Brian B. Ratliff

HMGB1 in renal ischemic injury

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 H(2)O(2) (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 EPs 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.

Dysfunctional Endothelial Progenitor Cells in Chronic Kidney Disease

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.

Endothelial Sirtuin 1 Deficiency Perpetrates Nephrosclerosis through Downregulation of Matrix Metalloproteinase-14: Relevance to Fibrosis of Vascular Senescence

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.

Adriamycin Nephropathy : A Failure of Endothelial Progenitor Cell-Induced Repair

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-1alpha and -beta 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.

Messengers without Borders: Mediators of Systemic Inflammatory Response in AKI

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.

Postobstructive regeneration of kidney is derailed when surge in renal stem cells during course of unilateral ureteral obstruction is halted.

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.

Endothelial progenitors encapsulated in bioartificial niches are insulated from systemic cytotoxicity and are angiogenesis competent

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.

Interaction between uric acid and HMGB1 translocation and release from endothelial cells

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.

Renal capsule as a stem cell niche

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/mm(2), 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.

High-mobility group box 1 is a novel deacetylation target of Sirtuin1

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 pro-inflammatory 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.