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A balanced diet with adequate nutrient intake is vital for normal fetal kidney development to occur. In contrast, maternal malnourishment during pregnancy impairs fetal kidney development due to a deficiency in nutrients that the mother and growing fetus have access to. Nutritional deficiency not only reduces the structural building blocks needed for nephron generation but also impacts various signaling pathways, growth factors, epigenetic modifications, gene expression, and oxidative stress that collectively blunts nephrogenesis. These effects of nutritional deficiency consequently reduce the stem cell pool that is available for nephron building by altering their self-renewal, survival, and patterned differentiation. As a result, the fetus is born following maternal malnourishment with significantly fewer nephrons, underdeveloped kidneys, potential kidney dysfunction, and a lifelong increased susceptibility to kidney and cardiovascular disease. Discussed in this chapter are the specific affects that vitamins, minerals, amino acids, lipids, and fatty acids have on stem cells, nephrogenesis, and kidney development.

(1) Background: Arterial hypertension (HTN) is one of the most relevant cardiovascular risk factors. Nowadays multiple pharmaceutical treatment options exist with novel interventional methods (e.g., baroreflex activation therapy (BAT)) as a last resort to treat patients with resistant HTN. Although pathophysiology behind resistant HTN is still not fully understood. There is evidence that selected biomarkers may be involved in the pathophysiology of HTN. (2) Methods: We investigated serum SDC4-levels in patients suffering from resistant HTN before and 6 months after BAT implantation. We collected 19 blood samples from patients with resistant HTN and blood pressure above target and measured serum SDC4-levels. (3) Results: Our results showed high serum SDC4-levels in patients with resistant HTN as compared to a healthy population. Patients with both, resistant HTN and diabetes mellitus type II, demonstrated higher serum SDC4-levels. &beta, blockers had lowering effects on serum SDC4-levels, whereas calcium channel blockers were associated with higher levels of serum SDC4. BAT implantation did not lead to a significant difference in serum SDC4-levels after 6 months of therapy. (4) Conclusion: Based on our results we propose SDC4 is elevated in patients suffering from resistant HTN. Thus, SDC4 might be a potential marker for endothelial dysfunction in patients with resistant hypertension.

During sepsis, the alarmin HMGB1 is released from tissues and promotes systemic inflammation that results in multi-organ damage, with the kidney particularly susceptible to injury. The severity of inflammation and pro-damage signaling mediated by HMGB1 appears to be dependent on the alarmin's redox state. Therefore, we examined HMGB1 redox in kidney cells during sepsis. Using intravital microscopy, CellROX labeling of kidneys in live mice indicated increased ROS generation in the kidney perivascular endothelium and tubules during lipopolysaccharide (LPS)-induced sepsis. Subsequent CellROX and MitoSOX labeling of LPS-stressed endothelial and kidney proximal tubule cells demonstrated increased ROS generation in these cells as sepsis worsens. Consequently, HMGB1 oxidation increased in the cytoplasm of kidney cells during its translocation from the nucleus to the circulation, with the degree of oxidation dependent on the severity of sepsis, as measured in in vivo mouse samples using a thiol assay and mass spectrometry (LC-MS/MS). The greater the oxidation of HMGB1, the greater the ability of the alarmin to stimulate pro-inflammatory cyto-/chemokine release (measured by Luminex Multiplex) and alter mitochondrial ATP generation (Luminescent ATP Detection Assay). Administration of glutathione and thioredoxin inhibitors to cell cultures enhanced HMGB1 oxidation during sepsis in endothelial and proximal tubule cells, respectively. In conclusion, as sepsis worsens, ROS generation and HMGB1 oxidation increases in kidney cells, which enhances HMGB1's pro-inflammatory signaling. Conversely, the glutathione and thioredoxin systems work to maintain the protein in its reduced state., Graphical abstract fx1, Highlights • Endotoxins (LPS) increase cellular oxidative stress during sepsis. • During its translocation, HMGB1 gets oxidized in the cytoplasm. • Thioredoxin and glutathione keep HMGB1 in a reduced redox state during sepsis. • HMGB1 oxidation enhances its stimulation of inflammatory cyto-/chemokine release.

BackgroundLow birth weight (LBW) neonates have impaired kidney development that leaves them susceptible to kidney disease and hypertension during adulthood. The study here identifies events that blunt nephrogenesis and kidney development in the murine LBW neonate.MethodsWe examined survival, kidney development, GFR, gene expression, and cyto-/chemokines in the LBW offspring of malnourished (caloric and protein-restricted) pregnant mice.ResultsMalnourished pregnant mothers gave birth to LBW neonates that had 40% reduced body weight and 54% decreased survival. Renal blood perfusion was reduced by 37%, whereas kidney volume and GFR were diminished in the LBW neonate. During gestation, the LBW neonatal kidney had 2.2-fold increased apoptosis, 76% decreased SIX2+ progenitor cells, downregulation of mesenchymal-to-epithelial signaling factors Wnt9b and Fgf8, 64% less renal vesicle formation, and 32% fewer nephrons than controls. At birth, increased plasma levels of IL-1β, IL-6, IL-12(p70), and granulocyte-macrophage colony-stimulating factor in the LBW neonate reduced SIX2+ progenitor cells.ConclusionIncreased pro-inflammatory cytokines in the LBW neonate decrease SIX2+ stem cells in the developing kidney. Reduced renal stem cells (along with the decreased mesenchymal-to-epithelial signaling) blunt renal vesicle generation, nephron formation, and kidney development. Subsequently, the mouse LBW neonate has reduced glomeruli volume, renal perfusion, and GFR.

Significance: A common link between all forms of acute and chronic kidney injuries, regardless of species, is enhanced generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during injury/disease progression. While low levels of ROS and RNS are required for prosurvival signaling, cell proliferation and growth, and vasoreactivity regulation, an imbalance of ROS and RNS generation and elimination leads to inflammation, cell death, tissue damage, and disease/injury progression. Recent Advances: Many aspects of renal oxidative stress still require investigation, including clarification of the mechanisms which prompt ROS/RNS generation and subsequent renal damage. However, we currently have a basic understanding of the major features of oxidative stress pathology and its link to kidney injury/disease, which this review summarizes. Critical Issues: The review summarizes the critical sources of oxidative stress in the kidney during injury/disease, including generation of ROS and RNS from mitochondria, NADPH oxidase, and inducible nitric oxide synthase. The review next summarizes the renal antioxidant systems that protect against oxidative stress, including superoxide dismutase and catalase, the glutathione and thioredoxin systems, and others. Next, we describe how oxidative stress affects kidney function and promotes damage in every nephron segment, including the renal vessels, glomeruli, and tubules. Future Directions: Despite the limited success associated with the application of antioxidants for treatment of kidney injury/disease thus far, preventing the generation and accumulation of ROS and RNS provides an ideal target for potential therapeutic treatments. The review discusses the shortcomings of antioxidant treatments previously used and the potential promise of new ones. Antioxid. Redox Signal. 25, 119–146.

