Search

Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipLow-dose streptozotocin was employed to induce diabetes in both HhipCompared with HhipTubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.

AIMS/HYPOTHESIS: We previously reported that renal tubule-specific deletion of Heterogeneous nuclear ribonucleoprotein F (Hnrnpf) results in upregulation of renal angiotensinogen (Agt) and downregulation of sodium-glucose co-transporter 2 (Sglt2) in Hnrnpf(RT) knockout (KO) mice. Non-diabetic Hnrnpf(RT) KO mice develop hypertension, renal interstitial fibrosis and glycosuria with no renoprotective effect from downregulated Sglt2 expression. Here, we investigated the effect of renal tubular Hnrnpf deletion on hyperfiltration and kidney injury in Akita mice, a model of type 1 diabetes. METHODS: Akita Hnrnpf(RT) KO mice were generated through crossbreeding tubule-specific (Pax8)-Cre mice with Akita floxed-Hnrnpf mice on a C57BL/6 background. Male non-diabetic control (Ctrl), Akita, and Akita Hnrnpf(RT) KO mice were studied up to the age of 24 weeks (n=8/group). RESULTS: Akita mice exhibited elevated systolic blood pressure as compared with Ctrl mice, which was significantly higher in Akita Hnrnpf(RT) KO mice than Akita mice. Compared with Akita mice, Akita Hnrnpf(RT) KO mice had lower blood glucose levels with increased urinary glucose excretion. Akita mice developed kidney hypertrophy, glomerular hyperfiltration (increased glomerular filtration rate), glomerulomegaly, mesangial expansion, podocyte foot process effacement, thickened glomerular basement membranes, renal interstitial fibrosis and increased albuminuria. These abnormalities were attenuated in Akita Hnrnpf(RT) KO mice. Treatment of Akita Hnrnpf(RT) KO mice with a selective A1 adenosine receptor inhibitor resulted in an increase in glomerular filtration rate. Renal Agt expression was elevated in Akita mice and further increased in Akita Hnrnpf(RT) KO mice. In contrast, Sglt2 expression was increased in Akita and decreased in Akita Hnrnpf(RT) KO mice. CONCLUSIONS/INTERPRETATION: The renoprotective effect of Sglt2 downregulation overcomes the renal injurious effect of Agt when these opposing factors coexist under diabetic conditions, at least partly via the activation of tubuloglomerular feedback.

The angiotensin II receptor type 2 (AT2R) may be a potential therapeutic target for the treatment of hypertension and chronic kidney disease (CKD). The expression and function of AT2R in the vasculature and kidney appear sexually dimorphic. We hypothesised that Agtr2 knockout dams (AT2RKO) with gestational diabetes would program their offspring for subsequent hypertension and CKD in a sex-dependent manner. Age- and sex-matched offspring of non-diabetic and diabetic dams of wild-type (WT) and AT2RKO mice were followed from 4 to 20 weeks of age and were monitored for development of hypertension and nephropathy; a mouse podocyte cell line (mPOD) was also studied. Body weight was progressively lower in female compared with male offspring throughout the lifespan. Female but not male offspring from diabetic AT2RKO dams developed insulin resistance. Compared with the offspring of non-diabetic dams, the progeny of diabetic dams had developed more hypertension and nephropathy (apparent glomerulosclerosis with podocyte loss) at 20 weeks of age; this programming was more pronounced in the offspring of AT2RKO diabetic dams, particularly female AT2RKO progeny. Female AT2RKO offspring had lower basal ACE2 glomerular expression, resulting in podocyte loss. The aberrant ACE2/ACE ratio was far more diminished in glomeruli of female progeny of diabetic AT2RKO dams than in male progeny. Knock-down of Agtr2 in mPODs confirmed the in vivo data. AT2R deficiency accelerated kidney programming in female progeny of diabetic dams, possibly due to loss of protective effects of ACE2 expression in the kidney.

Zinc is considered protective against atherosclerosis; however, the association between dietary zinc intake and cardiovascular disease remains debated. We investigated whether dietary zinc intake was associated with coronary artery calcium (CAC) progression in the Multi-Ethnic Study of Atherosclerosis (MESA). This analysis included 5186 participants aged 61.9 ± 10.2 years (48.8% men; 41.3% white, 25.0% black, 21.6% Hispanic, and 12.1% Chinese American) from the MESA. Dietary zinc intake was assessed by a self-administered, 120-item food frequency questionnaire at baseline (2000–2002). Baseline and follow-up CAC were measured by computed tomography. CAC progression was defined as CAC > 0 at follow-up for participants with CAC = 0 at baseline; and an annualized change of 10 or percent change of ≥ 10% for those with 0 0.05). In this multiethnic population free of clinically apparent cardiovascular disease, higher dietary zinc intake from non-red meat sources was independently associated with a lower risk of CAC progression. The MESA trial was registered at clinicaltrials.gov as NCT00005487.

