Retinoic acid improves nephrotoxic serum-induced glomerulonephritis through activation of podocyte retinoic acid receptor α

Abstract

Proliferation of glomerular epithelial cells, including podocytes, is a key histologic feature of crescentic glomerulonephritis. We previously found that retinoic acid (RA) inhibits proliferation and induces differentiation of podocytes by activating RA receptor-α (RARα) in a murine model of HIV-associated nephropathy. Here, we examined whether RA would similarly protect podocytes against nephrotoxic serum-induced crescentic glomerulonephritis and whether this effect was mediated by podocyte RARα. RA treatment markedly improved renal function and reduced the number of crescentic lesions in nephritic wild-type mice, while this protection was largely lost in mice with podocyte-specific ablation of Rara (Pod-Rara knockout). At a cellular level, RA significantly restored the expression of podocyte differentiation markers in nephritic wild-type mice, but not in nephritic Pod-Rara knockout mice. Furthermore, RA suppressed the expression of cell injury, proliferation, and parietal epithelial cell markers in nephritic wild-type mice, all of which were significantly dampened in nephritic Pod-Rara knockout mice. Interestingly, RA treatment led to the coexpression of podocyte and parietal epithelial cell markers in a small subset of glomerular cells in nephritic mice, suggesting that RA may induce transdifferentiation of parietal epithelial cells toward a podocyte phenotype. In vitro, RA directly inhibited the proliferation of parietal epithelial cells and enhanced the expression of podocyte markers. In vivo lineage tracing of labeled parietal epithelial cells confirmed that RA increased the number of parietal epithelial cells expressing podocyte markers in nephritic glomeruli. Thus, RA attenuates crescentic glomerulonephritis primarily through RARα-mediated protection of podocytes and in part through the inhibition of parietal epithelial cell proliferation and induction of their transdifferentiation into podocytes.

Keywords: crescentic glomerulonephritis; parietal epithelial cells; podocytes; proliferation; retinoic acid receptor-alpha; transdifferentiation.

Figure 1. Role of RA and RARα in NTS-induced GN

(a) Representative images of Periodic acid-Schiff (PAS)-stained kidney sections are shown for Pod-Rara^+/+ and Pod-Rara^−/− mice injected with vehicle (PBS), NTS only (NTS) or NTS with RA treatment (NTS+RA). Arrowheads show NTS-induced glomerular crescents (magnification: x200, scale bar: 50μm). (b) Quantification of percentage of glomeruli with crescents. (c) Development of proteinuria was assessed by urinary albumin to creatinine ratio (UACR). (d) Measurement of blood urea nitrogen levels. (*P<0.05 and ***P<0.001, compared to respective PBS-injected control; ^##P<0.01 and ^###P<0.001 compared to respective NTS-injected mice; ^§P<0.05 and ^§§P<0.01 compared to NTS+RA Pod-Rara^+/+; n=6 in each group, n.d., not detected.)

Figure 2. Role of RA and RARα in the expression of podocyte differentiation and injury markers

(a) Representative images of synaptopodin (green) and CD44 (red) immunofluorescence are shown (magnification: x400, scale bar: 50μm). (b, c) Quantification of immunofluorescence optical density for synaptopodin (b) and CD44 (c) are shown. AU, arbitrary units. n.d., not detected. (d, e) Real-time PCR analysis for synaptopodin (d) and CD44 (e) from isolated glomeruli are shown. (**P<0.01 and ***P<0.001 compared to respective PBS-injected control; ^##P<0.01 and ^###P<0.001 compared to respective NTS-injected mice; ^‡P<0.001 compared to NTS-injected Pod-Rara^{+/+; §}P<0.05 and ^§§§<0.001 compared to NTS+RA Pod-Rara^+/+. n.d., not detected, n=6 in each group).

Figure 3. Role of RA and RARα in glomerular cell proliferation in NTS-GN

(a) Immunohistochemical staining of kidney sections for Ki-67 are shown. Representative images of kidneys of mice in each group are shown (magnification: x400, scale bar: 50μm). (b) Quantification of Ki-67+ cells per glomerular cross section. (c) Real-time PCR analysis of Ki-67 from isolated glomeruli is shown. (***P<0.001 compared to respective PBS-injected control; ^##P<0.01 and ^###P<0.001 compared to respective NTS-injected mice; ^‡P<0.001 compared to NTS-injected Pod-Rara^{+/+; §§}P<0.01 compared to NTS+RA Pod-Rara^+/+. n.d., not detected, n=6 in each group).