Maternal undernutrition during pregnancy (MUN) often leads to low birth weight (LBW) neonates that have a reduced total nephron endowment, leaving these neonates susceptible to kidney disease throughout their lives. For reasons unknown, these LBW neonates have impaired kidney development due to a severe reduction in renal SIX2+ stem cells during nephrogenesis. Using a mouse model of MUN, we investigated SIX2+ stem cell reduction in the LBW neonate. Significant upregulation of the protein fetuin-B (measured by PCR and immunoblotting) in the MUN mother's placenta, organs and circulation yielded a 3-fold increase of this protein in the embryonic kidney. Recombinant fetuin-B, administered to healthy pregnant mothers at the concentration equivalent to that in the MUN mother, crossed the placenta and reduced both SIX2+ stem cells by 50% and nephron formation by 66% in embryonic kidneys (measured by immunofluorescence and the physical dissector/fractionator stereological method). Administration of fetuin-B to kidney explants yielded similar reductions in renal SIX2+ stem cells and nephron formation. Fetuin-B treatment of isolated embryonic renal SIX2+ stem cell primary cultures 1) increased NF-kB activity and apoptosis, 2) reduced cell proliferation due to upregulated p21 nuclear activity and subsequent cell cycle arrest, and 3) enhanced generation of reactive oxygen species (measured by fluorescence microscopy). In conclusion, MUN increases fetuin-B in the developing embryonic kidney. The increase in fetuin-B blunts nephrogenesis by reducing SIX2+ stem cells by promoting their apoptosis (via NF-kB upregulation), blunting their proliferative renewal (via p21 upregulation) and enhancing oxidative stress.

Maternal undernutrition (MUN) during pregnancy leads to low-birth weight (LBW) neonates that have a reduced kidney nephron endowment and higher morbidity as adults. Using a severe combined caloric and protein-restricted mouse model of MUN to generate LBW mice, we examined the progression of renal insufficiency in LBW adults. Through 6 mo of age, LBW males experienced greater albuminuria (ELISA analysis), a more rapid onset of glomerular hypertrophy, and a worse survival rate than LBW females. In contrast, both sexes experienced a comparable progressive decline in renal vascular density (immunofluorescence analysis), renal blood flow (Laser-Doppler flowmetry analysis), glomerular filtration rate (FITC-sinistrin clearance analysis), and a progressive increase in systemic blood pressure (measured via tail-cuff method). Isolated aortas from both LBW sexes demonstrated reduced vasodilation in response to ACh, indicative of reduced nitric oxide bioavailability and endothelial dysfunction. ELISA and immunofluorescence analysis revealed a significant increase of circulating reactive oxygen species and NADPH oxidase type 4 (NOX4) expression in both LBW sexes, although these increases were more pronounced in males. Although more effective in males, chronic tempol treatment did improve all observed pathologies in both sexes of LBW mice. Chronic NOX4 inhibition with GKT137831 was more effective than tempol in preventing pathologies in LBW males. In conclusion, despite some minor differences, LBW female and male adults have a reduced nephron endowment comparable with progressive renal and vascular dysfunction, which is associated with increased oxidative stress and subsequent endothelial dysfunction. Tempol treatment and/or NOX4 inhibition attenuates renal and vascular dysfunction in LBW adults.

We previously reported the delivery of endothelial progenitor cells (EPCs) embedded in hyaluronic acid-based (HA)-hydrogels protects renal function during acute kidney injury (AKI) and promotes angiogenesis. We attempted to further ameliorate renal dysfunction by coembedding EPCs with renal mesenchymal stem cells (MSCs), while examining their paracrine influence on cytokine/chemokine release and proinflammatory macrophages. A live/dead assay determined whether EPC-MSC coculturing improved viability during lipopolysaccharide (LPS) treatment, and HA-hydrogel-embedded delivery of cells to LPS-induced AKI mice was assessed for effects on mean arterial pressure (MAP), renal blood flow (RBF), circulating cytokines/chemokines, serum creatinine, proteinuria, and angiogenesis (femoral ligation). Cytokine/chemokine release from embedded stem cells was examined, including effects on macrophage polarization and release of proinflammatory molecules. EPC-MSC coculturing improved stem cell viability during LPS exposure, an effect augmented by MSC hypoxic preconditioning. The delivery of coembedded EPCs with hypoxic preconditioned MSCs to AKI mice demonstrated additive improvement (compared with EPC delivery alone) in medullary RBF and proteinuria, with comparable effects on serum creatinine, MAP, and angiogenesis. Exposure of proinflammatory M1 macrophages to EPC-MSC conditioned medium changed their polarization to anti-inflammatory M2. Incubation of coembedded EPCs-MSCs with macrophages altered their release of cytokines/chemokines, including enhanced release of anti-inflammatory interleukin (IL)-4 and IL-10. EPC-MSC delivery to endotoxemic mice elevated the levels of circulating M2 macrophages and reduced the circulating cytokines/chemokines. In conclusion, coembedding EPCs-MSCs improved their resistance to stress, impelled macrophage polarization from M1 to M2 while altering their cytokine/chemokines release, reduced circulating cytokines/chemokines, and improved renal and vascular function when MSCs were hypoxically preconditioned. Significance This report provides insight into a new therapeutic approach for treatment of sepsis and provides a new and improved strategy using hydrogels for the delivery of stem cells to treat sepsis and, potentially, other injuries and/or diseases. The delivery of two different stem cell lines (endothelial progenitor cells and mesenchymal stem cells; delivered alone and together) embedded in a protective bioengineered scaffolding (hydrogel) offers many therapeutic benefits for the treatment of sepsis. This study shows how hydrogel-delivered stem cells elicit their effects and how hydrogel embedding enhances the therapeutic efficacy of delivered stem cells. Hydrogel-delivered stem cells influence the components of the overactive immune system during sepsis and work to counterbalance the release of many proinflammatory and prodamage substances from immune cells, thereby improving the associated vascular and kidney damage.

Background Our laboratory has previously demonstrated that Sirt1endo-/- mice show endothelial dysfunction and exaggerated renal fibrosis, whereas mice with silenced endothelial transforming growth factor beta (TGF-β) signaling are resistant to fibrogenic signals. Considering the fact that the only difference between these mutant mice is confined to the vascular endothelium, this indicates that secreted substances contribute to these contrasting responses. Methods We performed an unbiased proteomic analysis of the secretome of renal microvascular endothelial cells (RMVECs) isolated from these two mutants. We cultured renal fibroblasts and RMVECs and used microfluidic devices for coculturing. Results Dickkopf-3 (DKK3), a putative ligand of the Wnt/β-catenin pathway, was present exclusively in the fibrogenic secretome. In cultured fibroblasts, DKK3 potently induced myofibroblast activation. In addition, DKK3 antagonized effects of DKK1, a known inhibitor of the Wnt pathway, in conversion of fibroblasts to myofibroblasts. In RMVECs, DKK3 induced endothelial-mesenchymal transition and impaired their angiogenic competence. The inhibition of endothelial outgrowth, enhanced myofibroblast formation and endothelial-mesenchymal transition were confirmed in coculture. In reporter DKK3-eGFP × Col3.6-GFPcyan mice, DKK3 was marginally expressed under basal conditions. Adriamycin-induced nephropathy resulted in upregulation of DKK3 expression in tubular and, to a lesser degree, endothelial compartments. Sulindac sulfide was found to exhibit superior Wnt pathway-suppressive action and decreased DKK3 signals and the extent of renal fibrosis. Conclusions In conclusion, this unbiased proteomic screen of the profibrogenic endothelial secretome revealed DKK3 acting as an agonist of the Wnt pathway, enhancing formation of myofibroblasts and endothelial-mesenchymal transition and impairing angiogenesis. A potent inhibitor of the Wnt pathway, sulindac sulfide, suppressed nephropathy-induced DKK3 expression and renal fibrosis.

