Caspase-1 and the inflammasome promote polycystic kidney disease progression.

We and others have previously shown that the presence of renal innate immune cells can promote polycystic kidney disease (PKD) progression. In this study, we examined the influence of the inflammasome, a key part of the innate immune system, on PKD. The inflammasome is a system of molecular sensors, receptors, and scaffolds that responds to stimuli like cellular damage or microbes by activating Caspase-1, and generating critical mediators of the inflammatory milieu, including IL-1ß and IL-18. We provide evidence that the inflammasome is primed in PKD, as multiple inflammasome sensors were upregulated in cystic kidneys from human ADPKD patients, as well as in kidneys from both orthologous (PKD1 RC/RC or RC/RC) and non-orthologous (jck) mouse models of PKD. Further, we demonstrate that the inflammasome is activated in female RC/RC mice kidneys, and this activation occurs in renal leukocytes, primarily in CD11c+ cells. Knock-out of Casp1, the gene encoding Caspase-1, in the RC/RC mice significantly restrained cystic disease progression in female mice, implying sex-specific differences in the renal immune environment. RNAseq analysis implicated the promotion of MYC/YAP pathways as a mechanism underlying the pro-cystic effects of the Caspase-1/inflammasome in females. Finally, treatment of RC/RC mice with hydroxychloroquine, a widely used immunomodulatory drug that has been shown to inhibit the inflammasome, protected renal function specifically in females and restrained cyst enlargement in both male and female RC/RC mice. Collectively, these results provide evidence for the first time that the activated Caspase-1/inflammasome promotes cyst expansion and disease progression in PKD, particularly in females. Moreover, the data suggest that this innate immune pathway may be a relevant target for therapy in PKD.

Ttc21b deficiency attenuates autosomal dominant polycystic kidney disease in a kidney tubular- and maturation-dependent manner.

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multiprotein complexes. Deletion of several IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult autosomal dominant polycystic kidney disease (ADPKD) mouse models. However, deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, we investigate the role of global deletion of an IFT-A gene, Ttc21b, in juvenile and adult mouse models of ADPKD. Both juvenile (postnatal day 21) and adult (six months of age) ADPKD mice exhibited kidney cysts, increased kidney weight/body weight ratios, lengthened kidney cilia, inflammation, and increased levels of the nutrient sensor, O-linked ß-N-acetylglucosamine (O-GlcNAc). Deletion of Ttc21b in juvenile ADPKD mice reduced cortical collecting duct cystogenesis and kidney weight/body weight ratios, increased proximal tubular and glomerular dilations, but did not reduce cilia length, inflammation, nor O-GlcNAc levels. In contrast, Ttc21b deletion in adult ADPKD mice markedly attenuated kidney cystogenesis and reduced cilia length, inflammation, and O-GlcNAc levels. Thus, unlike IFT-B, the effect of Ttc21b deletion in mouse models of ADPKD is development-specific. Unlike an IFT-A adaptor, deleting Ttc21b in juvenile ADPKD mice is partially ameliorative. Thus, our studies suggest that different microenvironmental factors, found in distinct nephron segments and in developing versus mature stages, modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a pathological feature of ADPKD.

MCP-1 promotes detrimental cardiac physiology, pulmonary edema, and death in the cpk model of polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by slowly expanding renal cysts that damage the kidney, typically resulting in renal failure by the fifth decade. The most common cause of death in these patients, however, is cardiovascular disease. Expanding cysts in PKD induce chronic kidney injury that is accompanied by immune cell infiltration, including macrophages, which we and others have shown can promote disease progression in PKD mouse models. Here, we show that monocyte chemoattractant protein-1 [MCP-1/chemokine (C-C motif) ligand 2 (CCL2)] is responsible for the majority of monocyte chemoattractant activity produced by renal PKD cells from both mice and humans. To test whether the absence of MCP-1 lowers renal macrophage concentration and slows disease progression, we generated genetic knockout (KO) of MCP-1 in a mouse model of PKD [congenital polycystic kidney (cpk) mice]. Cpk mice are born with rapidly expanding renal cysts, accompanied by a decline in kidney function and death by postnatal day 21. Here, we report that KO of MCP-1 in these mice increased survival, with some mice living past 3 mo. Surprisingly, however, there was no significant difference in renal macrophage concentration, nor was there improvement in cystic disease or kidney function. Examination of mice revealed cardiac hypertrophy in cpk mice, and measurement of cardiac electrical activity via ECG revealed repolarization abnormalities. MCP-1 KO did not affect the number of cardiac macrophages, nor did it alleviate the cardiac aberrancies. However, MCP-1 KO did prevent the development of pulmonary edema, which occurred in cpk mice, and promoted decreased resting heart rate and increased heart rate variability in both cpk and noncystic mice. These data suggest that in this mouse model of PKD, MCP-1 altered cardiac/pulmonary function and promoted death outside of its role as a macrophage chemoattractant.

