Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Anticystogenic activity of a small molecule PAK4 inhibitor may be a novel treatment for autosomal dominant polycystic kidney disease.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary renal disease with no currently available targeted therapies. Based on the established connection between ß-catenin signaling and renal ciliopathies, and on data from our and other laboratories showing striking similarities of this disease and cancer, we evaluated the use of an orally bioavailable small molecule, KPT-9274 (a dual inhibitor of the protein kinase PAK4 and nicotinamide phosphoribosyl transferase), for treatment of ADPKD. Treatment of PKD-derived cells with this compound not only reduces PAK4 steady-state protein levels and regulates ß-catenin signaling, but also inhibits nicotinamide phosphoribosyl transferase, the rate-limiting enzyme in a key NAD salvage pathway. KPT-9274 can attenuate cellular proliferation and induce apoptosis associated with a decrease in active (phosphorylated) PAK4 and ß-catenin in several Pkd1-null murine cell lines, with a less pronounced effect on the corresponding phenotypically normal cells. Additionally, KPT-9274 shows inhibition of cystogenesis in an ex vivo model of cyclic AMP-induced cystogenesis as well as in the early stage Pkd1flox/flox:Pkhd1-Cre mouse model, the latter showing confirmation of specific anti-proliferative, apoptotic, and on-target effects. NAD biosynthetic attenuation by KPT-9274, while critical for highly proliferative cancer cells, does not appear to be important in the slower growing cystic epithelial cells during cystogenesis. KPT-9274 was not toxic in our ADPKD animal model or in other cancer models. Thus, this small molecule inhibitor could be evaluated in a clinical trial as a viable therapy of ADPKD.

The cpk model of recessive PKD shows glutamine dependence associated with the production of the oncometabolite 2-hydroxyglutarate.

Since polycystic kidney disease (PKD) was first noted over 30 years ago to have neoplastic parallels, there has been a resurgent interest in elucidating neoplasia-relevant pathways in PKD. Taking a nontargeted metabolomics approach in the B6(Cg)-Cys1(cpk/)J (cpk) mouse model of recessive PKD, we have now characterized metabolic reprogramming in these tissues, leading to a glutamine-dependent TCA cycle shunt toward total 2-hydroxyglutarate (2-HG) production in cpk compared with B6 wild-type kidney tissue. After confirmation of increased 2-HG expression in immortalized collecting duct cpk cells as well as in human autosomal recessive PKD tissue using targeted analysis, we show that the increase in 2-HG is likely due to glutamine-sourced α-ketoglutarate. In addition, cpk cells require exogenous glutamine for growth such that inhibition of glutaminase-1 decreases cell viability as well as proliferation. This study is a demonstration of the striking parallels between recessive PKD and cancer metabolism. Our data, once confirmed in other PKD models, suggest that future therapeutic approaches targeting this pathway, such as using glutaminase inhibitors, have the potential to open novel treatment options for renal cystic disease.

Mapping the deletion endpoints in individuals with 22q11.2 deletion syndrome by droplet digital PCR.

BACKGROUND: Chromosome 22q11.2 deletion syndrome (22q11DS) is the most common human microdeletion syndrome and is associated with many cognitive, neurological and psychiatric disorders. The majority of individuals have a 3 Mb deletion while others have a nested 1.5 Mb deletion, but rare atypical deletions have also been described. To date, a study using droplet digital PCR (ddPCR) has not been conducted to systematically map the chromosomal breakpoints in individuals with 22q11DS, which would provide important genotypic insight into the various phenotypes observed in this syndrome. METHODS: This study uses ddPCR to assess copy number (CN) changes within the chromosome 22q11 deletion region and allows the mapping of the deletion endpoints. We used eight TaqMan assays interspersed throughout the deleted region of 22q11.2 to characterize the deleted region of chromosome 22 in 80 individuals known to have 22q11DS by FISH. Ten EvaGreen assays were used for finer mapping of the six identified individuals with 22q11DS atypical deletions and covering different regions of chromosome 22. RESULTS: ddPCR provided non-ambiguous CN measurements across the region, confirmed the presence of the deletion in the individuals screened, and led to the identification of five differently sized and located deletions. The majority of the participants (n = 74) had the large 3 Mb deletions, whereas three had the smaller 1.5 Mb deletions, and the remaining three had an interstitial deletion of different size. CONCLUSIONS: The lower cost, rapid execution and high reliability and specificity provided by ddPCR for CN measurements in the 22q11 region constitutes a significant improvement over the variable CN values generated by other technologies. The ability of the ddPCR approach, to provide a high resolution mapping of deletion endpoints may result in the identification of genes that are haplo-insufficient and play a role in the pathogenesis of 22q11DS. Finally, this methodology can be applied to the characterization of other microdeletions throughout the genome.

Biomarkers in IgA nephropathy.

IgA nephropathy is the most common primary glomerulonephritis and presents with gross hematuria and upper respiratory infection, with slow progression to end-stage renal disease in up to 50% of affected patients. Kidney biopsies are the gold standard method of diagnosis and frequently are not performed as the majority of individuals are asymptomatic. Thus, there is a need to discover and validate prognostic and predictive biomarkers that can be noninvasively obtained and are specific to this disease. Here we discuss the current state of research in this area and examine validated and clinically promising biofluid and tissue biomarkers of IgA nephropathy.

Decreased DGCR8 expression and miRNA dysregulation in individuals with 22q11.2 deletion syndrome.

Deletion of the 1.5-3 Mb region of chromosome 22 at locus 11.2 gives rise to the chromosome 22q11.2 deletion syndrome (22q11DS), also known as DiGeorge and Velocardiofacial Syndromes. It is the most common micro-deletion disorder in humans and one of the most common multiple malformation syndromes. The syndrome is characterized by a broad phenotype, whose characterization has expanded considerably within the last decade and includes many associated findings such as craniofacial anomalies (40%), conotruncal defects of the heart (CHD; 70-80%), hypocalcemia (20-60%), and a range of neurocognitive anomalies with high risk of schizophrenia, all with a broad phenotypic variability. These phenotypic features are believed to be the result of a change in the copy number or dosage of the genes located in the deleted region. Despite this relatively clear genetic etiology, very little is known about which genes modulate phenotypic variations in humans or if they are due to combinatorial effects of reduced dosage of multiple genes acting in concert. Here, we report on decreased expression levels of genes within the deletion region of chromosome 22, including DGCR8, in peripheral leukocytes derived from individuals with 22q11DS compared to healthy controls. Furthermore, we found dysregulated miRNA expression in individuals with 22q11DS, including miR-150, miR-194 and miR-185. We postulate this to be related to DGCR8 haploinsufficiency as DGCR8 regulates miRNA biogenesis. Importantly we demonstrate that the level of some miRNAs correlates with brain measures, CHD and thyroid abnormalities, suggesting that the dysregulated miRNAs may contribute to these phenotypes and/or represent relevant blood biomarkers of the disease in individuals with 22q11DS.