High-mobility group box 1 (HMGB1) undergoes acetylation, nuclear-to-cytoplasmic translocation, and release from stressed kidneys, unleashing a signaling cascade of events leading to systemic inflammation. Here, we tested whether the deacetylase activity of Sirtuin1 (SIRT1) participates in regulating nuclear retention of HMGB1 to ultimately modulate damage signaling initiated by HMGB1 secretion during stress. When immunoprecipitated acetylated HMGB1 was incubated with SIRT1, HMGB1 acetylation decreased by 57%. Proteomic analysis showed that SIRT1 deacetylates HMGB1 at four lysine residues (55, 88, 90, and 177) within the proinflammatory and nuclear localization signal domains of HMGB1. Genetic ablation or pharmacological inhibition of SIRT1 in endothelial cells increased HMGB1 acetylation and translocation. In vivo, deletion of SIRT1 reduced nuclear HMGB1 while increasing its acetylation and release into circulation during basal and ischemic conditions, causing increased renal damage. Conversely, resveratrol pretreatment led to decreased HMGB1 acetylation, its nuclear retention, decreased systemic release, and reduced tubular damage. Thus, a vicious cycle is set into motion in which the inflammation-induced repression of SIRT1 disables deacetylation of HMGB1, facilitates its nuclear-to-cytoplasmic translocation, and systemic release, thereby maintaining inflammation.

Factors that initiate cellular damage and trigger the inflammatory response cascade and renal injury are not completely understood after renal ischemia-reperfusion injury (IRI). High-mobility group box-1 protein (HMGB1) is a damage-associated molecular pattern molecule that binds to chromatin, but upon signaling undergoes nuclear-cytoplasmic translocation and release from cells. Immunohistochemical and Western blot analysis identified HMGB1 nuclear-cytoplasmic translocation and release from renal cells (particularly vascular and tubular cells) into the venous circulation after IRI. Time course analysis indicated HMGB1 release into the venous circulation progressively increased parallel to increased renal ischemic duration. Ethyl pyruvate (EP) treatment blocked H2O2(oxidative stress)-induced HMGB1 release from human umbilical vein endothelial cells in vitro, and in vivo resulted in nuclear retention and significant blunting of HMGB1 release into the circulation after IRI. EP treatment before IRI improved short-term serum creatinine and albuminuria, proinflammatory cyto-/chemokine release, and long-term albuminuria and fibrosis. The renoprotective effect of EP was abolished when exogenous HMGB1 was injected, suggesting EP's therapeutic efficacy is mediated by blocking HMGB1 translocation and release. To determine the independent effects of circulating HMGB1 after injury, exogenous HMGB1 was administered to healthy animals at pathophysiological dose. HMGB1 administration induced a rapid surge in systemic circulating cyto-/chemokines (including TNF-α, eotaxin, G-CSF, IFN-γ, IL-10, IL-1α, IL-6, IP-10, and KC) and led to mobilization of bone marrow CD34+Flk1+ cells into the circulation. Our results indicate that increased ischemic duration causes progressively enhanced HMGB1 release into the circulation triggering damage/repair signaling, an effect inhibited by EP because of its ability to block HMGB1 nuclear-cytoplasmic translocation.

We aimed to investigate the potential relationship between alarmins [acting via Toll-like receptor-4 (TLR4)], uric acid (UA), and high-mobility group box-1 protein (HMGB1) during acute kidney injury. UA, which is significantly increased in the circulation following renal ischemia-reperfusion injury (IRI), was used both in vitro and in vivo as an early response-signaling molecule to determine its ability to induce the secretion of HMGB1 from endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with UA resulted in increased HMGB1 mRNA expression, acetylation of nuclear HMGB1, and its subsequent nuclear-cytoplasmic translocation and release into the circulation, as determined by Western blotting and immunofluorescence. Treatment of HUVEC with UA and a calcium mobilization inhibitor (TMB-8) or a MEK/Erk pathway inhibitor (U0126) prevented translocation of HMGB1 from the nucleus, resulting in reduced cytoplasmic and circulating levels of HMGB1. Once released, HMGB1 in autocrine fashion promoted further HMGB1 release while also stimulating NF-κB activity and increased angiopoietin-2 expression and protein release. Transfection of HUVEC with TLR4 small interfering (si) RNA reduced HMGB1 levels during UA and HMGB1 treatment. In summary, UA after IRI mediates the acetylation and release of HMGB1 from endothelial cells by mechanisms that involve calcium mobilization, the MEK/Erk pathway, and activation of TLR4. Once released, HMGB1 promotes its own further cellular release while acting as an autocrine and paracrine to activate both proinflammatory and proreparative mediators.

Sepsis and its complications are associated with poor clinical outcomes. The circulatory system is a well-known target of lipopolysaccharide (LPS). Recently, several clinical studies documented mobilization of endothelial progenitor cells (EPCs) during endotoxemia, with the probability of patients' survival correlating with the rise in circulating EPCs. This fact combined with endotoxemia-induced vascular injury led us to hypothesize that the developing functional EPC incompetence could impede vascular repair and that adoptive transfer of EPCs could improve hemodynamics in endotoxemia. We used LPS injection to model endotoxemia. EPCs isolated from endotoxemic mice exhibited impaired clonogenic potential and LPS exerted Toll-like receptor 4-mediated cytotoxic effects toward EPCs, which was mitigated by embedding them in hyaluronic acid (HA) hydrogels. Therefore, intact EPCs were either delivered intravenously or embedded within pronectin-coated HA hydrogels. Adoptive transfer of EPCs in LPS-injected mice improved control of blood pressure and reduced hepatocellular and renal dysfunction. Specifically, EPC treatment was associated with the restoration of renal microcirculation and improved renal function. EPC therapy was most efficient when cells were delivered embedded in HA hydrogel. These findings establish major therapeutic benefits of adoptive transfer of EPCs, especially when embedded in HA hydrogels, in mice with LPS-induced endotoxemia, and they argue that hemodynamic and renal abnormalities of endotoxemia are in significant part due to developing incompetence of endogenous EPCs.

