Identification of MMP1 as a novel risk factor for intracranial aneurysms in ADPKD using iPSC models.

Cardiovascular complications are the leading cause of death in autosomal dominant polycystic kidney disease (ADPKD), and intracranial aneurysm (ICA) causing subarachnoid hemorrhage is among the most serious complications. The diagnostic and therapeutic strategies for ICAs in ADPKD have not been fully established. We here generated induced pluripotent stem cells (iPSCs) from seven ADPKD patients, including four with ICAs. The vascular cells differentiated from ADPKD-iPSCs showed altered Ca(2+) entry and gene expression profiles compared with those of iPSCs from non-ADPKD subjects. We found that the expression level of a metalloenzyme gene, matrix metalloproteinase (MMP) 1, was specifically elevated in iPSC-derived endothelia from ADPKD patients with ICAs. Furthermore, we confirmed the correlation between the serum MMP1 levels and the development of ICAs in 354 ADPKD patients, indicating that high serum MMP1 levels may be a novel risk factor. These results suggest that cellular disease models with ADPKD-specific iPSCs can be used to study the disease mechanisms and to identify novel disease-related molecules or risk factors.

The moyamoya disease susceptibility variant RNF213 R4810K (rs112735431) induces genomic instability by mitotic abnormality.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n=3, 61.0 ± 8.2%) compared with wild-type subjects (n=6, 13.1 ± 7.7%; p<0.01). Aneuploidy was observed more frequently in fibroblasts (p<0.01) and induced pluripotent stem cells (iPSCs) (p<0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p<0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p<0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.

Downregulation of Securin by the variant RNF213 R4810K (rs112735431, G>A) reduces angiogenic activity of induced pluripotent stem cell-derived vascular endothelial cells from moyamoya patients.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p<0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p<0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.

Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.

A method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed. Most cells in kidney are derived from an embryonic germ layer known as intermediate mesoderm. Here we show the establishment of an efficient system of homologous recombination in human pluripotent stem cells by means of bacterial artificial chromosome-based vectors and single-nucleotide polymorphism array-based detection. This system allowed us to generate human-induced pluripotent stem cell lines containing green fluorescence protein knocked into OSR1, a specific intermediate mesoderm marker. We have also established a robust induction protocol for intermediate mesoderm, which produces up to 90% OSR1(+) cells. These human intermediate mesoderm cells can differentiate into multiple cell types of intermediate mesoderm-derived organs in vitro and in vivo, thereby supplying a useful system to elucidate the mechanisms of intermediate mesoderm development and potentially providing a cell source for regenerative therapies of the kidney.

An autopsy case of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with intestinal bleeding in chronic renal failure.

A 50-year-old man who underwent hemodialysis (HD) at local outpatient HD center due to end-stage renal disease (ESRD) was transferred to our hospital because of pneumonia. He had severe emaciation and past history of congestive heart failure. Presenting symptoms almost consistently involved difficulty in hearing and recurrent attacks of migraine-like headaches. He was diagnosed with dilated cardiomyopathy, showing diastolic mechanical dyssynchrony by tissue Doppler echocardiography. On the day of death, he had hematemesis and hemorrhagic shock. Autopsy revealed perforation of duodenum, and genetic analysis using mitochondrial DNA from cardiac muscle and iliopsoas muscle revealed a 3243A > G mutation in the mitochondrial tRNA(Leu(UUR)) gene, which is related to mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Multiple organ failure due to the mutation of mitochondrial DNA with gastrointestinal bleeding is not a common.

Angio-embolization of renal artery pseudoaneurysm after renal biopsy: a case report.

Renal artery pseudoaneurysm is a rare clinical entity that has been reported after renal biopsy, percutaneous renal surgery, penetrating trauma, and rarely blunt renal trauma. We present the case of a 37-year-old man with ruptured renal artery pseudoaneurysm accompanied by massive gross hematuria, urinary clot retention, and bladder tamponade, which were the presenting signs seven hours after renal biopsy. Abdominal CT scan showed a large perinephric, intracapsular hematoma of left kidney. His angiogram revealed a left renal segmental artery pseudoaneurysm that measured 1 cm x 1 cm. He was successfully treated by selective embolization of the arterial branch supplying the pseudoaneurysm.