Xuewen Song

Polycystin-1 C-terminal tail associates with β-catenin and inhibits canonical Wnt signaling

Polycystin-1 (PC1), the product of the PKD1 gene mutated in the majority of autosomal dominant polycystic kidney disease (ADPKD) cases, undergoes a cleavage resulting in the intracellular release of its C-terminal tail (CTT). Here, we demonstrate that the PC1 CTT co-localizes with and binds to beta-catenin in the nucleus. This interaction requires a nuclear localization motif present in the PC1 CTT as well as the N-terminal portion of beta-catenin. The PC1 CTT inhibits the ability of both beta-catenin and Wnt ligands to activate T-cell factor (TCF)-dependent gene transcription, a major effector of the canonical Wnt signaling pathway. The PC1 CTT may produce this effect by reducing the apparent affinity of the interaction between beta-catenin and the TCF protein. DNA microarray analysis reveals that the canonical Wnt signaling pathway is activated in ADPKD patient cysts. Our results suggest a novel mechanism through which PC1 cleavage may impact upon Wnt-dependent signaling and thereby modulate both developmental processes and cystogenesis.

ADCK4 mutations promote steroid-Resistant nephrotic syndrome through CoQ10 biosynthesis disruption

Identification of single-gene causes of steroid-resistant nephrotic syndrome (SRNS) has furthered the understanding of the pathogenesis of this disease. Here, using a combination of homozygosity mapping and whole human exome resequencing, we identified mutations in the aarF domain containing kinase 4 (ADCK4) gene in 15 individuals with SRNS from 8 unrelated families. ADCK4 was highly similar to ADCK3, which has been shown to participate in coenzyme Q10 (CoQ10) biosynthesis. Mutations in ADCK4 resulted in reduced CoQ10 levels and reduced mitochondrial respiratory enzyme activity in cells isolated from individuals with SRNS and transformed lymphoblasts. Knockdown of adck4 in zebrafish and Drosophila recapitulated nephrotic syndrome-associated phenotypes. Furthermore, ADCK4 was expressed in glomerular podocytes and partially localized to podocyte mitochondria and foot processes in rat kidneys and cultured human podocytes. In human podocytes, ADCK4 interacted with members of the CoQ10 biosynthesis pathway, including COQ6, which has been linked with SRNS and COQ7. Knockdown of ADCK4 in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Interestingly, a patient with SRNS with a homozygous ADCK4 frameshift mutation had partial remission following CoQ10 treatment. These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10.

Systems biology of autosomal dominant polycystic kidney disease (ADPKD): computational identification of gene expression pathways and integrated regulatory networks

To elucidate the molecular pathways that modulate renal cyst growth in ADPKD, we performed global gene profiling on cysts of different size (<1 ml, n = 5; 10-20 ml, n = 5; >50 ml, n = 3) and minimally cystic tissue (MCT, n = 5) from five PKD1 human polycystic kidneys using Affymetrix HG-U133 Plus 2.0 arrays. We used gene set enrichment analysis to identify overrepresented signaling pathways and key transcription factors (TFs) between cysts and MCT. We found down-regulation of kidney epithelial restricted genes (e.g. nephron segment-specific markers and cilia-associated cystic genes such as HNF1B, PKHD1, IFT88 and CYS1) in the renal cysts. On the other hand, PKD1 cysts displayed a rich profile of gene sets associated with renal development, mitogen-mediated proliferation, cell cycle progression, epithelial-mesenchymal transition, hypoxia, aging and immune/inflammatory responses. Notably, our data suggest that up-regulation of Wnt/beta-catenin, pleiotropic growth factor/receptor tyrosine kinase (e.g. IGF/IGF1R, FGF/FGFR, EGF/EGFR, VEGF/VEGFR), G-protein-coupled receptor (e.g. PTGER2) signaling was associated with renal cystic growth. By integrating these pathways with a number of dysregulated networks of TFs (e.g. SRF, MYC, E2F1, CREB1, LEF1, TCF7, HNF1B/ HNF1A and HNF4A), our data suggest that epithelial dedifferentiation accompanied by aberrant activation and cross-talk of specific signaling pathways may be required for PKD1 cyst growth and disease progression. Pharmacological modulation of some of these signaling pathways may provide a potential therapeutic strategy for ADPKD.

