^{Department of Genetics and Cell Biology, Sector Molecular Cell Biology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands;}

^{Department of Pathology, Radboud University, Nijmegen, The Netherlands; and}

^{Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany}

^{Corresponding author.}

H.-J.G. and M.J.M. contributed equally to this work.

Correspondence: Dr. Marcus J. Moeller, Medizinische Klinik II, University Hospital of the RWTH Aachen University, Pauwelsstrasse 30, D-52074 Aachen, Germany. Email: ed.nehcaaku@relleomm

Received 2016 Jan 16; Accepted 2016 Oct 4.

Abstract

For several decades, glucocorticoids have been used empirically to treat rapid progressive GN. It is commonly assumed that glucocorticoids act primarily by dampening the immune response, but the mechanisms remain incompletely understood. In this study, we inactivated the glucocorticoid receptor (GR) specifically in kidney epithelial cells using Pax8-Cre/GR^{mice. Pax8-Cre/GR}^{mice did not exhibit an overt spontaneous phenotype. In mice treated with nephrotoxic serum to induce crescentic nephritis (rapidly progressive GN), this genetic inactivation of the GR in kidney epithelial cells exerted renal benefits, including inhibition of albuminuria and cellular crescent formation, similar to the renal benefits observed with high-dose prednisolone in control mice. However, genetic inactivation of the GR in kidney epithelial cells did not induce the immunosuppressive effects observed with prednisolone.}In vitro, prednisolone and the pharmacologic GR antagonist mifepristone each acted directly on primary cultures of parietal epithelial cells, inhibiting cellular outgrowth and proliferation. In wild-type mice, pharmacologic treatment with the GR antagonist mifepristone also attenuated disease as effectively as high-dose prednisolone without the systemic immunosuppressive effects. Collectively, these data show that glucocorticoids act directly on activated glomerular parietal epithelial cells in crescentic nephritis. Furthermore, we identified a novel therapeutic approach in crescentic nephritis, that of glucocorticoid antagonism, which was at least as effective as high-dose prednisolone with potentially fewer adverse effects.

Keywords: cortisol, glomerulonephritis, podocyte, parietal epithelial cells

Abstract

In the late 1940s, cortisone isolated from the adrenal cortex became available.¹ It was first used in nephrology in 1950 to empirically treat patients with nephrotic syndrome.² Despite their significant side effects, glucocorticoids have now become central in the treatment of most glomerulonephritides. An immune-mediated pathogenesis has been generally accepted for most glomerulopathies, and glucocorticoids are generally believed to act primarily via their immunosuppressive effects. However, experimental evidence in vivo is still needed to clarify the primary mechanism of action of glucocorticoids.

Other than the immune-mediated pathways, other crucial pathomechanisms in glomerulonephritides include injury or loss of podocytes, a major driver for progression to ESRD. A third and novel pathomechanism is activation of parietal epithelial cells (PECs), which may occur in a broad spectrum of glomerular diseases.³,⁴ Recently, it was shown that cellular crescents (i.e., proliferative lesions) in rapidly progressive glomerulonephritides (i.e., crescentic GN) are derived from activated PECs and to lesser extent, podocytes (Figure 1A).⁵ In rapid progressive GN, crescents ultimately obstruct the tubular outlet, and the affected nephron undergoes irreversible degeneration and scarring (Figure 1A, arrow). In most forms of GN, proliferative lesions can be detected to varying degrees and are generally associated with a poor prognosis. Thus, injured podocytes and activated PECs have been identified as a common pathomechanism and prime therapeutic target in glomerular diseases.⁶

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Figure 1.

GR expression in proliferative lesions. (A) Schematic of a glomerulus with a proliferative lesion (i.e., cellular crescent in crescentic GN; right). Obstruction of the tubular outlet by a crescent (arrow) triggers irreversible loss of the nephron and renal function. Blue, podocytes; light green, quiescent PECs. (B and B′) Immunofluorescence staining of a normal human glomerulus (LKIV stains PEC matrix of Bowman’s capsule in red; arrows with tails), podocytes (antisynaptopodin; green), and the GR (magenta). GR expression is visible in all glomerular cells, including podocyte (arrow) and PEC nuclei (arrowheads). (C–C2) Serial sections of a human biopsy with an extracapillary proliferative lesion (arrowheads in C and C′; Goodpasture syndrome). (C1) GR was expressed at low levels in podocytes, which were rarely observed in cellular crescents. GR colocalized with PEC activation marker CD44 (arrows in C2) and PEC matrix (LKIV; arrows with tails). GR (magenta) is expressed in all cell nuclei within sclerotic lesions. GR expression in murine glomerulus. Scale bars, 100 μm. (D and D′) Normal mouse glomerulus (red; PEC matrix [LKIV]; arrows with tails), podocytes (antisynaptopodin; green), and GR (magenta) show ubiquitous nuclear staining of the GR in all glomerular cells. Activated PECs express the GR in experimental GN in mice in proliferative and sclerotic lesions. (E1) Crescentic nephritis model (NTN): CD44–positive activated PECs (green; arrows) deposit PEC matrix (LKIV; red; arrows with tails) and populate a cellular lesion. Scale bars, 50 μm.

The glucocorticoid receptor (GR) binds glucocorticoids in the cytoplasm, dimerizes, and translocates to the nucleus, where it acts as a transcription factor.⁷ Glucocorticoid effects vary significantly between different cell types. For example, in neutrophils, GR activation can induce survival,⁸ whereas apoptosis is induced in lymphocytes (T, B, or NK cells).⁹ Because the GR is expressed in most tissues, glucocorticoids act on not only cells of the immune system but also, many cell types consistent with the broad range of mostly catabolic side effects.

Apart from crescentic GN, glucocorticoids are also effective in less inflammatory forms of GN (e.g., IgA nephropathy, membranous nephropathy, minimal change nephropathy, and FSGS). These clinical observations suggest direct effects of glucocorticoids on renal cells. This study set out to test this hypothesis. The focus was placed on proliferative lesions in GN (i.e., crescentic GN).

Sequences are in the 5′ to 3′ direction.

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Acknowledgments

We thank Prof. Günther Schütz (German Cancer Research Center, Deutsches Krebsforschungszentrum, Heidelberg, Germany) for GR floxed mice.

C.K. was supported by Else Kröner Fresenius Foundation grant A200/2013 and a START and Rotationsstellen Program of the Faculty of Medicine of the Rheinisch-Westfälische Technische Hochschule (RWTH) Aachen University. Support came from German Research Foundation (DFG) grants BO 3755/1-1 (to B.S.), SFB938 (to H.-J.G.), and 1118 (to H.-J.G.); grants TP17 (to J.F. and M.J.M.), TP25 (to J.F. and M.J.M.), and Q2 (to J.F. and M.J.M.) of Sonderforschungsbereich (SFB)/Transregio 57 of the DFG; and the consortium STOP-FSGS by the German Ministry for Science and Education (grant BMBF 01-GM1518A to M.J.M.). This work was supported by the Core Facility Two-Photon Imaging of the Interdisciplinary Center for Clinical Research Aachen within the Faculty of Medicine of RWTH Aachen University. J.F. and M.J.M. are members of the SFB/Transregio 57 DFG Consortium Mechanisms of Organ Fibrosis. M.J.M. was awarded a Heisenberg professorship (DFG grant MO 1082/7-1).

Acknowledgments

Footnotes

Published online ahead of print. Publication date available at www.jasn.org.

This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2016010060/-/DCSupplemental.

Footnotes

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