A conditionally immortalized Gli1-positive kidney mesenchymal cell line models myofibroblast transition.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-ß (TGF-ß). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-ß-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.

Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in PKD1 and PKD2 genes. Recent work suggests that epigenetic modulation of gene expression and protein function may play a role in ADPKD pathogenesis. In this study, we identified SMYD2, a SET and MYND domain protein with lysine methyltransferase activity, as a regulator of renal cyst growth. SMYD2 was upregulated in renal epithelial cells and tissues from Pkd1-knockout mice as well as in ADPKD patients. SMYD2 deficiency delayed renal cyst growth in postnatal kidneys from Pkd1 mutant mice. Pkd1 and Smyd2 double-knockout mice lived longer than Pkd1-knockout mice. Targeting SMYD2 with its specific inhibitor, AZ505, delayed cyst growth in both early- and later-stage Pkd1 conditional knockout mouse models. SMYD2 carried out its function via methylation and activation of STAT3 and the p65 subunit of NF-κB, leading to increased cystic renal epithelial cell proliferation and survival. We further identified two positive feedback loops that integrate epigenetic regulation and renal inflammation in cyst development: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2. These pathways provide mechanisms by which SMYD2 might be induced by cyst fluid IL-6 and TNF-α in ADPKD kidneys. The SMYD2 transcriptional target gene Ptpn13 also linked SMYD2 to other PKD-associated signaling pathways, including ERK, mTOR, and Akt signaling, via PTPN13-mediated phosphorylation.

TNFα signaling regulates cystic epithelial cell proliferation through Akt/mTOR and ERK/MAPK/Cdk2 mediated Id2 signaling.

Tumor necrosis factor alpha (TNFα) is present in cyst fluid and promotes cyst growth in autosomal dominant polycystic kidney disease (ADPKD). However, the cross-talk between TNFα and PKD associated signaling pathways remains elusive. In this study, we found that stimulation of renal epithelial cells with TNFα or RANKL (receptor activator of NF-κB ligand), a member of the TNFα cytokine family, activated either the PI3K pathway, leading to AKT and mTOR mediated the increase of Id2 protein, or MAPK and Cdk2 to induce Id2 nuclear translocation. The effects of TNFα/RANKL on increasing Id2 protein and its nuclear translocation caused significantly decreased mRNA and protein levels of the Cdk inhibitor p21, allowing increased cell proliferation. TNFα levels increase in cystic kidneys in response to macrophage infiltration and thus might contribute to cyst growth and enlargement during the progression of disease. As such, this study elucidates a novel mechanism for TNFα signaling in regulating cystic renal epithelial cell proliferation in ADPKD.

Macrophage migration inhibitory factor promotes cyst growth in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by renal cyst formation, inflammation, and fibrosis. Macrophages infiltrate cystic kidneys, but the role of these and other inflammatory factors in disease progression are poorly understood. Here, we identified macrophage migration inhibitory factor (MIF) as an important regulator of cyst growth in ADPKD. MIF was upregulated in cyst-lining epithelial cells in polycystin-1-deficient murine kidneys and accumulated in cyst fluid of human ADPKD kidneys. MIF promoted cystic epithelial cell proliferation by activating ERK, mTOR, and Rb/E2F pathways and by increasing glucose uptake and ATP production, which inhibited AMP-activated protein kinase signaling. MIF also regulated cystic renal epithelial cell apoptosis through p53-dependent signaling. In polycystin-1-deficient mice, MIF was required for recruitment and retention of renal macrophages, which promoted cyst expansion, and Mif deletion or pharmacologic inhibition delayed cyst growth in multiple murine ADPKD models. MIF-dependent macrophage recruitment was associated with upregulation of monocyte chemotactic protein 1 (MCP-1) and inflammatory cytokine TNF-α. TNF-α induced MIF expression, and MIF subsequently exacerbated TNF-α expression in renal epithelial cells, suggesting a positive feedback loop between TNF-α and MIF during cyst development. Our study indicates MIF is a central and upstream regulator of ADPKD pathogenesis and provides a rationale for further exploration of MIF as a therapeutic target for ADPKD.

Therapeutic targeting of BET bromodomain protein, Brd4, delays cyst growth in ADPKD.

In this study, we identified a BET bromodomain (BRD) protein, Brd4, not only as a novel epigenetic regulator of autosomal dominant polycystic kidney disease (ADPKD) but also as a novel client protein of Hsp90. We found that Brd4 was upregulated in Pkd1 mutant mouse renal epithelial cells and tissues. This upregulation of Brd4 appears to result from the chaperone activity of Hsp90 and escape proteasomal degradation. We further identify that Brd4 is an upstream regulator of the expression of c-Myc which has been upregulated in all rodent models of PKD and ADPKD patients with unknown mechanism. Inhibition of Brd4 in Pkd1 mutant renal epithelial cells with JQ1, a selective small-molecular inhibitor of BET BRD protein(s), (1) decreased the levels of c-Myc mRNA and protein; (2) increased the levels of p21 mRNA and protein, which was transcriptionally repressed by c-Myc; (3) decreased the phosphorylation of Rb; and (4) decreased cystic epithelial cell proliferation as shown by inhibition of S-phase entry. Most importantly, treatment with JQ1 strikingly delayed cyst growth and kidney enlargement, and preserved renal function in two early stage genetic mouse strains with Pkd1 mutations. This study not only provides one of the mechanisms of how c-Myc is upregulated in PKD but also suggests that targeting Brd4 with JQ1 may function as a novel epigenetic approach in ADPKD. The unraveled link between Brd4 and Hsp90 in ADPKD may also be a general mechanism for the upregulation of Brd4 in cancer cells and opens up avenues for combination therapies against ADPKD and cancer.