Signal transducer and activator of transcription-6 (STAT6) inhibition suppresses renal cyst growth in polycystic kidney disease

Autosomal-dominant (AD) polycystic kidney disease (PKD) is a leading cause of renal failure in the United States, and currently lacks available treatment options to slow disease progression. Mutations in the gene coding for polycystin-1 (PC1) underlie the majority of cases but the function of PC1 has remained poorly understood. We have previously shown that PC1 regulates the transcriptional activity of signal transducer and activator of transcription-6 (STAT6). Here we show that STAT6 is aberrantly activated in cyst-lining cells in PKD mouse models. Activation of the STAT6 pathway leads to a positive feedback loop involving auto/paracrine signaling by IL13 and the IL4/13 receptor. The presence of IL13 in cyst fluid and the overexpression of IL4/13 receptor chains suggests a mechanism of sustained STAT6 activation in cysts. Genetic inactivation of STAT6 in a PKD mouse model leads to significant inhibition of proliferation and cyst growth and preservation of renal function. We show that the active metabolite of leflunomide, a drug approved for treatment of arthritis, inhibits STAT6 in renal epithelial cells. Treatment of PKD mice with this drug leads to amelioration of the renal cystic disease similar to genetic STAT6 inactivation. These results suggest STAT6 as a promising drug target for treatment of ADPKD.

The Cleaved Cytoplasmic Tail of Polycystin-1 Regulates Src-Dependent STAT3 Activation

Polycystin-1 (PC1) mutations result in proliferative renal cyst growth and progression to renal failure in autosomal dominant polycystic kidney disease (ADPKD). The transcription factor STAT3 (signal transducer and activator of transcription 3) was shown to be activated in cyst-lining cells in ADPKD and PKD mouse models and may drive renal cyst growth, but the mechanisms leading to persistent STAT3 activation are unknown. A proteolytic fragment of PC1 corresponding to the cytoplasmic tail, PC1-p30, is overexpressed in ADPKD. Here, we show that PC1-p30 interacts with the nonreceptor tyrosine kinase Src, resulting in Src-dependent activation of STAT3 by tyrosine phosphorylation. The PC1-p30-mediated activation of Src/STAT3 was independent of JAK family kinases and insensitive to the STAT3 inhibitor suppressor of cytokine signaling 3. Signaling by the EGF receptor (EGFR) or cAMP amplified the activation of Src/STAT3 by PC1-p30. Expression of PC1-p30 changed the cellular response to cAMP signaling. In the absence of PC1-p30, cAMP dampened EGFR- or IL-6-dependent activation of STAT3; in the presence of PC1-p30, cAMP amplified Src-dependent activation of STAT3. In the polycystic kidney (PCK) rat model, activation of STAT3 in renal cystic cells depended on vasopressin receptor 2 (V2R) signaling, which increased cAMP levels. Genetic inhibition of vasopressin expression or treatment with a pharmacologic V2R inhibitor strongly suppressed STAT3 activation and reduced renal cyst growth. These results suggest that PC1, via its cleaved cytoplasmic tail, integrates signaling inputs from EGFR and cAMP, resulting in Src-dependent activation of STAT3 and a proliferative response.

Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease

Polycystic kidney disease (PKD) is a life-threatening disorder, commonly caused by defects in polycystin-1 (PC1) or polycystin-2 (PC2), in which tubular epithelia form fluid-filled cysts 1, 2. A major barrier to understanding PKD is the absence of human cellular models that accurately and efficiently recapitulate cystogenesis 3, 4. Previously, we have generated a genetic model of PKD using human pluripotent stem cells and derived kidney organoids 5, 6. Here we show that systematic substitution of physical components can dramatically increase or decrease cyst formation, unveiling a critical role for microenvironment in PKD. Removal of adherent cues increases cystogenesis 10-fold, producing cysts phenotypically resembling PKD that expand massively to 1-centimeter diameters. Removal of stroma enables outgrowth of PKD cell lines, which exhibit defects in PC1 expression and collagen compaction. Cyclic AMP, when added, induces cysts in both PKD organoids and controls. These biomaterials establish a highly efficient model of PKD cystogenesis that directly implicates the microenvironment at the earliest stages of the disease.

Polycystic kidney disease without an apparent family history

The absence of a positive family history (PFH) in 10%-25% of patients poses a diagnostic challenge for autosomal dominant polycystic kidney disease (ADPKD). In the Toronto Genetic Epidemiology Study of Polycystic Kidney Disease, 210 affected probands underwent renal function testing, abdominal imaging, and comprehensive PKD1 and PKD2 mutation screening. From this cohort, we reviewed all patients with and without an apparent family history, examined their parental medical records, and performed renal imaging in all available parents of unknown disease status. Subsequent reclassification of 209 analyzed patients revealed 72.2% (151 of 209) with a PFH, 15.3% (32 of 209) with de novo disease, 10.5% (22 of 209) with an indeterminate family history, and 1.9% (four of 209) with PFH in retrospect. Among the patients with de novo cases, we found two families with germline mosaicism and one family with somatic mosaicism. Additionally, analysis of renal imaging revealed that 16.3% (34 of 209) of patients displayed atypical PKD, most of which followed one of three patterns: asymmetric or focal PKD with PFH and an identified PKD1 or PKD2 mutation (15 of 34), asymmetric and de novo PKD with proven or suspected somatic mosaicism (seven of 34), or focal PKD without any identifiable PKD1 or PKD2 mutation (eight of 34). In conclusion, PKD without an apparent family history may be due to de novo disease, missing parental medical records, germline or somatic mosaicism, or mild disease from hypomorphic PKD1 and PKD2 mutations. Furthermore, mutations of a newly identified gene for ADPKD, GANAB, and somatic mosaicism need to be considered in the mutation-negative patients with focal disease.

