MTORC1-mediated inhibition of polycystin-1 expression drives renal cyst formation in tuberous sclerosis complex

Previous studies report a cross-talk between the polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC) genes. mTOR signalling is upregulated in PKD and rapamycin slows cyst expansion, whereas renal inactivation of the Tsc genes causes cysts. Here we identify a new interplay between the PKD and TSC genes, with important implications for the pathophysiology of both diseases. Kidney-specific inactivation of either Pkd1 or Tsc1 using an identical Cre (KspCre) results in aggressive or very mild PKD, respectively. Unexpectedly, we find that mTORC1 negatively regulates the biogenesis of polycystin-1 (PC-1) and trafficking of the PC-1/2 complex to cilia. Genetic interaction studies reveal an important role for PC-1 downregulation by mTORC1 in the cystogenesis of Tsc1 mutants. Our data potentially explain the severe renal manifestations of the TSC/PKD contiguous gene syndrome and open new perspectives for the use of mTOR inhibitors in autosomal dominant PKD caused by hypomorphic or missense PKD1 mutations

Emerging evidence of a link between the polycystins and the mTOR pathways

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by the formation of renal cysts. This disease can be caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC-1) and -2 (PC-2), respectively

Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

The localization of polycystin (PC)1) to the plasma membrane requires coexpression with PC2 and cleavage at the PC1 G protein-coupled receptor proteolytic site. Neither the PC1 binding capacity of PC2 nor its channel function is required for this effect

Experimental Verification of a Predicted Intronic MicroRNA in Human NGFR Gene with a Potential Pro-Apoptotic Function

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells

Protein Phosphatase-1α Interacts with and Dephosphorylates Polycystin-1

Polycystin signaling is likely to be regulated by phosphorylation. While a number of potential protein kinases and their target phosphorylation sites on polycystin-1 have been identified, the corresponding phosphatases have not been extensively studied. We have now determined that polycystin-1 is a regulatory subunit for protein phosphatase-1α (PP1α). Sequence analysis has revealed the presence of a highly conserved PP1-interaction motif in the cytosolic, C-terminal tail of polycystin-1; and we have shown that transfected PP1α specifically co-immunoprecipitates with a polycystin-1 C-tail construct. To determine whether PP1α dephosphorylates polycystin-1, a PKA-phosphorylated GST-polycystin-1 fusion protein was shown to be dephosphorylated by PP1α but not by PP2B (calcineurin). Mutations within the PP1-binding motif of polycystin-1, including an autosomal dominant polycystic kidney disease (ADPKD)-associated mutation, significantly reduced PP1α-mediated dephosphorylation of polycystin-1. The results suggest that polycystin-1 forms a holoenzyme complex with PP1α via a conserved PP1-binding motif within the polycystin-1 C-tail, and that PKA-phosphorylated polycystin-1 serves as a substrate for the holoenzyme

A new mouse model for renal lesions produced by intravenous injection of diphtheria toxin A-chain expression plasmid

BACKGROUND: Various animal models of renal failure have been produced and used to investigate mechanisms underlying renal disease and develop therapeutic drugs. Most methods available to produce such models appear to involve subtotal nephrectomy or intravenous administration of antibodies raised against basement membrane of glomeruli. In this study, we developed a novel method to produce mouse models of renal failure by intravenous injection of a plasmid carrying a toxic gene such as diphtheria toxin A-chain (DT-A) gene. DT-A is known to kill cells by inhibiting protein synthesis. METHODS: An expression plasmid carrying the cytomegalovirus enhancer/chicken β-actin promoter linked to a DT-A gene was mixed with lipid (FuGENE™6) and the resulting complexes were intravenously injected into adult male B6C3F1 mice every day for up to 6 days. After final injection, the kidneys of these mice were sampled on day 4 and weeks 3 and 5. RESULTS: H-E staining of the kidney specimens sampled on day 4 revealed remarkable alterations in glomerular compartments, as exemplified by mesangial cell proliferation and formation of extensive deposits in glomerular basement membrane. At weeks 3 and 5, gradual recovery of these tissues was observed. These mice exhibited proteinuria and disease resembling sub-acute glomerulonephritis. CONCLUSIONS: Repeated intravenous injections of DT-A expression plasmid DNA/lipid complex caused temporary abnormalities mainly in glomeruli of mouse kidney. The disease in these mice resembles sub-acute glomerulonephritis. These DT-A gene-incorporated mice will be useful as animal models in the fields of nephrology and regenerative medicine

The Exocyst Protein Sec10 Interacts with Polycystin-2 and Knockdown Causes PKD-Phenotypes

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown—including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation—suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes

Glomerular Filtration Is Required for Transfection of Proximal Tubular Cells in the Rat Kidney Following Injection od DNA Complexes into the Renal Artery

Gene transfer to the kidney can be achieved with various DNA vectors, resulting in transgene expression in glomerular or tubular districts. Controlling transgene destination is desirable for targeting defined renal cells for specific therapeutic purposes. We previously showed that injection of polyplexes into the rat renal artery resulted in transfection of proximal tubular cells. To investigate whether this process involves glomerular filtration of the DNA-containing particles, fluorescent polyethylenimine polyplexes were prepared, containing fluoresceinated poly-L-lysine. This allowed visualization of the route of the particles into the kidney. Our polyplexes were filtered through the glomerulus, since fluorescent proximal tubuli were observed. Conversely, fluorescent lipopolyplexes containing the cationic lipid DOTAP were never observed in tubular cells. Size measurements by laser light scattering showed that the mean diameter of polyplexes (93 nm) was smaller than that of lipopolyplexes (160 nm). The size of the transfecting particles is therefore a key parameter in this process, as expected by the constraints imposed by the glomerular filtration barrier. This information is relevant, in view of modulating the physico-chemical properties of DNA complexes for optimal transgene expression in tubular cells