Hedgehog interacting protein (Hhip) is essential for islet formation and beta-cell proliferation during pancreatic development; abnormally elevated Hhip expression has been linked to human pancreatitis. Here, we investigate the role of Hhip in modulating insulin secretion in adult Hhip mice (Hhip +/− vs. Hhip+/+) fed high fat diets (HFD). Both sexes of HFD-Hhip +/+ mice developed impaired glucose intolerance, that was only ameliorated in male HFD-Hhip +/− mice that had high levels of circulating plasma insulin, but not in female HFD-Hhip +/− mice. HFD stimulated Hhip gene expression, mainly in beta cells. Male HFD-Hhip +/+ mice had more large islets in which insulin content was reduced; islet architecture was disordered; and markers of oxidative stress (8-OHdG and Nox 2) were increased. In contrast, male HFD-Hhip +/− mice had more small islets with increased beta cell proliferation, enhanced GSIS, less oxidative stress and preserved islet integrity. In vitro, recombinant Hhip increased Nox2 and NADPH activity and decreased insulin-positive beta cells. siRNA-Hhip increased GSIS and abolished the stimulation of sodium palmitate (PA)-BSA on Nox2 gene expression. We conclude that pancreatic Hhip gene inhibits insulin secretion by altering islet integrity and promoting Nox2 gene expression in beta cells in response to HDF-mediated beta cell dysfunction, a novel finding.

We hypothesised that maternal diabetes impairs kidney formation in offspring via augmented expression of hedgehog interacting protein (HHIP). Our gene-array results were performed in neonatal kidneys from our murine model of maternal diabetes and indicated that Hhip expression was significantly modulated by maternal diabetes.We systematically examined the functional role of HHIP in kidney formation in our murine maternal diabetes model and elucidated the potential mechanisms related to dysnephrogenesis in vitro.The kidneys of the offspring of diabetic dams, compared with those of the offspring of control non-diabetic dams, showed retardation of development--small kidneys and less ureteric bud (UB) branching morphogenesis. Augmented HHIP expression was observed in the offspring of diabetic dams, initially localised to differentiated metanephric mesenchyme and UB epithelium and subsequently in maturing glomerular endothelial and tubulointerstitial cells. The heightened HHIP targeting TGF-β1 signalling was associated with dysmorphogenesis. In vitro, HHIP overexpression decreased sonic hedgehog and paired box gene 2 proteins (SHH and PAX2, respectively) and increased transcriptional nuclear factor-kappa B (NFκB, p50/p65), phosphorylation of p53, and TGF-β1 expression. In contrast, overexpression of PAX2 inhibited HHIP and NFκB and activated SHH, N-myc and p27(Kip1) expression. Moreover, high glucose stimulated HHIP expression, and then targeted TGF-β1 signalling. Thus, PAX2, via a negative autocrine feedback mechanism, attenuated the stimulatory effect of high glucose on HHIP expression.Maternal diabetes modulates kidney formation in young progeny mediated, at least in part, via augmented HHIP expression.

The genome structure of pear chloroplast DNA (cpDNA) is extremely highly conserved in comparison with that of other angiosperms, and therefore, relatively few phylogenetic analyses for pear (Pyrus spp.) have been carried out using cpDNA as a marker. In this study, we identified two hypervariable regions in intergenic spacers of cpDNA from 21 species in Pyrus. One of these regions is 857 bp in length and lies between the accD-psaI genes, and the other is a 904-bp region between the rps16-trnQ genes. The mutation rate of gaps for the two regions was 10 and 26 times higher, respectively, than the base change rate. Twenty-five haplotypes were revealed among 21 species in Pyrus by 36 mutations found in the two regions. These included 27 gaps and 9 base changes but excluded cpSSRs. Phylogenetic relationships between the 25 haplotypes were generated by haplotype network analysis. The 25 haplotypes represented three groups (types A–C) with two large deletions, one 228 bp in length between the accD-psaI genes and the other 141 bp between the rps16-trnQ genes. Types A and B consisted mostly of pears native to East and South Asia. Type C contained mainly Pyrus communis and wild relatives native to Europe, West and Central Asia, Russia, and Africa. Type B might have diverged from primitives such as pea pears in type A. Phylogenetic utility of structural alterations (gaps) occurring in the hypervariable regions of Pyrus cpDNA is discussed.