We developed an ex vivo approach characterizing renal mesenchymal stem cell (MSC) adhesion to kidney sections. Specificity of MSC adhesion was confirmed by demonstrating a) 3T3 cells displayed 10-fold lower adhesion, and b) MSC adhesion was CXCR4/stromal-derived factor-1 (SDF-1)-dependent. MSC adhesion was asymmetrical, with postischemic sections exhibiting more than twofold higher adhesion than controls, and showed preference to perivascular areas. Pretreating kidney sections with cyclic arginine-glycine-aspartic acid peptide resulted in increased MSC adhesion (by displacing resident cells), whereas blockade of CXCR4 with AMD3100 and inhibition of alpha4beta1(VLA4) integrin or vascular cellular adhesion molecule-1, reduced adhesion. The difference between adhered cells under cyclic arginine-glycine-aspartic acid peptide-treated and control conditions reflected prior occupancy of binding sites with endogenous cells. The AMD3100-inhibitable fraction of adhesion reflected CXCR4-dependent adhesion, whereas maximal adhesion was interpreted as kidney MSC-lodging capacity. MSC obtained from mice overexpressing caveolin-1 exhibited more robust adhesion than those obtained from knockout animals, consistent with CXCR4 dimerization in caveolae. These data demonstrate a) CXCR4/SDF-1-dependent adhesion increases in ischemia; b) CXCR4/SDF-1 activation is dependent on MSC surface caveolin-1; and c) occupancy of MSC binding sites is decreased, while d) capacity of MSC binding sites is expanded in postischemic kidneys. In conclusion, we developed a cell-bait strategy to unmask renal stem cell binding sites, which may potentially shed light on the MSC niche(s) and its characteristics.

Intrinsic stem cells (SC) participate in tissue remodeling and regeneration in various diseases and following toxic insults. Failure of tissue regeneration is in part attributed to lack of SC protection from toxic stress of noxious stimuli, thus prompting intense research efforts to develop strategies for SC protection and functional preservation for in vivo delivery. One strategy is creation of artificial SC niches in an attempt to mimic the requirements of endogenous SC niches by generating scaffolds with properties of extracellular matrix. Here, we investigated the use of hyaluronic acid (HA) hydrogels as an artificial SC niche and examined regenerative capabilities of encapsulated embryonic endothelial progenitor cells (eEPC) in three different in vivo models. Hydrogel-encapsulated eEPC demonstrated improved resistance to toxic insult (adriamycin) in vitro, thus prompting in vivo studies. Implantation of HA hydrogels containing eEPC to mice with adriamycin nephropathy or renal ischemia resulted in eEPC mobilization to injured kidneys (and to a lesser extent to the spleen) and improvement of renal function, which was equal or superior to adoptively transferred EPC by intravenous infusion. In mice with hindlimb ischemia, EPC encapsulated in HA hydrogels dramatically accelerated the recovery of collateral circulation with the efficacy superior to intravenous infusion of EPC. In conclusion, HA hydrogels protect eEPC against adriamycin cytotoxicity and implantation of eEPC encapsulated in HA hydrogels supports renal regeneration in ischemic and cytotoxic (adriamycin) nephropathy and neovascularization of ischemic hindlimb, thus establishing their functional competence and superior capabilities to deliver stem cells stored in and released from this bioartificial niche.

NO protection is crucial against angiotensin II (ANG II) mediated vasoconstriction in postnatal preglomerular resistance vessels. Although whole kidney NOS is developmentally regulated, NOS regulation in developing renal resistance vessels is unknown. The hypothesis was NOS expression and function in developing afferent arterioles are regulated by ANG II through AT1 and AT2 receptors. Afferent arterioles from porcine kidneys, ages newborn, 7, 21 d, and adult, were dissected using a polybead perfusion technique. Dissected afferent arterioles were treated with ANG II and with either the AT1 receptor inhibitor candesartan or the AT2 receptor inhibitor PD 123319 and evaluated for NOS isoform expression and NOS enzymatic activity. Although NOS activity and neuronal NOS (nNOS) expression were greater in the newborn than in the adult, endothelial NOS (eNOS) expression was greater in the adult. ANG II increased NOS activity and eNOS expression at all ages, but nNOS expression only in developing afferents. AT1 and AT2 receptor blockade significantly attenuated NOS activity and eNOS expression at all ages, but nNOS expression only in developing afferents. ANG II regulates nNOS and eNOS expression and NOS activity in afferent arterioles of the developing kidney via AT1 and AT2 receptors.

Primary and/or secondary injury of the renal microvascular endothelium is a common finding in various renal diseases. Besides well-known endothelial repair mechanisms, including endothelial cell (EC) proliferation and migration, homing of extrinsic cells such as endothelial progenitor cells (EPC) and hematopoietic stem cells (HSC) has been shown in various organs and may contribute to microvascular repair. However, these mechanisms have so far not been studied after selective microvascular injury in the kidney. The present study investigated the time course of EPC and HSC stimulation and homing following induction of selective EC injury in the mouse kidney along with various angiogenic factors potentially involved in EC repair and progenitor cell stimulation. Erythropoietin was used to stimulate progenitor cells in a therapeutic approach. We found that selective EC injury leads to a marked stimulation of EPCs, HSCs, and various angiogenic factors to orchestrate microvascular repair. Angiogenic factors started to increase as early as 30 min after disease induction. Progenitor cells could be first detected in the circulation and the spleen before they selectively homed to the diseased kidney. Injection of a high dose of erythropoietin 2 h after disease induction markedly attenuated vascular injury through nonhemodynamic mechanisms, possibly involving vascular endothelial growth factor release.

Renal resident stem cells were previously reported within the renal tubules and papillary area. The aim of the present study was to determine whether renal capsules harbor stem cells and whether this pool can be recruited to the renal parenchyma after ischemic injury. We demonstrated the presence of label-retaining cells throughout the renal capsule, at a density of ∼10 cells/mm2, and their close apposition to the blood vessels. By flow cytometry, in vitro cultured cells derived from the renal capsule were positive for mesenchymal stem cell (MSC) markers (CD29+, vimentin+, Sca-1+, nestin+) but did not express hematopoietic and endothelial stem cell markers. Moreover, renal capsule-derived cells also exhibited self-renewal, clonogenicity, and multipotency in differentiation conditions, all favoring stem cell characteristics and identifying them with MSC. In situ labeling of renal capsules with CM-DiI CellTracker demonstrated in vivo a directed migration of CM-DiI-labeled cells to the ischemic renal parenchyma, with the rate of migration averaging 30 μm/h. Decapsulation of the kidneys during ischemia resulted in a modest, but statistically significant, deceleration of recovery of plasma creatinine compared with ischemic kidneys with intact renal capsule. Comparison of these conditions allows the conclusion that renal capsular cells may contribute ∼25–30% of the recovery from ischemia. In conclusion, the data suggest that the renal capsule may function as a novel stem cell niche harboring MSC capable of participating in the repair of renal injury.

