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Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease. ADPKD is characterized by cyst development that leads to abnormal kidney structure. Renal tubules are a fundamental unit of architecture, so controls of tubular growth and formation are important for proper kidney function. The molecular mechanisms of tubulogenesis are being actively studied as the basis of diagnosis and treatment of ADPKD. Mxi1 is a member of the MAD family of proteins that functions in terminal differentiation, inhibition of cell cycle progression and tumor suppression, while the Myc protein, which is antagonized by Mxi1, causes renal cystogenesis. Based on these molecular relationships, the present study implicated Mxi1 with ADPKD be demonstrating that curtailed Mxi1 gene expression caused cyst formation in Mxi1-deficient mice. To ascertain whether Mxi1 affects renal epithelial cell tubulogenesis, three-dimensional cultures (3D culture) of mIMCD-3 cells and stably Mxi1 over-expressed mIMCD-3 cells were established. The results indicated that over-expression of the Mxi1 gene plays a role in the regulation of tubulogenesis by regulating some genes participating in renal epithelial branching tubulogenesis such as matrix metalloproteinase 9 (MMP9), integrins, fibronectin, and E-cadherin. The results support the suggestion that over-expression of Mxi1 can suppress renal epithelial tubulogenesis. In particular, MMP9 is greatly affected by the expression level of Mxi1. It can be concluded that mIMCD-3 cells that stably over-express Mxi1 fail to form renal epithelial tubules because of abnormally reduced expression of MMP9.

Cyst formation is a major characteristic of ADPKD and iscaused by the abnormal proliferation of epithelial cells. Renalcyst formation disrupts renal function and induces diversecomplications. The mechanism of cyst formation is unclear.mIMCD-3 cells were established to develop simple epithelialcell cysts in 3-D culture. We confirmed previously that Mxi1plays a role in cyst formation in Mxi1-deficient mice. Cysts inMxi1 transfectanted cells were showed by collagen or mebiolgels in 3-D cell culture system. Causative genes of ADPKDwere measured by q RT-PCR. Herein, Mxi1 transfectants rarelyformed a simple epithelial cyst and induced cell death.Overexpression of Mxi1 resulted in a decrease in the PKD1,PKD2 and c-myc mRNA relating to the pathway of cystformation. These data indicate that Mxi1 influences cystformation of mIMCD-3 cells in 3-D culture and that Mxi1 maycontrol the mechanism of renal cyst formation. [BMB reports2012; 45(3): 189-193]

ADPKD (Autosomal Dominant Polycystic Kidney Disease) is characterized by the progressive expansion of multiple cystic lesions in the kidneys. ADPKD is caused by mutations in Ed-pl. consider PKD1 and PKD2. Recently a relation between c-myc and the pathogenesis of ADPKD was reported. In addition, c-Myc is a downstream effector of PKD1. To identify the gene regulated by PKD2 and c-Myc, we performed gene expression profiling in PKD2 and c-Myc overexpressing cells using a human 8K cDNA microarray. NCAM (neuronal cell adhesion molecule) levels were significantly reduced in PKD2 overexpressing systems in vitro and in vivo. These results suggest that NCAM is an important molecule in the cystogenesis induced by PKD2 overexpression.

The Mxi1 proteins are biochemical and biological antagonists of c-myc oncoprotein. It has been reported that the overexpression pattern of c-myc might be similar to a molecular feature of early and late stages of human autosomal dominant polycystic kidney disease. We identified the cyst phenotype in Mxi1-deficient mice aged 6–12 months using H&E staining. Some chemokines containing a protein domain similar to human IL-8, which is associated with the inflammatory response, were subsequently selected from the up-regulated genes. We confirmed the expression level of these chemokines and measured protein concentrations of IL-8 using ELISA in the Mxi1-knockdown cells. IL-8 was found to be significantly increased in Mxi1-knockdown cells. We found that p38 MAP kinase activation was involved in the signal transduction of the Mxi1-inactivated secretion of IL-8. Therefore, we could suggest that the inactivation of Mxi1 leads to the inflammatory response and has the potential to induce polycystic renal disease.

PCD (programmed cell death) is important mechanism for development, homeostasis and disease. To analyze the gene expression pattern in brain cells undergoing PCD in response to serum deprivation, we analyzed the cDNA microarray consisting of 2,300 genes and 7 housekeeping genes of cortical cells derived from mouse embryonic brain. Cortical cells were induced apoptosis by serum deprivation for 8 hours. We identified 69 up-regulated genes and 21 down-regulated genes in apoptotic cells. Based on the cDNA microarray data four genes were selected and analyzed by RT-PCR and northern blotting. To characterize the role of UNC-51-like kinase (ULK2) gene in PCD, we investigated cell death effect by ULK2. And we examined expression of several genes that related with PCD. Especially GAPDH was increased by ULK2. Theses findings indicated that ULK2 is involved in apoptosis through p53 pathway.

Cardiac systolic function is significantly decreased in a proportion of patients with Hunter syndrome. This study was performed to evaluate the change in myocardial function associated with enzyme replacement therapy (ERT) in a mouse model of cardiomyopathy associated with Hunter syndrome. Thirty 9-week-old iduronate-2-sulfatase (IDS) knockout mice received either intravenous injection of human recombinant IDS (ERT group, N=15) or saline (control group, N=15) for 5 weeks. Echocardiography was performed at baseline and after treatment. Echocardiographic parameters of left ventricular (LV) systolic function and 2-dimensional radial and circumferential strain were assessed. At follow-up, there was a significant increase in LV fractional shortening and radial and circumferential strain in the ERT group only. Notable myocardial fibrosis was observed in the control group only. In the murine model of Hunter syndrome, ERT exerts beneficial effects on cardiac function, which can be evaluated by serial echocardiographic evaluation including 2-dimensional strain analysis.

Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

The pathogenic mechanisms of human autosomal dominant polycystic kidney disease (ADPKD) have been well known to include the mutational inactivation of PKD2. Although haploinsufficiency and loss of heterozygosity at the Pkd2 locus can cause cyst formation in mice, polycystin-2 is frequently expressed in the renal cyst of human ADPKD, raising the possibility that deregulated activation of PKD2 may be associated with the cystogenesis of human ADPKD. To determine whether increased PKD2 expression is physiologically pathogenic, we generated PKD2-overexpressing transgenic mice. These mice developed typical renal cysts and an increase of proliferation and apoptosis, which are reflective of the human ADPKD phenotype. These manifestations were first observed at six months, and progressed with age. In addition, we found that ERK activation was induced by PKD2 overexpression via B-Raf signaling, providing a possible molecular mechanism of cystogenesis. In PKD2 transgenic mice, B-Raf/MEK/ERK sequential signaling was up-regulated. Additionally, the transgenic human polycystin-2 partially rescues the lethality of Pkd2 knock-out mice and therefore demonstrates that the transgene generated a functional product. Functional strengthening or deregulated activation of PKD2 may be a direct cause of ADPKD. The present study provides evidence for an in vivo role of overexpressed PKD2 in cyst formation. This transgenic mouse model should provide new insights into the pathogenic mechanism of human ADPKD.