, SMAD-dependent, and non-canonical TGF-β pathways, we investigated NPNT transcription, translation, secretion, and regulation through miRs in podocytes. TGF-β decreased NPNT mRNA and protein in cultured human podocytes. TGF-β-dependent regulation of NPNT was meditated through intracellular signaling pathways. Under baseline conditions, non-canonical pathways predominantly regulated NPNT post-transcriptionally. Podocyte NPNT secretion, however, was not dependent on canonical or non-canonical TGF-β pathways. The canonical TGF-β pathway was also dispensable for NPNT regulation after TGF-β stimulation, as TGF-β was still able to downregulate NPNT in the presence of SMAD inhibitors. In contrast, in the presence of different non-canonical pathway inhibitors, TGF-β stimulation did not further decrease NPNT expression. Moreover, distinct non-canonical TGF-β pathways mediated TGF-β-induced upregulation of NPNT-targeting miR-378a-3p. Thus, we conclude that post-transcriptional fine-tuning of NPNT expression in podocytes is mediated predominantly through non-canonical TGF-β pathways.

Emerging Technologies to Study the Glomerular Filtration Barrier

Kidney disease is characterized by loss of glomerular function with clinical manifestation of proteinuria. Identifying the cellular and molecular changes that lead to loss of protein in the urine is challenging due to the complexity of the filtration barrier, constituted by podocytes, glomerular endothelial cells, and glomerular basement membrane. In this review, we will discuss how technologies like single cell RNA sequencing and bioinformatics-based spatial transcriptomics, as well as in vitro systems like kidney organoids and the glomerulus-on-a-chip, have contributed to our understanding of glomerular pathophysiology. Knowledge gained from these studies will contribute toward the development of personalized therapeutic approaches for patients affected by proteinuric diseases.

Insulin-activated store-operated Ca2+ entry via Orai1 induces podocyte actin remodeling and causes proteinuria

Podocyte, the gatekeeper of the glomerular filtration barrier, is a primary target for growth factor and Ca2+ signaling whose perturbation leads to proteinuria. However, the effects of insulin action on store-operated Ca2+ entry (SOCE) in podocytes remain unknown. Here, we demonstrated that insulin stimulates SOCE by VAMP2-dependent Orai1 trafficking to the plasma membrane. Insulin-activated SOCE triggers actin remodeling and transepithelial albumin leakage via the Ca2+-calcineurin pathway in podocytes. Transgenic Orai1 overexpression in mice causes podocyte fusion and impaired glomerular filtration barrier. Conversely, podocyte-specific Orai1 deletion prevents insulin-stimulated SOCE, synaptopodin depletion, and proteinuria. Podocyte injury and albuminuria coincide with Orai1 upregulation at the hyperinsulinemic stage in diabetic (db/db) mice, which can be ameliorated by the suppression of Orai1-calcineurin signaling. Our results suggest that tightly balanced insulin action targeting podocyte Orai1 is critical for maintaining filter integrity, which provides novel perspectives on therapeutic strategies for proteinuric diseases, including diabetic nephropathy.

Atypical Renal Clearance of Nanoparticles Larger Than the Kidney Filtration Threshold

In recent years, several publications reported that nanoparticles larger than the kidney filtration threshold were found intact in the urine after being injected into laboratory mice. This theoretically should not be possible, as it is widely known that the kidneys prevent molecules larger than 6–8 nm from escaping into the urine. This is interesting because it implies that some nanoparticles can overcome the size limit for renal clearance. What kinds of nanoparticles can “bypass” the glomerular filtration barrier and cross into the urine? What physical and chemical characteristics are essential for nanoparticles to have this ability? And what are the biomolecular and cellular mechanisms that are involved? This review attempts to answer those questions and summarize known reports of renal-clearable large nanoparticles.

Sodium-Glucose Cotransporter-2 Inhibitors in Patients with Hereditary Podocytopathies, Alport Syndrome, and FSGS: A Case Series to Better Plan a Large-Scale Study