Adriamycin-associated nephropathy (AAN) remains poorly understood. We hypothesized that adriamycin affects endothelial progenitor cells (EPCs), leading to impaired regeneration. We analyzed renal hematopoietic stem cells (HSCs) and EPCs in mice with AAN and examined the potential contribution of adoptive transfer of intact EPCs to the repair processes. FACS analyses revealed that populations of HSCs and EPCs were scarcely represented in control kidneys and did not change numerically in kidneys obtained from mice with AAN. The observed defect in engraftment was attributable to the decreased viability and increased senescence of EPCs. Adoptive transfer of intact EPCs improved proteinuria and renal function, with a threefold decrease in mortality. Infusion of EPCs to adriamycin-treated mice reduced plasma levels of interleukin-1α and -β and granulocyte-colony stimulating factor as well as increased the level of vascular endothelial growth factor with concomitant improvement of vascular density and reduction of apoptosis. An additional mechanism of tissue repair is proposed based on tunneling nanotube formation between EPCs and endothelial cells exposed to adriamycin, leading to the multiple rounds of exchange between EPCs and mature cells. In conclusion, AAN is associated with development of EPC incompetence; adoptive transfer of intact EPCs blunts morphological and functional manifestations of AAN; and the proposed mechanisms of repair by EPCs include direct incorporation into blood vessels, paracrine signaling, and tunneling nanotube renewal of mitochondrial pool in endothelial cells.

Nitric oxide (NO), produced by nitric oxide synthase (NOS), critically counteracts angiotensin-II-enhanced vascular resistance in the immature kidney, perhaps due to the developmental regulation of NOS expression and function in the postnatal preglomerular resistance vessels (PRV). Our experiments measured the messenger ribonucleic acid (mRNA) gene expression of neuronal NOS (nNOS), endothelial NOS (eNOS), and components of the renin-angiotensin system (renin, AT1 and AT2 receptors), by real-time RT-PCR, as well as NOS enzymatic activity by citrulline assay in PRVs (afferent, interlobular, and arcuate arterioles) obtained from swine ages newborn, 7 and 21 days, and adult. NOS enzymatic activity was upregulated in PRVs immediately after birth but decreased to adult levels with maturation. Neuronal NOS, renin, and AT2 receptor expression in PRVs were upregulated in the newborn and decreased with age to lowest levels in the adult. In contrast, eNOS and AT1 receptor expression were downregulated at birth but increased to the highest levels in the adult. Upregulated NOS enzymatic activity in newborn PRVs supports the critical neonatal role for NO renal vascular vasodilation. Upregulated nNOS gene expression, concomitant with downregulated eNOS gene expression in neonatal PRVs, suggests that the nNOS isoform may be responsible for counteracting angiotensin II increased vascular resistance in immature porcine PRVs.

Endothelial cell dysfunction is associated with bioavailable nitric oxide deficiency and an excessive generation of reactive oxygen species. We modeled this condition by chronically inhibiting nitric oxide generation with subpressor doses of N(G)-monomethyl-L-arginine (L-NMMA) in C57B6 and Tie-2/green fluorescent protein mouse strains. L-NMMA-treated mice exhibited a slight reduction in vasorelaxation ability, as well as detectable abnormalities in soluble adhesion molecules (soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1, and matrix metalloproteinase 9), which represent surrogate indicators of endothelial dysfunction. Proteomic analysis of the isolated microvasculature using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy revealed abnormal expression of a cluster of mitochondrial enzymes, which was confirmed using immunodetection. Aconitase-2 and enoyl-CoA-hydratase-1 expression levels were decreased in L-NMMA-treated animals; this phenotype was absent in nitric oxide synthase-1 and -3 knockout mice. Depletion of aconitase-2 and enoyl-CoA-hydratase-1 resulted in the inhibition of the Krebs cycle and enhanced pyruvate shunting toward the glycolytic pathway. To assess mitochondrial mass in vivo, co-localization of green fluorescent protein and MitoTracker fluorescence was detected by intravital microscopy. Quantitative analysis of fluorescence intensity showed that L-NMMA-treated animals exhibited lower fluorescence of MitoTracker in microvascular endothelia as a result of reduced mitochondrial mass. These findings provide conclusive and unbiased evidence that mitochondriopathy represents an early manifestation of endothelial dysfunction, shifting cell metabolism toward "metabolic hypoxia" through the selective depletion of both aconitase-2 and enoyl-CoA-hydratase-1. These findings may contribute to an early preclinical diagnosis of endothelial dysfunction.

Deficiency of nitric oxide (NO) represents a consistent manifestation of endothelial dysfunction (ECD), and the accumulation of asymmetric dimethylarginine occurs early in renal disease. Here, we confirmed in vitro and in vivo the previous finding that a fragment of collagen XVIII, endostatin, was upregulated by chronic inhibition of NO production and sought to support a hypothesis that primary ECD contributes to nephrosclerosis in the absence of other profibrotic factors. To emulate more closely the indolent course of ECD, the study was expanded to an in vivo model with NG-monomethyl-l-arginine(l-NMMA; mimics effects of asymmetric dimethylarginine) administered to mice in the drinking water at subpressor doses of 0.3 and 0.8 mg/ml for 3–6 mo. This resulted in subtle but significant morphological alterations detected in kidneys of mice chronically treated with l-NMMA: 1) consistent perivascular expansion of interstitial matrix components at the inner stripe of the outer medulla and 2) collagen XVIII/endostatin abundance. Ultrastructural abnormalities were detected in l-NMMA-treated mice: 1) increased activity of the interstitial fibroblasts; 2) occasional detachment of endothelial cells from the basement membrane; 3) splitting of the vascular basement membrane; 4) focal fibrosis; and 5) accumulation of lipofuscin by interstitial fibroblasts. Preembedding labeling of microvasculature with anti-CD31 antibodies showed infiltrating leukocytes and agglomerating platelets attaching to the visibly intact or denuded capillaries. Collectively, the data indicate that the mouse model of subpressor chronic administration of l-NMMA is not a robust one (endothelial pathology visible only ultrastructurally), and yet it closely resembles the natural progression of endothelial dysfunction, microvascular abnormalities, and associated tubulointerstitial scarring.

Glomerular maturation increases from immature superficial to advanced juxtamedullary nephrons, while nephrogenesis continues postnatally in porcine kidneys. Endothelial NOS, eNOS, shows significant postnatal renal developmental regulation, perhaps mediated by Angiotensin II (AII). The objective was to compare eNOS mRNA gene expression between superficial and juxtamedullary glomeruli obtained from piglets and adult pigs utilizing laser capture microdissection during basal conditions and, to determine the role of the AII AT1 receptor, AT1, after chronic AT1 inhibition (AT1X) with candesartan. Superficial glomerular eNOS expression was lowest in newborns (NB) and at 7 days, and was highest in 14, 21 day old piglets and adults. Juxtamedullary glomerular eNOS, while similar in NB, 14, 21 day and adult, dipped to the lowest level at 7 days. Juxtamedullary glomerular eNOS expression in the NB was 7 fold greater than in superficial glomeruli. AT1X did not change eNOS expression in adult glomeruli. AT1X significantly reduced NB eNOS expression in both superficial, 90+/-10%, and juxtamedullary glomeruli, 89+/-5% respectively. In conclusion, eNOS gene expression demonstrates significant differences between NB superficial and juxtamedullary glomeruli, significant postnatal developmental regulation of both glomerular locations, and this expression may be mediated in the NB by AII via the AT1 receptor.

