LRG1 loss effectively restrains glomerular TGF-ß signaling to attenuate diabetic kidney disease.

Transforming growth factor (TGF)-ß signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-ß signaling regulators can substantially influence TGF-ß's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-ß-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-ß-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-ß signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-ß signaling to attenuate DKD.

A Small Molecule Agonist of Krüppel-Like Factor 15 in Proteinuric Kidney Disease.

BACKGROUND: Podocyte loss is the major driver of primary glomerular diseases such as Focal Segmental Glomerulosclerosis. While systemic glucocorticoids remain the initial and primary therapy for these diseases, high-dose and chronic use of glucocorticoids is riddled with systemic toxicities. Krüppel-Like Factor 15 (KLF15) is a glucocorticoid-responsive gene, which is essential for the restoration of mature podocyte differentiation markers and stabilization of actin cytoskeleton in the setting of cell stress. Induction of KLF15 attenuates podocyte injury and glomerulosclerosis in the setting of cell stress. METHODS: A cell-based high-throughput screen with a subsequent structure-activity relationship study was conducted to identify novel agonists of KLF15 in human podocytes. Next, the agonist was tested in cultured human podocytes under cell stress and in three independent proteinuric models (lipopolysaccharide, nephrotoxic serum nephritis, HIV-1 transgenic mice). A combination of RNA-sequencing and molecular modeling with experimental validation was conducted to demonstrate the direct target of the agonist. RESULTS: The high-throughput screen with structure-activity relationship study identified BT503, a urea-based compound, as a novel agonist of KLF15, independent of glucocorticoid signaling. BT503 demonstrated protective effects in cultured human podocytes and in three independent proteinuric murine models. Subsequent molecular modeling with experimental validation shows that BT503, targets the IKK complex by directly binding to IKKß to inhibit canonical NF-κB signaling, which, in turn, restores KLF15 under cell stress, thereby rescuing podocyte loss and ameliorating kidney injury. CONCLUSIONS: By developing and validating a cell-based high-throughput screen in human podocytes, we identified a novel agonist for KLF15 with salutary effects in proteinuric murine models through direct inhibition of IKKß kinase activity.

Podocyte-derived soluble RARRES1 drives kidney disease progression through direct podocyte and proximal tubular injury.

Retinoic acid receptor responder protein-1 (RARRES1) is a podocyte-enriched transmembrane protein whose increased expression correlates with human glomerular disease progression. RARRES1 promotes podocytopenia and glomerulosclerosis via p53-mediated podocyte apoptosis. Importantly, the cytopathic actions of RARRES1 are entirely dependent on its proteolytic cleavage into a soluble protein (sRARRES1) and subsequent podocyte uptake by endocytosis, as a cleavage mutant RARRES1 exerted no effects in vitro or in vivo. As RARRES1 expression is upregulated in human glomerular diseases, here we investigated the functional consequence of podocyte-specific overexpression of RARRES1 in mice in the experimental focal segmental glomerulosclerosis and diabetic kidney disease. We also examined the effects of long-term RARRES1 overexpression on slowly developing aging-induced kidney injury. As anticipated, the induction of podocyte overexpression of RARRES1 (Pod-RARRES1WT) significantly worsened glomerular injuries and worsened kidney function in all three models, while overexpression of RARRES1 cleavage mutant (Pod-RARRES1MT) did not. Remarkably, direct uptake of sRARRES1 was also seen in proximal tubules of injured Pod-RARRES1WT mice and associated with exacerbated tubular injuries, vacuolation, and lipid accumulation. Single-cell RNA sequence analysis of mouse kidneys demonstrated RARRES1 led to a marked deregulation of lipid metabolism in proximal tubule subsets. We further identified matrix metalloproteinase 23 (MMP23) as a highly podocyte-specific metalloproteinase and responsible for RARRES1 cleavage in disease settings, as adeno-associated virus 9-mediated knockdown of MMP23 abrogated sRARRES1 uptake in tubular cells in vivo. Thus, our study delineates a previously unrecognized mechanism by which a podocyte-derived protein directly facilitates podocyte and tubular injury in glomerular diseases and suggests that podocyte-specific functions of RARRES1 and MMP23 may be targeted to ameliorate glomerular disease progression in vivo.

Podocyte-targeted therapies - progress and future directions.

Podocytes are the key target cells for injury across the spectrum of primary and secondary proteinuric kidney disorders, which account for up to 90% of cases of kidney failure worldwide. Seminal experimental and clinical studies have established a causative link between podocyte depletion and the magnitude of proteinuria in progressive glomerular disease. However, no substantial advances have been made in glomerular disease therapies, and the standard of care for podocytopathies relies on repurposed immunosuppressive drugs. The past two decades have seen a remarkable expansion in understanding of the mechanistic basis of podocyte injury, with prospects increasing for precision-based treatment approaches. Dozens of disease-causing genes with roles in the pathogenesis of clinical podocytopathies have been identified, as well as a number of putative glomerular permeability factors. These achievements, together with the identification of novel targets of podocyte injury, the development of potential approaches to harness the endogenous podocyte regenerative potential of progenitor cell populations, ongoing clinical trials of podocyte-specific pharmacological agents and the development of podocyte-directed drug delivery systems, contribute to an optimistic outlook for the future of glomerular disease therapy.

Rationale and Design of a Phase 2, Double-blind, Placebo-Controlled, Randomized Trial Evaluating AMP Kinase-Activation by Metformin in Focal Segmental Glomerulosclerosis.

Introduction: Focal segmental glomerulosclerosis (FSGS), the most common primary glomerular disease leading to end-stage kidney disease (ESKD), is characterized by podocyte injury and depletion, whereas minimal change disease (MCD) has better outcomes despite podocyte injury. Identifying mechanisms capable of preventing podocytopenia during injury could transform FSGS to an "MCD-like" state. Preclinical data have reported conversion of an MCD-like injury to one with podocytopenia and FSGS by inhibition of AMP-kinase (AMPK) in podocytes. Conversely, in FSGS, AMPK-activation using metformin (MF) mitigated podocytopenia and azotemia. Observational studies also support beneficial effects of MF on proteinuria and chronic kidney disease (CKD) outcomes in diabetes. A randomized controlled trial (RCT) to test MF in podocyte injury with FSGS has not yet been conducted. Methods: We report the rationale and design of phase 2, double-blind, placebo-controlled RCT evaluating the efficacy and safety of MF as adjunctive therapy in FSGS. By randomizing 30 patients with biopsy-confirmed FSGS to MF or placebo (along with standard immunosuppression), we will study mechanistic biomarkers that correlate with podocyte injury or depletion and evaluate outcomes after 6 months. We specifically integrate novel urine, blood, and tissue markers as surrogates for FSGS progression along with unbiased profiling strategies. Results and Conclusion: Our phase 2 trial will provide insight into the potential efficacy and safety of MF as adjunctive therapy in FSGS-a crucial step to developing a larger phase 3 study. The mechanistic assays here will guide the design of other FSGS trials and contribute to understanding AMPK activation as a potential therapeutic target in FSGS. By repurposing an inexpensive agent, our results will have implications for FSGS treatment in resource-poor settings.