Tubular deficiency of ABCA1 augments cholesterol- and Na+-dependent effects on systemic blood pressure in male mice.

Dyslipidemia, with changes in plasma membrane (PM) composition, is associated with hypertension, while rising PM cholesterol induces Na+ channel activity. We hypothesize that ablation of renal tubular ABCA1, a cholesterol efflux protein, leads to cholesterol- and Na+-dependent changes in blood pressure (BP). Transgenic mice (TgPAX8rtTA;tetO-Cre/+) expressing a doxycycline (dox)-inducible CRE recombinase were bred with mice expressing floxed ABCA1 to generate renal tubules deficient in ABCA1 (ABCA1FF). Tail-cuff systolic BP (SBP) was measured in mice on specific diets. Immunoblotting was performed on whole and PM protein lysates of kidney from mice completing experimental diets. Cortical PM of ABCA1FF showed reduced ABCA1 (60 ± 28%; n = 10, P < 0.05) compared with wild-type littermates (WT; n = 9). Tail-cuff SBP of ABCA1FF (n = 11) was not only greater post dox, but also during cholesterol or high Na+ feeding (P < 0.05) compared with WT mice (n = 15). A Na+-deficient diet abolished the difference, while 6 wk of cholesterol diet raised SBP in ABCA1FF compared with mice before cholesterol feeding (P < 0.05). No difference in α-ENaC protein abundance was noted in kidney lysate; however, γ-ENaC increased in ABCA1FF mice versus WT mice. In kidney membranes, NKCC2 abundance was greater in ABCA1FF versus WT mice. Cortical lysates of ABCA1FF mouse kidneys expressed less renin and angiotensin I receptor than WT mouse kidneys. Furosemide injection induced a greater diuretic effect in ABCA1FF (n = 7; 45.2 ± 8.7 µL/g body wt) versus WT (n = 7; 33.1 ± 6.9 µL/g body wt; P < 0.05) but amiloride did not. Tubular ABCA1 deficiency induces cholesterol-dependent rise in SBP and modest Na+ sensitivity of SBP, which we speculate is partly related to Na+ transporters and channels.NEW & NOTEWORTHY Cholesterol has been linked to greater Na+ channel activity in kidney cells, which may predispose to systemic hypertension. We showed that when ABCA1, a protein that removes cholesterol from tissues, is ablated from mouse kidneys, systemic blood pressure is greater than normal mice. Dietary cholesterol further increases blood pressure in transgenic mice, whereas low dietary salt intake reduced blood pressure to that of normal mice. Thus, we speculate that diseases and pharmaceuticals that reduce renal ABCA1 expression, like diabetes and calcineurin inhibitors, respectively, contribute to the prominence of hypertension in their clinical presentation.

Unilateral Nephrectomy Stimulates ERK and Is Associated With Enhanced Na Transport.

Nephron loss initiates compensatory hemodynamic and cellular effects on the remaining nephrons. Increases in single nephron glomerular filtration rate and tubular flow rate exert higher fluid shear stress (FSS) on tubules. In principal cell (PC) culture models FSS induces ERK, and ERK is implicated in the regulation of transepithelial sodium (Na) transport, as well as, proliferation. Thus, we hypothesize that high tubular flow and FSS mediate ERK activation in the cortical collecting duct (CCD) of solitary kidney which regulates amiloride sensitive Na transport and affects CCD cell number. Immunoblotting of whole kidney protein lysate was performed to determine phospho-ERK (pERK) expression. Next, sham and unilateral nephrectomized mice were stained with anti-pERK antibodies, and dolichos biflorus agglutinin (DBA) to identify PCs with pERK. Murine PCs (mpkCCD) were grown on semi-permeable supports under static, FSS, and FSS with U0126 (a MEK1/2 inhibitor) conditions to measure the effects of FSS and ERK inhibition on amiloride sensitive Na short circuit current (Isc). pERK abundance was greater in kidney lysate of unilateral vs. sham nephrectomies. The total number of cells in CCD and pERK positive PCs increased in nephrectomized mice (9.3 ± 0.4 vs. 6.1 ± 0.2 and 5.1 ± 0.5 vs. 3.6 ± 0.3 cell per CCD nephrectomy vs. sham, respectively, n > 6 per group, p < 0.05). However, Ki67, a marker of proliferation, did not differ by immunoblot or immunohistochemistry in nephrectomy samples at 1 month compared to sham. Next, amiloride sensitive Isc in static mpkCCD cells was 25.3 ± 1.7 µA/cm2 (n = 21), but after exposure to 24 h of FSS the Isc increased to 41.4 ± 2.8 µA/cm2 (n = 22; p < 0.01) and returned to 19.1 ± 2.1 µA/cm2 (n = 18, p < 0.01) upon treatment with U0126. Though FSS did not alter α- or γ-ENaC expression in mpkCCD cells, γ-ENaC was reduced in U0126 treated cells. In conclusion, pERK increases in whole kidney and, specifically, CCD cells after nephrectomy, but pERK was not associated with active proliferation at 1-month post-nephrectomy. In vitro studies suggest high tubular flow induces ERK dependent ENaC Na absorption and may play a critical role in Na balance post-nephrectomy.

Cellular cholesterol modifies flow-mediated gene expression.