In the intricate maze of proposed mechanisms, modifiers, modulators, and sensitizers for acute kidney injury (AKI) and diverse causes inducing it, this chapter focuses on several common and undisputable strands which do exist.Structurally, the loss of the brush border, desquamation of tubular epithelial cells, and obstruction of the tubular lumen are commonly observed, albeit to various degrees. These morphologic hallmarks of AKI are accompanied by functional defects, most consistently reflected in the decreased glomerular filtration rate and variable degree of reduction in renal blood flow, accompanied by changes in the microcirculation. Although all renal resident cells participate in AKI, the brunt falls on the epithelial and endothelial cells, the fact that underlies the development of tubular epithelial and vascular compromise.This chapter further summarizes the involvement of several cell organelles in AKI: mitochondrial involvement in perturbed energy metabolism, lysosomal involvement in degradation of misfolded proteins and damaged organelles, and peroxisomal involvement in the regulation of oxidative stress and metabolism, all of which become defective. Common molecular pathways are engaged in cellular stress response and their roles in cell death or survival. The diverse families of nephrotoxic medications and the respective mechanisms they induce AKI are discussed. The mechanisms of action of some nephrotoxins are analysed, and also of the preventive therapies of ischaemic or pharmacologic pre-conditioning.An emerging concept of the systemic inflammatory response triggered by AKI, which can potentially aggravate the local injury or tend to facilitate the repair of the kidney, is presented. Rational therapeutic strategies should be based on these well-established pathophysiological hallmarks of AKI.

Sepsis is a systemic inflammatory syndrome induced by bacterial infection that can lead to multiorgan failure. Endothelial surface glycocalyx (ESG) decorating the inner wall of blood vessels is a regulator of multiple vascular functions. Here, we tested a hypothesis that patchy degradation of ESG occurs early in sepsis and is a result of exocytosis of lysosome-related organelles. Time-lapse video microscopy revealed that exocytosis of Weibel-Palade bodies and secretory lysosomes occurred a few minutes after application of lipopolysaccharides to endothelial cells. Two therapeutic maneuvers, a nitric oxide intermediate, NG-hydroxy-l-arginine, and culture media conditioned by endothelial progenitor cells reduced the motility of lysosome-related organelles. Confocal and stochastic optical reconstruction microscopy confirmed the patchy loss of ESG simultaneously with the exocytosis of lysosome-related organelles and Weibel-Palade bodies in cultured endothelial cells and mouse aorta. The loss of ESG was blunted by pretreatment with NG-hydroxy-l-arginine or culture media conditioned by endothelial progenitor cells. Moreover, these treatments resulted in a significant reduction in deaths of septic mice. Our data support the hypothesis assigning to stress-induced exocytosis of these organelles the role of a hair-trigger for local degradation of ESG that initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later rates of morbidity and mortality.

We are witnessing the emergence of a novel type of biological regulation, namely, the communication between cells via their secreted substances, the secretome. This brief overview is based on the available published data and our own experience. We discuss three vignettes illustrating the importance of communication via the secretome: (1) the secretome of stem cells and its effects in sepsis and systemic inflammatory response; (2) the profibrotic secretomes partially responsible for development of fibrotic complications; and (3) the contribution of senescence-associated secretory products to the propagation of the senescence phenotype. Considering the richness of secretomes of different cells under diverse conditions, it becomes imperative to gain insights into their individual components in an attempt to harness cell secretomes for therapeutic purposes.

We recently demonstrated the use of in vitro expanded kidney-derived mesenchymal stem cells (KMSC) protected peritubular capillary endothelial cells in acute renal ischemia-reperfusion injury. Herein, we isolated and characterized microparticles (MPs) from KMSC. We investigated their in vitro biologic effects on human endothelial cells and in vivo renoprotective effects in acute ischemia-reperfusion renal injury. MPs were isolated from the supernatants of KMSC cultured in anoxic conditions in serum-deprived media for 24 hours. KMSC-derived MPs demonstrated the presence of several adhesion molecules normally expressed on KMSC membranes, such as CD29, CD44, CD73, alpha 4, 5, and 6 integrins. Quantitative real time PCR confirmed the presence of 3 splicing variants of VEGF-A (120, 164, 188), bFGF and IGF-1 in isolated MPs. MPs labeled with PKH26 red fluorescence dye were incorporated by cultured human umbilical vein endothelial cells (HUVEC) via surface molecules such as CD44, CD29, and alpha 4, 5, and 6 integrins. MP dose dependently improved in vitro HUVEC proliferation and promoted endothelial tube formation on growth factor reduced Matrigel. Moreover, apoptosis of human microvascular endothelial cell was inhibited by MPs. Administration of KMSC-derived MPs into mice with acute renal ischemia was followed by selective engraftment in ischemic kidneys and significant improvement in renal function. This was achieved by improving proliferation, of peritubular capillary endothelial cell and amelioration of peritubular microvascular rarefaction. Our results support the hypothesis that KMSC-derived MPs may act as a source of proangiogenic signals and confer renoprotective effects in ischemic kidneys.

NFAT5 is a transcription factor that protects the kidney from hypertonic stress and also is activated by hypoxia. We hypothesized that NFAT5 mitigates the extent of renal damage induced by ischemia–reperfusion injury (IRI). Mice were subjected to IRI by unilateral clamping of the left renal pedicle for 30 minutes followed by reperfusion. After 3 hours of reperfusion, the level of NFAT5 mRNA was similar in contralateral and clamped kidneys. However, after 48 hours, NFAT5 mRNA accumulation increased ≈3-fold in both outer medulla and medullary thick ascending limb tubules. NFAT1 levels were elevated at 3 hours but did not increase further at 48 hours. Mice were then either pretreated for 72 hours with an intrarenal injection of a lentivirus short-hairpin RNA construct to silence NFAT5 (enhanced green fluorescent protein-U6-N5-ex8) or a control vector (enhanced green fluorescent protein-U6) before induction of IRI. Neutrophil gelatinase–associated lipocalin and kidney ischemia molecule-1 mRNA levels increased after IRI and further increased after knockdown of NFAT5, suggesting that silencing of NFAT5 exacerbates renal damage during IRI. In contrast, silencing of NFAT1 had no effect on the levels of neutrophil gelatinase–associated lipocalin or kidney ischemia molecule-1 mRNA. Hematoxylin and eosin staining revealed patchy denudation of renal epithelial cells and tubular dilation when NFAT5 was silenced. The number of TUNEL-positive cells in the outer and inner medulla of the clamped kidney increased nearly 2-fold after knockdown of NFAT5 and was associated with an increase in the number of caspase-3–positive cells. Collectively, the data suggest that NFAT5 is part of a protective mechanism that limits renal damage induced by IRI.