Figure 4. Role of RA and RARα in the expression PECs and podocyte markers

(a) Immunofluorescence staining of nephrin and Claudin-1. Representative images from mice in each group are shown (magnification: x400, scale bar: 50μm). (b, c) Semi-quantification of immunofluorescence optical density for nephrin (b) and Claudin-1 (c) are shown. AU, arbitrary units. (d, e) Real-time PCR analysis was performed for nephrin and Claudin-1. (f) Quantification of nephrin/Claudin-1-double positive cells in glomeruli of NTS and vehicle-injected mice. Nephrin and Claudin-1-double positive cells were detected only in the nephritic mice treated with RA. (*P<0.05, **P<0.01, and ***P<0.001 compared to respective PBS-injected control; ^##P<0.01 and ^###P<0.001 compared to respective NTS-injected mice; ^§P<0.05 and ^§§P<0.01 compared to NTS+RA Pod-Rara^+/+. n.d., not detected, n.s. not significant, n=6 in each group).

Figure 5. RA inhibits proliferation of PECs and enhances expression of podocyte marker synaptopodin in vitro

(a) Immortalized mouse PECs were cultured in 37°C without IFN-γ for 14 days to induce their differentiation and were incubated with either vehicle (DMSO), atRA (5μM), or AM580 (200nM) for 96 hours. Crystal violet cell proliferation assay was performed to determine the effect of RA on PECs. Growth curve of differentiated PECs are shown as relative fold change in crystal violet stain (OD_570nm) compared to 0h (no factors added) (*P<0.05 and ***P<0.001 compared to DMSO control; n=6). (b) Immortalized mouse PECs were cultured either at 33°C with IFN- γ or 37°C without IFN- γ to induce differentiation for 14 days. Following 14 days, cells were additionally treated with either vehicle (DMSO) atRA (5μM), or AM580 (200nM) for 72 hours. Real-time PCR analysis for PEC marker, Pax2, and podocyte markers, podocin, nephrin and synaptodopodin, are shown (*P<0.05, **P<0.01 and ***P<0.001 compared to 33°C-grown undifferentiated controls; ^##P<0.01 and ^###P<0.001 compared to DMSO-treated differentiated PECs. n=6). (c) Representative image of Pax2/synaptopodin immunofluorescence in differentiated PECs treated with vehicle or atRA (5μM) for 72 hours. Quantification of Pax2/synaptopodin co-expressing PECs/field is shown on the right (***P<0.001 compared to DMSO control).

Figure 6. Lineage tracing of PECs shows increased PECs expressing podocyte marker synaptodopodin in vivo following RA admnistration in NTS-GN mice

PEC-rtTA; TetO-Cre; CAGs-tdTomato transgenic mice were fed with DOX for 4 weeks before injection of NTS to label PECs with tdTomato RFP. (a) Representative images of glomeruli in mice with or without DOX induction show RFP-labeled PECs only with the DOX induction (magnification: x400, scale bar 20μM). RA treatment induced migration of PECs into glomerular tufts in NTS-treated mice. Co-localization of RFP with synaptopodin (in green) confirms the transdifferentiation of PECs into podocytes in NTS+RA glomeruli. (b) Quantification of RFP/synaptopodin (synpo)-double positive cells/glomerular cross section (n.d., not detected, n=6 per group).

Figure 7. Expression of RARα in human crescentic GN kidneys

Immunohistochemical analysis of RARα in biopsy samples of human crescentic GN and minimal change disease (MCD) kidneys. Representative image from crescentic GN biopsy samples (n=5, 3 IgA nephropathy, 2 anti-GBM patients) in comparison to those of MCD (n=3). There is no significant change in RARα expression pattern between crescentic GN compared to MCD kidneys (Magnification 400x, scale bar: 50μm).

Figure 8. Schema of RA effects in crescentic GN

Podocyte injury is the initial event leading to the development of GN. Podocytes may undergo proliferation and/or stimulate PEC proliferation through paracrine effects. RA improves renal function and reduces the number of crescents in NTS-GN mice primarily by protecting podocytes from initial injury and inhibiting proliferation of glomerular cells (podocytes and/or PECs), and possibly by inducing transdifferentiation of PECs into podocytes.