Characterization of the Intrarenal Renin-Angiotensin System in Experimental Alport Syndrome

Blockade of the renin-angiotensin system attenuates the progression of experimental and clinical Alport syndrome (AS); however, the underlying mechanism(s) remains largely unknown. We evaluated the renin-angiotensin system in 4- and 7-week-old homozygous for collagen, type IV, α3 gene (Col4A3(-/-)) and wild-type mice, a model of AS characterized by proteinuria and progressive renal injury. Renal angiotensin (Ang) II levels increased, whereas renal Ang-(1-7) levels decreased in 7-week-old Col4a3(-/-) mice compared with age-matched controls; these changes were partially reversed by recombinant angiotensin-converting enzyme 2 (ACE2) treatment. The expression of both the angiotensinogen and renin protein increased in Col4a3(-/-) compared with wild-type mice. Consistent with the Ang-(1-7) levels, the expression and activity of kidney ACE2 decreased in 7-week-old Col4a3(-/-) mice. The urinary excretion rate of ACE2 paralleled the decline in tissue expression. Expression of an Ang II-induced gene, heme oxygenase-1, was up-regulated in the kidneys of 7-week-old Col4a3(-/-) mice compared with wild-type mice by microarray analysis. Heme oxygenase-1 (HO-1) protein expression was increased in kidneys of Col4a3(-/-) mice and normalized by treatment with ACE inhibitor. Urinary HO-1 excretion paralleled renal HO-1 expression. In conclusion, progressive kidney injury in AS is associated with changes in expression of intrarenal renin Ang system components and Ang peptides. HO-1 and ACE2 may represent novel markers of AS-associated kidney injury, whereas administration of recombinant ACE2 and/or Ang-(1-7) may represent novel therapeutic approaches in AS.

Evidence for Pathogenicity of Atypical Splice Mutations in Autosomal Dominant Polycystic Kidney Disease

BACKGROUND AND OBJECTIVES Mutation-based molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) is complicated by locus and allelic heterogeneity, large multi-exon gene structure and duplication in PKD1, and a high level of unclassified variants. Comprehensive screening of PKD1 and PKD2 by two recent studies have shown that atypical splice mutations account for 3.5% to 5% of ADPKD. We evaluated the role of bioinformatic prediction of atypical splice mutations and determined the pathogenicity of an atypical PKD2 splice variant from a multiplex ADPKD (TOR101) family. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Using PubMed, we identified 17 atypical PKD1 and PKD2 splice mutations. We found that bioinformatics analysis was often useful for evaluating the pathogenicity of these mutations, although RT-PCR is needed to provide the definitive proof. RESULTS Sequencing of both PKD1 and PKD2 in an affected subject of TOR101 failed to identify a definite mutation, but revealed several UCVs, including an atypical PKD2 splice variant. Linkage analysis with microsatellite markers indicated that TOR101 was PKD2-linked and IVS8 + 5G-->A was shown to cosegregate only with affected subjects. RT-PCR of leukocyte mRNA from an affected subject using primers from exons 7 and 9 revealed six splice variants that resulted from activation of different combinations of donor and acceptor cryptic splice sites, all terminating with premature stop codons. CONCLUSIONS The data provide strong evidence that IVS8 + 5G-->A is a pathogenic mutation for PKD2. This case highlights the importance of functional analysis of UCVs.

Molecular diagnosis of autosomal dominant polycystic kidney disease

ABSTRACT Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease that accounts for 5–10% of end-stage renal disease in developed countries. Mutations in PKD1 and PKD2 account for a majority of cases. Mutation screening of PKD1 is technically challenging largely due to the complexity resulting from duplication of its first 33 exons in six highly homologous pseudogenes (i.e. PKD1P1-P6). Protocol using locus-specific long-range and nested PCR has enabled comprehensive PKD1 mutation screening but is labor-intensive and costly. Here, the authors review how recent advances in Next Generation Sequencing are poised to transform and extend molecular diagnosis of ADPKD. Areas covered: Key original research articles and reviews of the topic published in English identified through PubMed from 1957–2017. Expert commentary: The authors review current and evolving approaches using targeted resequencing or whole genome sequencing for screening typical as well as challenging cases (e.g. cases with no detectable PKD1 and PKD2 mutations which may be due to somatic mosaicism or other cystic disease; and complex genetics such as bilineal disease).