Sirtuin 1 (SIRT1) depletion in vascular endothelial cells mediates endothelial dysfunction and premature senescence in diverse cardiovascular and renal diseases. However, the molecular mechanisms underlying these pathologic effects remain unclear. Here, we examined the phenotype of a mouse model of vascular senescence created by genetically ablating exon 4 of Sirt1 in endothelial cells (Sirt1(endo-/-)). Under basal conditions, Sirt1(endo-/-) mice showed impaired endothelium-dependent vasorelaxation and angiogenesis, and fibrosis occurred spontaneously at low levels at an early age. In contrast, induction of nephrotoxic stress (acute and chronic folic acid-induced nephropathy) in Sirt1(endo-/-) mice resulted in robust acute renal functional deterioration followed by an exaggerated fibrotic response compared with control animals. Additional studies identified matrix metalloproteinase-14 (MMP-14) as a target of SIRT1. In the kidneys of Sirt1(endo-/-) mice, impaired angiogenesis, reduced matrilytic activity, and retention of the profibrotic cleavage substrates tissue transglutaminase and endoglin accompanied MMP-14 suppression. Furthermore, restoration of MMP-14 expression in SIRT1-depeleted mice improved angiogenic and matrilytic functions of the endothelium, prevented renal dysfunction, and attenuated nephrosclerosis. Our findings establish a novel mechanistic molecular link between endothelial SIRT1 depletion, downregulation of MMP-14, and the development of nephrosclerosis.

Aims: We examined that (a) how the endotoxic stress affects peroxisomal function and autophagic degradation of peroxisomes—pexophagy, (b) how a superimposed dysfunction of lysosomes and pexophagy modifies responses to lipopolysaccharide (LPS), and (c) the mechanisms of peroxisomal contribution to renal injury. To accomplish this, we used lysosome-defective Lyst-mice in vivo and primary endothelial cells in vitro, and compared the responses with wild-type (WT) littermates. Results: LPS induced pexophagic degradation, followed by proliferation of peroxisomes in WT mice, which was abolished in Lyst-mice. Lyst-mice exhibited impaired activation of catalase, which together with preserved hydrogen peroxide-generating β-oxidation resulted in redox disequilibrium. LPS treatment induced a heightened inflammatory response, increased oxidative damage, and aggravated renal injury in Lyst-mice. Similarly, as in vivo, LPS-activated lysosomal (LYS) pexophagy and transiently repressed peroxisomes in vitro, supported by reduced peroxisomal density in the vicinity of lysosomes. Peroxisomal dynamics was also abolished in lysosome-defective cells, which accumulated peroxisomes with compromised functions and intraorganellar redox imbalance. Innovation: We demonstrated that pexophagy is a default response to endotoxic injury. However, when LYS dysfunction (a frequent companion of chronic diseases) is superimposed, recycling and functioning of peroxisomes are impaired, and an imbalance between hydrogen peroxide-generating β-oxidation and hydrogen peroxide-detoxifying catalase ensues, which ultimately results in peroxisomal burnout. Conclusion: Our data strongly suggest that pexophagy, a cellular mechanism per se, is essential in functional maintenance of peroxisomes during LPS exposure. Inhibition of pexophagy results in accumulation of impaired peroxisomes, redox disequilibrium, and aggravated renal damage. Antioxid. Redox Signal. 19, 211–230.

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.

The list of signals sent by an injured organ to systemic circulation, so-called danger signals, is growing to include multiple metabolites and secreted moieties, thus revealing a highly complex and integrated network of interlinked systemic proinflammatory and proregenerative messages. Emerging new data indicate that, apart from the well established local inflammatory response to AKI, danger signaling unleashes a cascade of precisely timed, interdependent, and intensity-gradated mediators responsible for development of the systemic inflammatory response. This fledgling realization of the importance of the systemic inflammatory response to the localized injury and inflammation is at the core of this brief overview. It has a potential to explain the additive effects of concomitant diseases or preexisting chronic conditions that can prime the systemic inflammatory response and exacerbate it out of proportion to the actual degree of acute kidney damage. Although therapies for ameliorating AKI per se remain limited, a potentially powerful strategy that could reap significant benefits in the future is to modulate the intensity of danger signals and consequently the systemic inflammatory response, while preserving its intrinsic proregenerative stimuli.

Nitric oxide (NO) vasodilation critically modulates renal hemodynamics in the neonate compared with the adult. Based on the postnatal expression pattern of renal neuronal nitric oxide synthase (nNOS), the hypothesis was that nNOS is the major NOS isoform regulating renal hemodynamics in the immature, but not mature, kidney.NOS inhibitors did not alter mean arterial pressure (MAP) in either group. Intrarenal S-methyl-L-thiocitrulline (L-SMTC) in newborns significantly reduced renal blood flow (RBF) 38 ± 4%, glomerular filtration rate (GFR) 42 ± 6%, and increased renal vascular resistance (RVR) 37 ± 7%, whereas intrarenal L-nitro-arginine methyl ester (L-NAME) affected RBF, GFR, and RVR equivalent to L-SMTC treatment. When L-NAME was administered after L-SMTC treatment, newborn renal hemodynamic changes were not further altered from what was observed when L-SMTC was administered alone. In contrast, in the adult, only intrarenal L-NAME, and not L-SMTC, affected renal hemodynamic responses.In conclusion, these studies demonstrate that nNOS is an important regulator of renal hemodynamics in the newborn kidney, but not in the adult.Experiments compared renal hemodynamic responses with intrarenal infusion of L-NAME, an inhibitor of all NOS isoforms, with the selective nNOS inhibitor L-SMTC in the newborn piglet and the adult pig.

Exocytosis of Weibel-Palade bodies (WPB) represents a distinct response of endothelial cells to stressors, and local release of WPB contents leads to systemic escalation of this response. We synthesized a glycine-(Nα-Et)lysine-proline-arginine (ITF 1697) peptide that has a potential to inhibit exocytosis of WPB and protect microcirculation. Here, we confirmed an inhibitory effect of ITF 1697 using intravital videoimaging and point-tracking of individual organelles. In an in vivo study, mice were implanted with Alzet osmotic pumps (10 μg ITF 1697·kg−1·min−1 at volume of 1 μl/h) and subjected to renal ischemia (IRI). IRI resulted in marked renal injury and elevation of serum creatinine in mice treated with a vehicle. In contrast, renal injury and elevation of creatinine were significantly ameliorated in mice subjected to IRI and receiving ITF 1697. ITF 1697 prevented a systemic response to IRI: a significant surge in the levels of eotaxin and IL-8 (KC; both components of WPB), IL-1α, IL-1β, and RANTES was all prevented or blunted by the administration of ITF 1697, whereas the levels of an anti-inflammatory, IL-10, and macrophage inflammatory protein-1α were upregulated in ITF 1697-treated animals. En face staining of aortic endothelial cells showed that WPB were depleted after 40–180 min post-IRI, and this was significantly blunted in aortic preparations obtained from mice treated with ITF 1697. WPB exocytosis contributed to IRI-associated mobilization of endothelial progenitor cells and hematopoietic stem cells, and ITF 1697 blunted their mobilization. Unexpectedly, 1 mo after IRI, mice treated with ITF 1697 showed a significantly more pronounced degree of scarring than nontreated animals. In conclusion, 1) application of ITF 1697 inhibits exocytosis of WPB and IRI; 2) the systemic inflammatory response of IRI is in part due to the exocytosis of WPB and its blockade blunts it; and 3) ITF 1697 improves short-term renal function after IRI, but not the long-term fibrotic complications.