SIRT2 regulates ciliogenesis and contributes to abnormal centrosome amplification caused by loss of polycystin-1.

The mechanisms underlying many of the human disease phenotypes associated with ciliary dysfunction and abnormal centrosome amplification have yet to be fully elucidated. Here, we present for the first time that SIRT2, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, regulates ciliogenesis and centrosome amplification. Overexpression of SIRT2 in renal epithelial cells appeared to disrupt cilia formation, causing decreased numbers of cells with cilia and decreased cilia length, while inhibition of SIRT2 activity by nicotinamide treatment or knockdown of SIRT2 with siRNA was shown to block cilia disassembly during the cell cycle. Overexpression of SIRT2 in zebrafish decreased cilia numbers in Kupffer's vesicle, while morpholino knock down of SIRT2 increased cilia length. Aberrant centrosome amplification and polyploidy were seen with overexpression of SIRT2 in mouse inner medullary collecting duct 3 cells, similar to that observed following Pkd1 knockdown. SIRT2 was up-regulated in both Pkd1 mutant and knockdown cells. Depletion of SIRT2 prevented the abnormal centrosome amplification and polyploidy associated with loss of polycystin-1 (PC1) alone. Thus, we conclude that the aberrant centrosome amplification and polyploidy in Pkd1 mutant or depleted cells was mediated through overexpression of SIRT2. Our results suggest a novel function of SIRT2 in cilia dynamics and centrosome function, and in ciliopathy-associated disease progression.

Smac-mimetic-induced epithelial cell death reduces the growth of renal cysts.

Past efforts to pharmacologically disrupt the development and growth of renal cystic lesions focused primarily on normalizing the activity of a specific signaling molecule, but the effects of stimulating apoptosis in the proliferating epithelial cells have not been well studied. Although benign, ADPKD renal cysts created by the sustained proliferation of epithelial cells resemble tumors, and malignant cell death can be achieved by cotreatment with TNF-α and a mimetic of second mitochondria-derived activator of caspase (Smac). Notably, TNF-α accumulates to high levels in ADPKD cyst fluid. Here, we report that an Smac-mimetic selectively induces TNF-α-dependent cystic renal epithelial cell death, leading to the removal of cystic epithelial cells from renal tissues and delaying cyst formation. In vitro, a Smac-mimetic (GT13072) induced the degradation of cIAP1 that is required but not sufficient for cell death. Cotreatment with TNF-α augmented the formation and activation of the RIPK1-dependent death complex and the degradation and cleavage of FLIP, an inhibitor of caspase-8, in renal cystic epithelial cells. This approach produced death specifically in Pkd1 mutant epithelial cells, with no effect on normal renal epithelial cells. Moreover, treatment with the Smac-mimetic slowed cyst and kidney enlargement and preserved renal function in two genetic strains of mice with Pkd1 mutations. Thus, our mechanistic data characterize an apoptotic pathway, activated by the selective synergy of an Smac-mimetic and TNF-α in renal cyst fluid, that attenuates cyst development, providing an innovative translational platform for the rational development of novel therapeutics for ADPKD.

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease.

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide-dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.

HDAC6 regulates epidermal growth factor receptor (EGFR) endocytic trafficking and degradation in renal epithelial cells.

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.

Inhibition of histone deacetylases targets the transcription regulator Id2 to attenuate cystic epithelial cell proliferation.

The pan-histone deacetylase (HDAC) inhibitor, trichostatin A, was found to reduce cyst progression and slow the decline of kidney function in Pkd2 knockout mice, model of autosomal dominant polycystic kidney disease (ADPKD). Here we determine whether HDAC inhibition acts by regulating cell proliferation to prevent cyst formation, or by other mechanisms. The loss of Pkd1 caused an upregulation of the inhibitor of differentiation 2 (Id2), a transcription regulator, triggering an Id2-mediated downregulation of p21 in mutant mouse embryonic kidney cells in vitro. Using mouse embryonic kidney cells, mutant for Pkd1, we found that trichostatin A decreased Id2, which resulted in upregulation of p21. Further, phosphorylated retinoblastoma (Rb), usually regulated by Cdk2/Cdk4 activity, was also reduced in these cells. Since these latter enzymes are under the control of p21, these studies suggest that the proliferation of cyst epithelial cells that is reduced by trichostatin A might result from p21 upregulation, or alternatively through the Rb-E2F pathway. Additional studies showed that Id2 directly bound to Rb, releasing the transcription activator E2F from transcriptionally inactive Rb-E2F complexes. HDAC inhibition was able to reverse this process by downregulation of Id2. Furthermore, treatment of pregnant Pkd1 mice with trichostatin A prevented cyst formation in the developing embryonic kidneys, showing that this inhibition is effective in vivo during early cyst formation. Thus, HDAC inhibition targets Id2-mediated pathways to downregulate cystic epithelial cell proliferation and hence cystogenesis.