Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1.

Polycystic kidney disease (ADPKD) results from failure of the kidney to properly maintain three-dimensional structure after loss of either polycystin-1 or -2. Mice with kidney selective inactivation of Pkd1 during embryogenesis develop profound renal cystic disease and die from renal failure within 3 weeks of birth. In this model, cysts form exclusively from cells in which Cre recombinase is active, but the apparent pace of cyst expansion varies by segment and cell type. Intercalated cells do not participate in cyst expansion despite the presence of cilia up to at least postnatal day 21. Cystic segments show a persistent increase in proliferation as determined by bromodeoxyuridine (BrdU) incorporation; however, the absolute proliferative index is dependent on the underlying proliferative potential of kidney tubule cells. Components of the extracellular regulated kinase (MAPK/ERK) pathway from Ras through MEK1/2 and ERK1/2 to the effector P90(RSK) are activated in both perinatal Pkd1 and adult Pkd2 ortholgous gene disease models. The pattern of MAPK/ERK activation is focal and does not correlate with the pattern of active proliferation identified by BrdU uptake. The possibility of a causal relationship between ERK1/2 activation and cyst cell proliferation was assessed in vivo in the acute perinatal Pkd1 model of ADPKD using MEK1/2 inhibitor U0126. U0126 treatment had no effect on progression of cyst formation in this model at doses sufficient to reduce phospho-ERK1/2 in cystic kidneys. Cysts in ADPKD exhibit both increased proliferation and activation of MAPK/ERK, but cyst growth is not prevented by inhibition of ERK1/2 activation.

Neutrophil gelatinase-associated lipocalin suppresses cyst growth by Pkd1 null cells in vitro and in vivo.

Cyst growth in patients with autosomal dominant polycystic kidney disease is thought to be due to increased tubular cell proliferation. One model to explain this altered proliferation suggests that the polycystin proteins PC1 and PC2 localize to apical cilia and serve as an integral part of the flow-sensing pathway thus modulating the proliferative response. We measured proliferation and apoptosis in proximal tubule derived cell lines lacking PC1. These cells showed increased rates of proliferation, a decreased rate of apoptosis, compared to control heterozygous cell lines, and spontaneously formed cysts rather than tubules in an in vitro tubulogenesis assay. Addition of neutrophil gelatinase associated lipocalin (NGAL), a small secreted protein that binds diverse ligands, to the cells lacking PC1 inhibited proliferation and increased apoptosis leading to slower cyst growth in vitro. Sustained over-expression at low level of NGAL by an adenoviral delivery system suppressed cyst enlargement without improving renal function in the Pkd1 mutant mice. Our studies show that renal epithelial cells lacking PC1 have an inherent tendency to hyper-proliferate forming cysts in vitro independent of a flow stimulus. The potential benefit of attenuating cyst growth with NGAL remains to be determined.

Polycystin-2 traffics to cilia independently of polycystin-1 by using an N-terminal RVxP motif.

Primary cilia play a key role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The affected proteins, polycystin-1 (PC1) and polycystin-2 (PC2), interact with each other and are expressed in cilia. We found that COOH-terminal truncated PC2 (PC2-L703X), lacking the PC1 interaction region, still traffics to cilia. We examined PC2 expression in several tissues and cells lacking PC1 and found that PC2 is expressed in cilia independently of PC1. We used N-terminal deletion constructs to narrow the domain necessary for cilia trafficking to the first 15 amino acids of PC2 and identified a conserved motif, R6VxP, that is required for cilial localization. The N-terminal 15 amino acids are also sufficient to localize heterologous proteins in cilia. PC2 has endogenous cilia trafficking information and is present in cilia of cells lining cysts that result from mutations in PKD1.