Hereditary diseases of the glomerular filtration barrier are characterized by a more vulnerable glomerular basement membrane and dysfunctional podocytes. Recent clinical trials have demonstrated the nephroprotective effect of sodium-glucose cotransporter-2 inhibitors (SGLT2i) in chronic kidney disease (CKD). SGLT2-mediated afferent arteriole vasoconstriction is hypothesized to correct the hemodynamic overload of the glomerular filtration barrier in hereditary podocytopathies. To test this hypothesis, we report data in a case series of patients with Alport syndrome and focal segmental glomerulosclerosis (FSGS) with respect of the early effect of SGLT2i on the kidney function. Mean duration of treatment was 4.5 (±2.9) months. Mean serum creatinine before and after SGLT-2i initiation was 1.46 (±0.42) and 1.58 (±0.55) mg/dL, respectively, with a median estimated glomerular filtration rate of 64 (±27) before and 64 (±32) mL/min/1.73 m2 after initiation of SGLT2i. Mean urinary albumin-creatinine ratio in mg/g creatinine before SGLT-2i initiation was 1827 (±1560) and decreased by almost 40% to 1127 (±854) after SGLT2i initiation. To our knowledge, this is the first case series on the effect and safety of SGLT2i in patients with hereditary podocytopathies. Specific large-scale trials in podocytopathies are needed to confirm our findings in this population with a tremendous unmet medical need for more effective, early on, and safe nephroprotective therapies.

Urinary Extracellular Vesicles: Uncovering the Basis of the Pathological Processes in Kidney-Related Diseases

Intercellular communication governs multicellular interactions in complex organisms. A variety of mechanisms exist through which cells can communicate, e.g., cell-cell contact, the release of paracrine/autocrine soluble molecules, or the transfer of extracellular vesicles (EVs). EVs are membrane-surrounded structures released by almost all cell types, acting both nearby and distant from their tissue/organ of origin. In the kidney, EVs are potent intercellular messengers released by all urinary system cells and are involved in cell crosstalk, contributing to physiology and pathogenesis. Moreover, urine is a reservoir of EVs coming from the circulation after crossing the glomerular filtration barrier—or originating in the kidney. Thus, urine represents an alternative source for biomarkers in kidney-related diseases, potentially replacing standard diagnostic techniques, including kidney biopsy. This review will present an overview of EV biogenesis and classification and the leading procedures for isolating EVs from body fluids. Furthermore, their role in intra-nephron communication and their use as a diagnostic tool for precision medicine in kidney-related disorders will be discussed.

Modeling the Glomerular Filtration Barrier and Intercellular Crosstalk

The glomerulus is a compact cluster of capillaries responsible for blood filtration and initiating urine production in the renal nephrons. A trilaminar structure in the capillary wall forms the glomerular filtration barrier (GFB), composed of glycocalyx-enriched and fenestrated endothelial cells adhering to the glomerular basement membrane and specialized visceral epithelial cells, podocytes, forming the outermost layer with a molecular slit diaphragm between their interdigitating foot processes. The unique dynamic and selective nature of blood filtration to produce urine requires the functionality of each of the GFB components, and hence, mimicking the glomerular filter in vitro has been challenging, though critical for various research applications and drug screening. Research efforts in the past few years have transformed our understanding of the structure and multifaceted roles of the cells and their intricate crosstalk in development and disease pathogenesis. In this review, we present a new wave of technologies that include glomerulus-on-a-chip, three-dimensional microfluidic models, and organoids all promising to improve our understanding of glomerular biology and to enable the development of GFB-targeted therapies. Here, we also outline the challenges and the opportunities of these emerging biomimetic systems that aim to recapitulate the complex glomerular filter, and the evolving perspectives on the sophisticated repertoire of cellular signaling that comprise the glomerular milieu.

Early mineralocorticoid receptor antagonism in diabetic nephropathy limits albuminuria by preserving the glomerular endothelial glycocalyx

The glomerular endothelial glycocalyx (GEnGlx) forms the first part of the glomerular filtration barrier (GFB). We have previously shown that excess mineralocorticoid receptor (MR) activation causes GEnGlx damage and albuminuria. Damage to the GEnGlx occurs early in the pathogenesis of diabetic nephropathy (DN). Here we sought to determine whether MR antagonism with spironolactone could prevent the development of albuminuria in diabetes, by preserving the GEnGlx to maintain the GFB. Streptozotocin-induced diabetic Wistar rats developed increased glomerular albumin permeability (Ps alb) and albuminuria, with associated GEnGlx loss and increases in plasma and urine active matrix metalloproteinase 2 (MMP2). MR antagonism reduced urine active MMP2, preserved the GEnGlx, restored Ps alb to control values and prevented diabetes-induced albuminuria progression. Enzymatic degradation of the GEnGlx, with hyaluronidase, reversed the effect of MR antagonism in diabetic rats, confirming the importance of GEnGlx preservation in this model. Using this model we validated a novel fluorescent profile peak-to-peak confocal imaging technique and applied it to assess GEnGlx damage on renal biopsies from patients with DN and compared them to healthy controls. We confirmed that GEnGlx loss occurs in human DN and may contribute to the disease phenotype. Taken together our work suggests GEnGlx preservation as an important novel mechanism for reno-protection by MR inhibitors in diabetes.