Publisher Summary With the rapidly increasing demand for stem cells, the problems related to their storage and preservation, maintenance, and expansion become ever more acute. The requirements for stem cell banking would place the need for preserving quiescence and stemness at the top of the list, whereas the requirements for transplantation of stem and progenitor cells would give preference to the possibility of increasing the mass of stem cells without losing their properties. The constellation of different requirements should orbit around the physiological ways of preserving stem cells, i.e. stem cell niches. The niches are defined as the umbrella microenvironment supporting cell attachment and quiescence by sheltering cells from proliferation and differentiation signals, enhancing cell survival, regulating stem cell division and renewal, and coordinating the population of resident stem cells to meet the actual requirements of an organ. The way in which these diverse functions are accomplished by the niches may vary from one type of stem cells and their niche to another, yet the general outline is preserved. It includes specific components of the extracellular matrix (ECM) that create a scaffold and a boundary to the niche, cell-cell and cell-matrix adhesion molecules, locally stored growth and/or quiescence factors, and a set of instructions for proliferation and/or migration that can be triggered on demand. The vicinity of vascular beds, often covered with sinusoidal endothelial cells, provides a portal for entry into the circulation.

Putative endothelial progenitor cells play a role in organ regeneration, and their incompetence may be important in the development of chronic kidney disease. The mechanisms of this incompetence are broad and range from poor mobilization, viability, and engraftment to impaired differentiation into mature endothelial cells. By contrasting the role of endothelial progenitor cells in tissue regeneration with their developing incompetence in chronic kidney disease, we emphasize the importance of designing rational pharmacologic strategies to tackle such incompetence in the broader search for therapies to attenuate chronic disease.

Unilateral ureteral obstruction (UUO), a model of tubulointerstitial scarring (TIS), has a propensity toward regeneration of renal parenchyma after release of obstruction (RUUO). No information exists on the contribution of stem cells to this process. We performed UUO in FVB/N mice, reversed it after 10 days, and examined kidneys 3 wk after RUUO. UUO resulted in attenuation of renal parenchyma. FACS analysis of endothelial progenitor (EPC), mesenchymal stem (MSC) and hematopoietic stem (HSC) cells obtained from UUO kidneys by collagenase-dispersed single-cell suspension showed significant increase in EPC, MSC, and HSC compared with control. After RUUO cortical parenchyma was nearly restored, and TIS score improved by 3 wk. This reversal process was associated with return of stem cells toward baseline level. When animals were chronically treated with nitric oxide synthase (NOS) inhibitor at a dose that did not induce hypertension but resulted in endothelial dysfunction, TIS scores were not different from control UUO, but EPC number in the kidney decreased significantly; however, parenchymal regeneration in these mice was similar to control. Blockade of CXCR4-mediated engraftment resulted in dramatic worsening of UUO and RUUO. Similar results were obtained in caveolin-1-deficient but not -overexpressing mice, reflecting the fact that activation of CXCR4 occurs in caveolae. The present data show increase in EPC, HSC, and MSC population during UUO and a tendency for these cells to decrease to control level during RUUO. These processes are minimally affected by chronic NOS inhibition. Blockade of CXCR4-stromal cell-derived factor-1 (SDF-1) interaction by AMD3100 or caveolin-1 deficiency significantly reduced the UUO-associated surge in stem cells and prevented parenchymal regeneration after RUUO. We conclude that the surge in stem cell accumulation during UUO is a prerequisite for regeneration of renal parenchyma.

We sought to characterize a minor renal cryoinjury that allows investigation into renal damage processes and subsequent endogenous repair mechanisms. To achieve this, we induced a small cryoinjury to mice, in which the transient superficial application of a liquid nitrogen-cooled cryoprobe to the exposed kidney induces a localized lesion that did not impair renal function. The resulting cryoinjury was examined by immunohistochemistry and Laser-Doppler flowmetry. Within hours of cryoinjury induction, tubular and vascular necrotic damage was observed, while blood flow in the directly injured area was reduced by 65%. The injured area demonstrated a peak in tubular and perivascular cell proliferation at 4 days postinjury, while apoptosis and fibrosis peaked at day 7. Infiltration of macrophages into the injury was first observed at day 4, and peaked at day 7. Vascular density in the direct injured area was lowest at day 7. As compared to the direct injured area, the (peripheral) penumbral region surrounding the directly injured area demonstrated enhanced cellular proliferation (2.5-6-fold greater), vascular density (1.6-2.9 fold greater) and blood perfusion (twofold greater). After 4 weeks, the area of damage was reduced by 73%, fibrosis decreased by 50% and blood flow in the direct injured area was reestablished by 63% with almost complete perfusion restoration in the injury's penumbral region. In conclusion, kidney cryoinjury provides a flexible facile model for the study of renal damage and associated endogenous repair processes.

Background The assessment of hydration status remains a challenging task in hemodialysis (HD) management. There are only limited data available on the relevance of clinical decisions in the estimation of dialysis overhydration (OH). The objective of this study was to examine the significance of clinical judgment in the assessment of pre-dialysis OH. Methods We compared the performance of three methods of OH assessment: (1) clinical judgment guided by a single clinical examination with (2) multifrequency bioimpedance analysis (BIA) and (3) complex systematic clinical approach. We additionally studied the associations of these methods with selected laboratory and imaging parameters. Results Any of the single parameters alone reached a sufficient level of accuracy for reliable prediction of OH. Clinical judgment was the single most important factor in OH estimation, and also had the highest contribution when in combination with other parameters. BIA reliably measured extracellular fluid, but the automatically calculated OHBIA exhibited a substantial degree of inaccuracy that precludes the use of BIA as a standard at present. The combination of clinical judgment with additional clinical parameters had the highest prediction accuracy for OH. Among the parameters studied, vena cava collapsibility index and calf circumference showed the strongest association with OH. Echocardiography, cardiothoracic index, atrial natriuretic peptide levels and spirometry did not have acceptable sensitivity. Conclusion The systematic clinical approach combining physician and patient inputs, laboratory and imaging data enables an individualized decision and a superior accuracy in OH assessment. Electronic supplementary material The online version of this article (doi:10.1007/s10157-012-0745-9) contains supplementary material, which is available to authorized users.