Marcus Johannes Moeller

Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes. We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GR^fl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone. Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GR^fl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin aminonucleoside–induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria. Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids.

BACKGROUND/AIMS: Podocytes are lost in most glomerular diseases, leading to glomerulosclerosis and progressive kidney disease. It is generally assumed, that podocytes are exposed to the filtration flow and thus to significant shear forces driving their detachment from the glomerular basement membrane (GBM). In this context, foot process effacement has been proposed as potential adaptive response to increase adhesion of podocytes to the GBM. METHODS: We have tested these hypotheses using optical clearing and high-resolution 3-dimensional morphometric analysis in the isolated perfused murine kidney. We investigated the dynamics of podocyte detachment at different perfusion pressures (50, 300 and more than 450 mmHg) in healthy young or old mice (20 vs. 71 weeks of age), or mice injected with anti-GBM serum to induce global foot process effacement. RESULTS: Results show that healthy podocytes in young mice are tightly attached onto the GBM and even supramaximal pressures did not cause significant detachment. Compared to young mice, in aged mice and mice with anti-GBM nephritis and foot process effacement, gradual progressive loss of podocytes had occurred already before perfusion. High perfusion pressures resulted in a relatively minor additional loss of podocytes in aged mice. In mice with anti-GBM nephritis significant additional podocyte loss occurred at this early time point when increasing perfusion pressures to 300 mmHg or higher. CONCLUSION: This work provides the first experimental evidence that podocytes are extraordinarily resistant to acutely increased perfusion pressures in an ex vivo isolated kidney perfusion model. Only in glomerular disease, significant numbers of injured podocytes detached following acute increases in perfusion pressure.

Here, we show that inducible overexpression of Cre recombinase in glomerular podocytes but not in parietal epithelial cells may trigger focal segmental glomerulosclerosis (FSGS) in juvenile transgenic homocygous Pod-rtTA/LC1 mice. Administration of doxycycline shortly after birth, but not at any other time point later in life, resulted in podocyte injury and development of classical FSGS lesions in these mice. Sclerotic lesions were formed as soon as 3 wk of age, and FSGS progressed with low variability until 13 wk of age. In addition, our experiments identified Cre toxicity as a potentially relevant limitation for studies in podocytes of transgenic animals. In summary, our study establishes a novel genetic model for FSGS in mice, which exhibits low variability and manifests already at a young age.

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

Vascular endothelial growth factor A (VEGF) is an essential growth factor during glomerular development and postnatal homeostasis. VEGF is secreted in high amounts by podocytes into the primary urine, back-filtered across the glomerular capillary wall to act on endothelial cells. So far it has been assumed that VEGF back-filtration is driven at a constant rate exclusively by diffusion. In the present work, glomerular VEGF back-filtration was investigated in vivo using a novel extended model based on endothelial fenestrations as surrogate marker for local VEGF concentrations. Single nephron glomerular filtration rate (SNGFR) and/or local filtration flux were manipulated by partial renal mass ablation, tubular ablation, and in transgenic mouse models of systemic or podocytic VEGF overexpression or reduction. Our study shows positive correlations between VEGF back-filtration and SNGFR as well as effective filtration rate under physiological conditions along individual glomerular capillaries in rodents and humans. Our results suggest that an additional force drives VEGF back-filtration, potentially regulated by SNGFR.