Nanostructured thermal barrier coatings (TBCs) were deposited by plasma spraying using agglomerated nanostructured YSZ powder on Inconel 738 substrate with cold-sprayed nanostructured NiCrAlY powder as bond coat. The isothermal oxidation and thermal cycling tests were applied to examine failure modes of plasma-sprayed nanostructured TBCs. For comparison, the TBC consisting of conventional microstructure YSZ and conventional NiCrAlY bond coat was also deposited and subjected to the thermal shock test. The results showed that nanostructured YSZ coating contained two kinds of microstructures; nanosized zirconia particles embedded in the matrix and microcolumnar grain structures of zirconia similar to those of conventional YSZ. Although, after thermal cyclic test, a continuous, uniform thermally grown oxide (TGO) was formed, cracks were observed at the interface between TGO/BC or TGO/YSZ after thermal cyclic test. However, the failure of nanostructured and conventional TBCs mainly occurred through spalling of YSZ. Compared with conventional TBCs, nanostructured TBCs exhibited better thermal shock resistance.

We previously demonstrated that angiotensin II (Ang II) stimulates paired homeo box-2 (Pax-2) via the Ang II type 2 receptor (AT(2)R). The Pax-2 gene and N-myc play pivotal roles in renal morphogenesis via their effects on cell proliferation and differentiation in embryonic mesenchymal cells and embryonic mouse kidneys. Since AT(2)R knock-out (KO) mice have a phenotype that is similar to that of humans with congenital renal and urinary tract anomalies (CAKUT) and develop hypertension in adulthood, these mice and wild-type controls were used for this study. Embryonic kidneys isolated from E12 to term gestation were cultured in Dulbecco's modified Eagle's medium (DMEM) with or without Ang II (10(-6) M) for 24 h ex vivo. Renal morphogenesis was histologically assessed. Mean glomerular tuft volume was determined by the method of Weibel and Gomez with the aid of image analysis software. Pax-2 and N-myc gene expression were determined by immunostaining as well as by Western blotting and real-time-quantitative polymerase chain reaction (RT-qPCR). Glomerular size was significantly smaller, and Pax-2 and N-myc expression down-regulated, in kidneys of AT(2)R KO mice compared with those of wild-type mice. In ex vivo studies, Ang II stimulated Pax-2 and N-myc mRNA expression in embryonic kidneys of wild-type mice, but this stimulatory effect was absent in embryonic kidneys of AT(2)R KO mice. Taken together, these data indicate that intrarenal AT(2)R plays an important role in nephrogenesis. Deficiency of AT(2)R may impair both Pax-2 and N-myc expression, eventually resulting in glomerular hyperfiltration that may, ultimately, lead to later development of hypertension.

Self-incompatibility (SI) has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility (GSI). In the present study, four PpsS-haplotypes (Prunus pseudocerasus S-haplotypes) comprising at least two genes, i.e., PpsS-RNase (P. pseudocerasus S-RNase) and PpsSFB (P. pseudocerasus S-haplotype-specific F-box) have been successfully isolated in tetraploid P. pseudocerasus Lindl. CV. Nanjing Chuisi ("NC") which exhibited self-compatibility (SC), and its S-genotype was determined as S-1/S-3'/S-5/S-7. These PpsS-RNases, which were expressed exclusively in style, shared the typical structural features with S-RNases from other Prunus species exhibiting GSI. All PpsSFBs showed similar structure characteristics of SFBs from other Prunus species, and matched with the necessary conditions for pollen S-determinant. No mutations leading to dysfunction of S-haplotype were found in their full-length c-DNA sequences, except for PpsS-3'-haplotype which was not amplified by PCR. These four S-haplotypes complied with tetrasomic inheritance. Diploid pollen grains with S-genotypes S-7/S-1, S-7/S-5 and S-1/S-5 can grow the full length of the style after self-pollination, while pollen grains with S-3'/S-7, S-3'/S-1 and S-3'/S-5 cannot. These results suggest that PpsS-haplotypes-1, -5 and -7 are functional, and that competitive interaction between two of them confer self-compatibility on cultivar "NC". Furthermore, in terms of recognition specificity, diploid pollen grains carrying PpsS-3'-haplotype are equal to monoploid pollen grains carrying the other functional S-haplotype.

