Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: In patients with autosomal dominant polycystic kidney disease (ADPKD), most of whom have a mutation in PKD1 or PKD2, abnormally large numbers of macrophages accumulate around kidney cysts and promote their growth. Research by us and others has suggested that monocyte chemoattractant protein-1 (Mcp1) may be a signal for macrophage-mediated cyst growth. METHODS: To define the role of Mcp1 and macrophages in promoting cyst growth, we used mice with inducible knockout of Pkd1 alone (single knockout) or knockout of both Pkd1 and Mcp1 (double knockout) in the murine renal tubule. Levels of Mcp1 RNA expression were measured in single-knockout mice and controls. RESULTS: In single-knockout mice, upregulation of Mcp1 precedes macrophage infiltration. Macrophages accumulating around nascent cysts (0-2 weeks after induction) are initially proinflammatory and induce tubular cell injury with morphologic flattening, oxidative DNA damage, and proliferation-independent cystic dilation. At 2-6 weeks after induction, macrophages switch to an alternative activation phenotype and promote further cyst growth because of an additional three-fold increase in tubular cell proliferative rates. In double-knockout mice, there is a marked reduction in Mcp1 expression and macrophage numbers, resulting in less initial tubular cell injury, slower cyst growth, and improved renal function. Treatment of single-knockout mice with an inhibitor to the Mcp1 receptor Ccr2 partially reproduced the morphologic and functional improvement seen with Mcp1 knockout. CONCLUSIONS: Mcp1 is upregulated after knockout of Pkd1 and promotes macrophage accumulation and cyst growth via both proliferation-independent and proliferation-dependent mechanisms in this orthologous mouse model of ADPKD.

Glycogen synthase kinase-3ß promotes cyst expansion in polycystic kidney disease.

Polycystic kidney diseases (PKDs) are inherited disorders characterized by the formation of fluid filled renal cysts. Elevated cAMP levels in PKDs stimulate progressive cyst enlargement involving cell proliferation and transepithelial fluid secretion often leading to end-stage renal disease. The glycogen synthase kinase-3 (GSK3) family of protein kinases consists of GSK3α and GSK3ß isoforms and has a crucial role in multiple cellular signaling pathways. We previously found that GSK3ß, a regulator of cell proliferation, is also crucial for cAMP generation and vasopressin-mediated urine concentration by the kidneys. However, the role of GSK3ß in the pathogenesis of PKDs is not known. Here we found that GSK3ß expression and activity were markedly upregulated and associated with cyst-lining epithelia in the kidneys of mice and humans with PKD. Renal collecting duct-specific gene knockout of GSK3ß or pharmacological inhibition of GSK3 effectively slowed down the progression of PKD in mouse models of autosomal recessive or autosomal dominant PKD. GSK3 inactivation inhibited cAMP generation and cell proliferation resulting in reduced cyst expansion, improved renal function, and extended life span. GSK3ß inhibition also reduced pERK, c-Myc, and cyclin-D1, known mitogens in proliferation of cystic epithelial cells. Thus, GSK3ß has a novel functional role in PKD pathophysiology, and its inhibition may be therapeutically useful to slow down cyst expansion and progression of PKD.

Using Imaging Mass Cytometry to Define Cell Identities and Interactions in Human Tissues.

In the evolving landscape of highly multiplexed imaging techniques that can be applied to study complex cellular microenvironments, this review characterizes the use of imaging mass cytometry (IMC) to study the human kidney. We provide technical details for antibody validation, cell segmentation, and data analysis specifically tailored to human kidney samples, and elaborate on phenotyping of kidney cell types and novel insights that IMC can provide regarding pathophysiological processes in the injured or diseased kidney. This review will provide the reader with the necessary background to understand both the power and the limitations of IMC and thus support better perception of how IMC analysis can improve our understanding of human disease pathogenesis and can be integrated with other technologies such as single cell sequencing and proteomics to provide spatial context to cellular data.

Targeting the vasopressin type-2 receptor for renal cell carcinoma therapy.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

A cAMP and CREB-mediated feed-forward mechanism regulates GSK3ß in polycystic kidney disease.

Glycogen synthase kinase 3ß (GSK3ß), a serine/threonine protein kinase, is commonly known to be regulated at the level of its activity. However, in some diseases including polycystic kidney disease (PKD), GSK3ß expression is increased and plays a pathophysiological role. The current studies aimed to determine the mechanism for the increased GSK3ß expression in PKD and its significance to disease progression. In mouse models of PKD, increases in renal GSK3ß corresponded with increases in renal cAMP levels and disease progression. In vivo and in vitro studies revealed that GSK3ß is a cAMP-responsive gene, and elevated cAMP levels, as seen in PKD, can increase GSK3ß expression. In normal mice, vasopressin signaling induced by water deprivation increased GSK3ß expression, which decreased following rehydration. Examination of the GSK3ß promoter revealed five potential binding sites for the transcription factor, cAMP response element binding protein (CREB). CREB was found to bind to GSK3ß promoter and essential for cAMP-mediated regulation of GSK3ß. Importantly, this regulation was demonstrated to be part of a feed-forward loop in which cAMP through CREB regulates GSK3ß expression, and GSK3ß in turn positively regulates cAMP generation. GSK3ß or CREB inhibition reduced transepithelial fluid secretion and cyst expansion in vitro Thus, disruption at any point of this destructive cycle may be therapeutically useful to reduce cyst expansion and preserve renal function in PKD.