Rapamycin Ameliorates PKD Resulting from Conditional Inactivation of Pkd1

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD

Pkd1 and Pkd2 are required for normal placental development.

Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of inherited renal failure that results from mutations in PKD1 and PKD2. The disorder is characterized by focal cyst formation that involves somatic mutation of the wild type allele in a large fraction of cysts. Consistent with a two-hit mechanism, mice that are homozygous for inactivating mutations of either Pkd1 or Pkd2 develop cystic kidneys, edema and hemorrhage and typically die in midgestation. Cystic kidney disease is unlikely to be the cause of fetal loss since renal function is not required to complete gestation. One hypothesis is that embryonic demise is due to leaky vessels or cardiac pathology.In these studies we used a series of genetically modified Pkd1 and Pkd2 murine models to investigate the cause of embryonic lethality in mutant embryos. Since placental defects are a frequent cause of fetal loss, we conducted histopathologic analyses of placentas from Pkd1 null mice and detected abnormalities of the labyrinth layer beginning at E12.5. We performed placental rescue experiments using tetraploid aggregation and conditional inactivation of Pkd1 with the Meox2 Cre recombinase. We found that both strategies improved the viability of Pkd1 null embryos. Selective inactivation of Pkd1 and Pkd2 in endothelial cells resulted in polyhydramnios and abnormalities similar to those observed in Pkd1(-/-) placentas. However, endothelial cell specific deletion of Pkd1 or Pkd2 did not yield the dramatic vascular phenotypes observed in null animals.Placental abnormalities contribute to the fetal demise of Pkd(-/-) embryos. Endothelial cell specific deletion of Pkd1 or Pkd2 recapitulates a subset of findings seen in Pkd null animals. Our studies reveal a complex role for polycystins in maintaining vascular integrity

Network Analysis of a <em>Pkd1</em>-Mouse Model of Autosomal Dominant Polycystic Kidney Disease Identifies HNF4α as a Disease Modifier

<div><p>Autosomal Dominant Polycystic Kidney Disease (ADPKD; MIM ID's 173900, 601313, 613095) leads to end-stage kidney disease, caused by mutations in <em>PKD1</em> or <em>PKD2</em>. Inactivation of <em>Pkd1</em> before or after P13 in mice results in distinct early- or late-onset disease. Using a mouse model of ADPKD carrying floxed <em>Pkd1</em> alleles and an inducible Cre recombinase, we intensively analyzed the relationship between renal maturation and cyst formation by applying transcriptomics and metabolomics to follow disease progression in a large number of animals induced before P10. Weighted gene co-expression network analysis suggests that <em>Pkd1</em>-cystogenesis does not cause developmental arrest and occurs in the context of gene networks similar to those that regulate/maintain normal kidney morphology/function. Knowledge-based Ingenuity Pathway Analysis (IPA) software identifies HNF4α as a likely network node. These results are further supported by a meta-analysis of 1,114 published gene expression arrays in <em>Pkd1</em> wild-type tissues. These analyses also predict that metabolic pathways are key elements in postnatal kidney maturation and early steps of cyst formation. Consistent with these findings, urinary metabolomic studies show that <em>Pkd1</em> cystic mutants have a distinct profile of excreted metabolites, with pathway analysis suggesting altered activity in several metabolic pathways. To evaluate their role in disease, metabolic networks were perturbed by inactivating <em>Hnf4α</em> and <em>Pkd1</em>. The <em>Pkd1/Hnf4α</em> double mutants have significantly more cystic kidneys, thus indicating that metabolic pathways could play a role in <em>Pkd1</em>-cystogenesis.</p> </div

Histology and gene expression patterns in early-onset model.

<p>A) Representative kidney histology (H&E stain; scale bar: 500 mm); B) PCA plot: genotype and age explain most of the clustering in the complete set of 70 kidneys in the early-onset model (control: red; mutant: blue; size of spheres: age). C and D) Heatmap plot of mutant-signature (genes in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003053#pgen.1003053.s002" target="_blank">Table S1</a>) in the test (C) and validation (D) groups. E) Heatmap plot of ME2 (genes in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003053#pgen.1003053.s004" target="_blank">Table S3</a>) cluster in the validation group.</p

Metabolic pathways significantly different in the urine of mutant mice.

<p>Metabolic pathways significantly different in the urine of mutant mice.</p

Urinary metabolomics shows distinct patterns in cystic versus control mice.

<p>(A) PCA plot of positive mass spectra identified in urine (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003053#pgen.1003053.s014" target="_blank">Table S13</a>), showing separation of control and <i>Pkd1</i> mutant samples, irrespective of <i>Hnf4α</i> genotype, along the first principal component. (B) Normalized levels of acetylcarnitine in urine samples of <i>Pkd1/Hnf4α</i> mice at different ages. (C) MS/MS fragmentation spectra of acetylcarnitine authentic standard versus a representative urine sample.</p

Metabolic pathways could underlie kidney maturation and determine the susceptibility to rapid cyst formation in the early-onset model of ADPKD.

<p>A) Knowledge-based IPA network (score: 24) using genes in early-onset mutant-signature (genes in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003053#pgen.1003053.s002" target="_blank">Table S1</a>). HNF4α(blue arrow) is a node (green/red: genes down/up-regulated in P12 mutant kidneys; solid lines: direct interactions; dashed lines: indirect interactions). B) Dendrograms of module eigengenes showing blocks of correlated eigengenes (meta-modules: in rectangles) suggest that gene correlation networks are preserved in mutant animals but change during P12 to P14 kidney maturation. The metabolism-related MEturquoise cluster (arrow; genes in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003053#pgen.1003053.s006" target="_blank">Table S5</a>) is part of meta-module at P12 but not at P14.</p