Most Rosaceae fruit trees such as Japanese plum and sweet cherry have a gametophytic self-incompatibility (GSI) system controlled by a single S locus containing at least two linked genes with multiple alleles, i.e., S-RNase as a pistil determinant and SFB (S-haplotype-specific F-box gene) as a candidate for the pollen S determinant. For identification of S genotypes, many methods based on polymerase chain reaction (PCR) utilizing polymorphism in length of the S-RNase and SFB gene have been developed. In this study, we developed two dot-blot analysis methods for S-haplotype identification utilizing allele-specific oligonucleotides based on the SFB-HVa region, which has high sequence polymorphism. Dot-blotting of allele-specific oligonucleotides hybridized with digoxigenin-labeled PCR products allowed S genotyping of plants with nine S haplotypes (S-a, S-b, S-c, S-e, S-f, S-h, S-k, S-7 and S-10) in Japanese plum and ten S haplotypes (S-1, S-2, S-3, S-4, S-4′, S-5, S-6, S-7, S-9 and S-16) in sweet cherry (dot-blot-S-genotyping). In addition, dot-blotting of PCR products of SFB probed with the allele-specific oligonucleotides, occasionally utilizing competitive hybridization, was successful in screening for a desirable S haplotype in sweet cherry (dot-blot-S-screening).

Both paired homeo box-2 (Pax-2) and N-myc genes play pivotal roles in renal morphogenesis via their effects on cell proliferation and differentiation, but whether and how they interact have not been addressed. In the present study, we investigated such a potential interaction using embryonic renal cells in vitro. Mouse embryonic mesenchymal (MK4) cells stably transfected with Pax-2 cDNA in sense (+) or antisense (-) orientation were used for experiments. Pax-2 promoter activity was monitored by luciferase assay. Reactive oxygen species (ROS) generation, cell proliferation, and cell apoptosis were evaluated. We found that Pax-2 and N-myc gene expression were upregulated and downregulated in Pax-2 (+) and Pax-2 (-) stable transformants, respectively. ROS generation and apoptosis were significantly reduced both in Pax-2 (+) transformants compared with Pax-2 (-) transformants and in naïve MK4 cells cultured in either normal- (5 mM) or high-glucose (25 mM) medium. Transient transfection of N-myc cDNA into Pax-2 (-) stable transformants restored Pax-2 gene expression and prevented ROS generation induced by high glucose. Our data demonstrate that Pax-2 gene overexpression prevents hyperglycemia-induced apoptosis, and N-myc appears to provide a positive autocrine feedback on Pax-2 gene expression in embryonic mesenchymal cells.

The S-allele-associated proteins (S-proteins) in the styles of the Japanese pear (Pyrus serotina Rehd. var. culta Rehd.) were purified by cation exchange chromatography. Their inhibitory action on the growth of incompatible pollen tubes (pollen tubes bearing the same S- allele as in the style from which the S-proteins were prepared) was characterized in vitro. Germination and tube growth of self-pollen (pollen from the same cultivar from which the S-proteins were prepared) decreased dose-dependently when the S-protein was added to the medium. Tube length was reduced to 10% that of compatible pollen tubes (pollen tubes bearing the S-allele different from that in the style from which the S-proteins were prepared) at 1.5 µg µl1. S-proteins from Shinsui (S 4 S 5 ) also inhibited growth of cross-incompatible Kosui (S 4 S 5 ) pollen tubes, but not of compatible Chojuro (S 2 S 3 ) pollen tubes. After inactivation of RNase of the S- protein, the inhibitory action of the S-protein disappeared. These results indicate that the S-protein acts directly to inhibit growth of incompatible pollen tubes in Japanese pear styles, and that the RNase activity of the protein is essential for the biological function. However, small amounts of proteins that co-migrated with the S-protein may also play some roles in the inhibition. This is the first report on the selective inhibitory action of S-proteins in Rosaceae.

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5′-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a β1/β2-adrenergic receptor (AR) agonist) and iodoclonidine (an α2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (α1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (β-AR blocker), atenolol (β1-AR blocker), yohimbine (α2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (β2-AR blocker) and prazosin (α1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP 254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5′-flanking region of the ANG gene and subsequently stimulates the gene expression.