Novel Retinoic Acid Receptor Alpha Agonists for Treatment of Kidney Disease

Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN

Tubular HIPK2 is a key contributor to renal fibrosis

We previously used global Hipk2-null mice in various models of kidney disease to demonstrate the central role of homeodomain-interacting protein kinase 2 (HIPK2) in renal fibrosis development. However, renal tubular epithelial cell–specific (RTEC-specific) HIPK2 function in renal fibrogenesis has yet to be determined. Here, we show that modulation of tubular HIPK2 expression and activity affects renal fibrosis development in vivo. The loss of HIPK2 expression in RTECs resulted in a marked diminution of renal fibrosis in unilateral ureteral obstruction (UUO) mouse models and HIV-associated nephropathy (HIVAN) mouse models, which was associated with the reduction of Smad3 activation and downstream expression of profibrotic markers. Conversely, WT HIPK2 overexpression in RTECs accentuated the extent of renal fibrosis in the setting of UUO, HIVAN, and folic acid–induced nephropathy in mice. Notably, kinase-dead HIPK2 mutant overexpression or administration of BT173, an allosteric inhibitor of HIPK2-Smad3 interaction, markedly attenuated the renal fibrosis in these mouse models of kidney disease, indicating that HIPK2 requires both the kinase activity and its interaction with Smad3 to promote TGF-β–mediated renal fibrosis. Together, these results establish an important RTEC-specific role of HIPK2 in kidney fibrosis and further substantiate the inhibition of HIPK2 as a therapeutic approach against renal fibrosis

Expression of podocyte differentiation markers in vivo.

<p>Glomeruli were isolated from both WT and Tg26 mice treated with vehicle or BD4 for 6 weeks. Total RNA was isolated from the glomeruli for real-time PCR analysis for the podocyte differentiation markers including synaptopodin, nephrin, and WT-1 (<b>A</b>) as well as for RARα and RARβ (<b>B</b>). The ratio of these genes normalized to GAPDH was calculated. n = 6, *p<0.01 compared to vehicle-treated WT mice (WT-vehicle), **p<0.05 compared to vehicle-treated Tg26 mice (Tg26-vehicle), ***p<0.05 compared to vehicle-treated WT mice (WT-vehicle).</p

Sequences of the primers.

<p>Sequences of the primers.</p

Expression of podocyte differentiation markers in vitro.

<p><b>A.</b> Mouse podocytes cultured at 37°C for 10 days and then stimulated with either ATRA, or Am580, or BD4 at 0.1 µM and 1 µM for 24 hours in serum free medium. Total RNA was extracted and analyzed by real-time PCR to determine the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1). <b>B.</b> Podocytes stimulated with either ATRA, or Am580, or BD4 at 1 µM for 24 hours in serum free medium. Real-time PCR analysis of RARα and RARβ expression were performed. The ratios of these genes normalized to GAPDH expression are expressed. N = 4, p*<0.01 compared to cells treated with DMSO.</p

Cell toxicity assay.

<p>Mouse podocytes cultured at 37°C for 10 days and then treated with ATRA, Am580, or BD4 at the different concentrations for 24 hours in serum free medium. Cell death was quantified by trypan blue staining, n = 6, *p<0.01 compared to cells treated with DMSO, **p<0.05 compared to cells treated with either ATRA or Am580 at corresponding concentrations.</p

Effects of BD4 on mouse body weight, BUN, proteinuria, and kidney histology.

<p>Tg26 and the WT littermates were treated with BD4 or control DMSO for 6 weeks. Body weight was recorded when mice were sacrificed. Blood samples were collected for measurement of BUN and urine samples were collected for determination of albumin and creatinine. Histology of these kidneys was analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027945#s4" target="_blank">method</a>, n = 6, </p><p>*p<0.01 compared to Tg26 mice treated with vehicle.</p