Identification of Free Nitric Oxide Radicals in Rat Bone Marrow: Implications for Progenitor Cell Mobilization in Hypertension

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention

Chronic kidney failure mineral bone disorder leads to a permanent loss of hematopoietic stem cells through dysfunction of the stem cell niche

In chronic kidney disease (CKD), endothelial injury, is associated with disease progression and an increased risk for cardiovascular complications. Circulating cells with vascular reparative functions are hematopoietic and also reduced in CKD. To explore the mechanistic basis behind these observations, we have investigated hematopoietic stem cell (HSC) homeostasis in a mouse model for non-progressive CKD-mineral and bone disorder with experimentally induced chronic renal failure (CRF). In mice subjected to 12 weeks of CRF, bone marrow HSC frequencies were decreased and transplantation of bone marrow cells from CRF donors showed a decrease in long-term HSC repopulation compared to controls. This loss was directly associated with a CRF-induced defect in the HSC niche affecting the cell cycle status of HSC and could not be restored by the PTH-reducing agent cinacalcet. In CRF, frequencies of quiescent (G0) HSC were decreased coinciding with an increase in hematopoietic progenitor cells (HPC) in the S-and G2-phases of cell cycle. Moreover, in CRF mice, HSC-niche supporting macrophages were decreased compared to controls concomitant to impaired B lymphopoiesis. Our data point to a permanent loss of HSC and may provide insight into the root cause of the loss of homeostatic potential in CKD

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor β (TGF-β) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation

Relative contribution of NOS isoforms to basal NO production in BM of Lewis rats.

<p>The basal NO level that was obtained in absence of a stimulus was taken as 100%. Contribution of the NOS isotypes was determined using two concentrations of the NOS isoform-specific inhibitor 1400 W. 1400 W was used at final concentrations of 0.1 µM for iNOS inhibition and 10 µM for inhibition of both iNOS and nNOS. With L-NAME (500 µM, final column), the signal intensity was below the detection threshold. Values represent biological triplicates.</p

MAP and BM-derived MNIC yields in control and hypertensive Wistar rats.

<p>b.d.: below detection</p>*<p>Two BM samples, where MNIC remained below the detection threshold are excluded from this average.</p

X-band EPR spectra of MNIC at 77°K.

<p>(A). The EPR spectrum from BM cell suspension of <i>ca</i> 100 mln cells. These cells were collected from a DOCA-treated Wistar male rat and stimulated for NO production with Ca-ionophore A23187. The intensity of the spectrum was not changed and was quantified as 60±7 pmol MNIC. (B) The reference spectrum of a strong calibration sample with 4 nmol MNIC, showing the characteristic triplet structure centered around g = 2.035 with a hyperfine splitting of 25.2 G = 2A_N¹⁴. The intensity of the reference spectrum was scaled down by a factor 25.</p

Spearman rank correlation between NO and MMP9 levels in BM of Wistar rats.

<p>A positive and statistically significant (0.016 cells, total MMP9 amount was quantified in the supernatants from the same BM samples. Errors in MNIC yields and MMP9 (not indicated) are ca 10% and 5% respectively.</p

MNIC yields (pmol/10⁸ cells) in rat BM suspensions and EC cultures.

*<p>Technical footnote: Since proper eNOS activity requires EC attachment and cell-cell contact, NO trapping was done on monolayers of HUVEC and bEnd.3 cells firmly attached to the bottom of a culture T₇₅ flasks, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057761#pone.0057761-Vanin1" target="_blank">[21]</a>. A single flask with <i>ca</i> 7.5×10⁶ confluent bEnd.3 cells has adequate signal to noise ratio <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057761#pone.0057761-Vanin1" target="_blank">[21]</a>, whereas HUVEC, having less eNOS, requires at least two flasks with total 15×10⁶ cells.</p

The effect of NOS inhibitors and/or activators on NO level in BM of Wistar rats.

<p>X-band EPR spectra at 77°K of frozen BM cell suspensions from Wistar rats after 30 min of NO trapping with Fe²⁺-DETC. The samples (53±2×10⁶ BM cells) were reduced by 50 mM dithionite to remove the overlapping signal from paramagnetic Cu²⁺-DETC. A basal yield of 21±2 pmol MNIC is obtained in absence of stimulus (top), clearly recognizable by its characteristic hyperfine triplet structure at g = 2.035. Stimulation with ionomycin or A23187 calcium ionophores raises the MNIC yields to 45±4 and 49±5 pmol. In presence of the NOS inhibitor L-NAME, the MNIC yields remained below the detection threshold of <i>ca</i> 10 pmol. The EPR spectra show significant background signals not related to MNIC, as visible in the bottom spectra. Such background signals derive from paramagnetic iron centers, and some residual Cu²⁺-DETC as commonly found in biological samples <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057761#pone.0057761-vanFaassen1" target="_blank">[18]</a>.</p

Chronic kidney failure mineral bone disorder leads to a permanent loss of hematopoietic stem cells through dysfunction of the stem cell niche

In chronic kidney disease (CKD), endothelial injury, is associated with disease progression and an increased risk for cardiovascular complications. Circulating cells with vascular reparative functions are hematopoietic and also reduced in CKD. To explore the mechanistic basis behind these observations, we have investigated hematopoietic stem cell (HSC) homeostasis in a mouse model for non-progressive CKD-mineral and bone disorder with experimentally induced chronic renal failure (CRF). In mice subjected to 12 weeks of CRF, bone marrow HSC frequencies were decreased and transplantation of bone marrow cells from CRF donors showed a decrease in long-term HSC repopulation compared to controls. This loss was directly associated with a CRF-induced defect in the HSC niche affecting the cell cycle status of HSC and could not be restored by the PTH-reducing agent cinacalcet. In CRF, frequencies of quiescent (G0) HSC were decreased coinciding with an increase in hematopoietic progenitor cells (HPC) in the S-and G2-phases of cell cycle. Moreover, in CRF mice, HSC-niche supporting macrophages were decreased compared to controls concomitant to impaired B lymphopoiesis. Our data point to a permanent loss of HSC and may provide insight into the root cause of the loss of homeostatic potential in CKD.Biomaterials & Tissue Biomechanic