Autocrine IL-10 activation of the STAT3 pathway is required for pathological macrophage differentiation in polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by slow expansion of fluid-filled cysts derived from tubules within the kidney. Cystic expansion results in injury to surrounding parenchyma and leads to inflammation, scarring and ultimately loss of renal function. Macrophages are a key element in this process, promoting cyst epithelial cell proliferation, cyst expansion and disease progression. Previously, we have shown that the microenvironment established by cystic epithelial cells can 'program' macrophages, inducing M2-like macrophage polarization that is characterized by expression of markers that include Arg1 and Il10 Here, we functionally characterize these macrophages, demonstrating that their differentiation enhances their ability to promote cyst cell proliferation. This observation indicates a model of reciprocal pathological interactions between cysts and the innate immune system: cyst epithelial cells promote macrophage polarization to a phenotype that, in turn, is especially efficient in promoting cyst cell proliferation and cyst growth. To better understand the genesis of this macrophage phenotype, we examined the role of IL-10, a regulatory cytokine shown to be important for macrophage-stimulated tissue repair in other settings. Herein, we show that the acquisition of the pathological macrophage phenotype requires IL-10 secretion by the macrophages. Further, we demonstrate a requirement for IL-10-dependent autocrine activation of the STAT3 pathway. These data suggest that the IL-10 pathway in macrophages plays an essential role in the pathological relationship between cysts and the innate immune system in PKD, and thus could be a potential therapeutic target.

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.

Macrophages promote polycystic kidney disease progression.

Renal M2-like macrophages have critical roles in tissue repair, stimulating tubule cell proliferation and, if they remain, fibrosis. M2-like macrophages have also been implicated in promoting cyst expansion in mouse models of autosomal dominant polycystic kidney disease (ADPKD). While renal macrophages have been documented in human ADPKD, there are no studies in autosomal recessive polycystic kidney disease (ARPKD). Here we evaluated the specific phenotype of renal macrophages and their disease-impacting effects on cystic epithelial cells. We found an abundance of M2-like macrophages in the kidneys of patients with either ADPKD or ARPKD and in the cystic kidneys of cpk mice, a model of ARPKD. Renal epithelial cells from either human ADPKD cysts or noncystic human kidneys promote differentiation of naive macrophages to a distinct M2-like phenotype in culture. Reciprocally, these immune cells stimulate the proliferation of renal tubule cells and microcyst formation in vitro. Further, depletion of macrophages from cpk mice indicated that macrophages contribute to PKD progression regardless of the genetic etiology. Thus, M2-like macrophages are two-pronged progression factors in PKD, promoting cyst cell proliferation, cyst growth, and fibrosis. Agents that block the emergence of these cells or their effects in the cystic kidney may be effective therapies for slowing PKD progression.

Why kidneys fail in autosomal dominant polycystic kidney disease.

The weight of evidence gathered from studies in humans with hereditary polycystic kidney disease (PKD)1 and PKD2 disorders, as well as from experimental animal models, indicates that cysts are primarily responsible for the decline in glomerular filtration rate that occurs fairly late in the course of the disease. The processes underlying this decline include anatomic disruption of glomerular filtration and urinary concentration mechanisms on a massive scale, coupled with compression and obstruction by cysts of adjacent nephrons in the cortex, medulla and papilla. Cysts prevent the drainage of urine from upstream tributaries, which leads to tubule atrophy and loss of functioning kidney parenchyma by mechanisms similar to those found in ureteral obstruction. Cyst-derived chemokines, cytokines and growth factors result in a progression to fibrosis that is comparable with the development of other progressive end-stage renal diseases. Treatment of renal cystic disorders early enough to prevent or reduce cyst formation or slow cyst growth, before the secondary changes become widespread, is a reasonable strategy to prolong the useful function of kidneys in patients with autosomal dominant polycystic kidney disease.

Rho GTPase activity modulates Wnt3a/beta-catenin signaling.

Wnt proteins constitute a family of secreted signaling molecules that regulate highly conserved pathways essential for development and, when aberrantly activated, drive oncogenesis in a number of human cancers. A key feature of the most widely studied Wnt signaling cascade is the stabilization of cytosolic beta-catenin, resulting in beta-catenin nuclear translocation and transcriptional activation of multiple target genes. In addition to this canonical, beta-catenin-dependent pathway, Wnt3A has also been shown to stimulate RhoA GTPase. While the importance of activated Rho to non-canonical Wnt signaling is well appreciated, the potential contribution of Wnt3A-stimulated RhoA to canonical beta-catenin-dependent transcription has not been examined and is the focus of this study. We find that activated Rho is required for Wnt3A-stimulated osteoblastic differentiation in C3H10T1/2 mesenchymal stem cells, a biological phenomenon mediated by stabilized beta-catenin. Using expression microarrays and real-time RT-PCR analysis, we show that Wnt3A-stimulated transcription of a subset of target genes is Rho-dependent, indicating that full induction of these Wnt targets requires both beta-catenin and Rho activation. Significantly, neither beta-catenin stabilization nor nuclear translocation stimulated by Wnt3A is affected by inhibition or activation of RhoA. These findings identify Rho activation as a critical element of the canonical Wnt3A-stimulated, beta-catenin-dependent transcriptional program.

MLK3 limits activated Galphaq signaling to Rho by binding to p63RhoGEF.

Mixed lineage kinase 3 (MLK3) is a MAP3K that activates the JNK-dependent MAPK pathways. Here, we show that MLK3 is required for cell migration in a manner independent of its role as a MAP3K or MLK3 kinase activity. Rather, MLK3 functions in a regulated way to limit levels of the activated GTPase Rho by binding to the Rho activator, p63RhoGEF/GEFT, which, in turn, prevents its activation by Galphaq. These findings demonstrate a scaffolding role for MLK3 in controlling the extent of Rho activation that modulates cell migration. Moreover, they suggest that MLK3 functions as a network hub that links a number of signaling pathways.