Downregulation of heme oxygenase-1 (HO-1), cyclooxygenase-2 (COX2), and nitric oxide synthase-2 (NOS2) in the kidneys of Dahl rodents causes salt sensitivity, while restoring their expression aids in Na+ excretion and blood pressure reduction. Loading cholesterol into collecting duct (CD) cells represses fluid shear stress (FSS)-mediated COX2 activity. Thus, we hypothesized that cholesterol represses flow-responsive genes necessary to effectuate Na+ excretion. To this end, CD cells were used to test whether FSS induces these genes and if cholesterol loading represses them. Mice fed either 0% or 1% cholesterol diet were injected with saline, urine volume and electrolytes were measured, and renal gene expression determined. FSS-exposed CD cells demonstrated increases in HO-1 mRNA by 350-fold, COX2 by 25-fold, and NOS2 by 8-fold in sheared cells compared with static cells (P < 0.01). Immunoblot analysis of sheared cells showed increases in HO-1, COX2, and NOS2 protein, whereas conditioned media contained more HO-1 and PGE2 than static cells. Cholesterol loading repressed the sheared mediated protein abundance of HO-1 and NOS2 as well as HO-1 and PGE2 concentrations in media. In cholesterol-fed mice, urine volume was less at 6 h after injection of isotonic saline (P < 0.05). Urinary Na+ concentration, urinary K+ concentration, and osmolality were greater, whereas Na+ excretion was less, at the 6-h urine collection time point in cholesterol-fed versus control mice (P < 0.05). Renal cortical and medullary HO-1 (P < 0.05) and NOS2 (P < 0.05) mRNA were repressed in cholesterol-fed compared with control mice. Cholesterol acts to repress flow induced natriuretic gene expression, and this effect, in vivo, may contribute to renal Na+ avidity.

The Influence of Microdeletions and Microduplications of 16p11.2 on Global Transcription Profiles.

Copy number variants (CNVs) of a 600 kb region on 16p11.2 are associated with neurodevelopmental disorders and changes in brain volume. The authors hypothesize that abnormal brain development associated with this CNV can be attributed to changes in transcriptional regulation. The authors determined the effects of 16p11.2 dosage on gene expression by transcription profiling of lymphoblast cell lines derived from 6 microdeletion carriers, 15 microduplication carriers and 15 controls. Gene dosage had a significant influence on the transcript abundance of a majority (20/34) of genes within the CNV region. In addition, a limited number of genes were dysregulated in trans. Genes most strongly correlated with patient head circumference included SULT1A, KCTD13, and TMEM242. Given the modest effect of 16p11.2 copy number on global transcriptional regulation in lymphocytes, larger studies utilizing neuronal cell types may be needed in order to elucidate the signaling pathways that influence brain development in this genetic disorder.

Inferring tumor progression from genomic heterogeneity.

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA).

We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

DBC2 is essential for transporting vesicular stomatitis virus glycoprotein.

DBC2 is a tumor suppressor gene linked to breast and lung cancers. Although DBC2 belongs to the RHO GTPase family, it has a unique structure that contains a Broad-Complex/Tramtrack/Bric a Brac (BTB) domain at the C terminus instead of a typical CAAX motif. A limited number of functional studies on DBC2 have indicated its participation in diverse cellular activities, such as ubiquitination, cell-cycle control, cytoskeleton organization and protein transport. In this study, the role of DBC2 in protein transport was analyzed using vesicular stomatitis virus glycoprotein (VSVG) fused with green fluorescent protein. We discovered that DBC2 knockdown hinders the VSVG transport system in 293 cells. Previous studies have demonstrated that VSVG is transported via the microtubule motor complex. We demonstrate that DBC2 mobility depends also on an intact microtubule network. We conclude that DBC2 plays an essential role in microtubule-mediated VSVG transport from the endoplasmic reticulum to the Golgi apparatus.

DBC2 significantly influences cell-cycle, apoptosis, cytoskeleton and membrane-trafficking pathways.

The tumor suppressor DBC2 belongs to a previously uncharacterized gene family, RHOBTB (Bric-a-brac, Tramtrack, Broad-complex). The biological roles of RHOBTB proteins, including DBC2, remain unclear. To understand the physiological functions of DBC2, a global approach was applied. Expression of DBC2 was manipulated in HeLa cells and RNA profiling of the cells was performed by microarray analyses. DBC2 was introduced into HeLa cells by a mammalian expression vector with a constitutive promoter. DBC2 knockdown was achieved by RNA interference with small interfering RNA. RNA profiles of these samples were performed by microarray analysis using Affymetrix GeneChip HG-U133A 2.0. The microarray data were analyzed by Microarray Suite 5.0 (MAS 5.0) and Robust Multichip Average (RMA). A list of genes whose expression was significantly altered (p<0.001) was generated and overlaid onto a cellular pathway map in the Ingenuity Systems' Pathway Knowledge Base (Winter'04 Release). Two networks were found to react substantially to DBC2 expression; namely, more than half of participating genes are affected. One of the networks regulates cell growth through cell-cycle control and apoptosis. The other network is related to cytoskeleton and membrane trafficking. Our findings suggest that the biological roles of DBC2 are related directly and/or indirectly to these cellular machineries.

DBC2, a candidate for a tumor suppressor gene involved in breast cancer.

A previously uncharacterized gene, DBC2 (deleted in breast cancer), was cloned from a homozygously deleted region at human chromosome 8p21. DBC2 contains a highly conserved RAS domain and two putative protein interacting domains. Our analyses indicate that DBC2 is the best candidate tumor suppressor gene from this region. It lies within the epicenter of the deletions and is homozygously deleted in 3.5% (7/200) of breast tumors. Mutation analysis of DBC2 led to discovery of two instances of somatic missense mutations in breast tumor specimens, whereas no missense mutations were found in other candidates from the region. Unlike other genes in the region, expression of DBC2 is often extinguished in breast cancer cells or tissues. Moreover, our functional analysis revealed that DBC2 expression in breast cancer cells lacking DBC2 transcripts causes growth inhibition. By contrast, expression of a somatic mutant discovered in a breast cancer specimen does not suppress the growth of breast cancer cells.