Lloyd G. Cantley

Bone marrow stem cells contribute to repair of the ischemically injured renal tubule

The paradigm for recovery of the renal tubule from acute tubular necrosis is that surviving cells from the areas bordering the injury must migrate into the regions of tubular denudation and proliferate to re-establish the normal tubular epithelium. However, therapies aimed at stimulating these events have failed to alter the course of acute renal failure in human trials. In the present study, we demonstrate that Lin-Sca-1+ cells from the adult mouse bone marrow are mobilized into the circulation by transient renal ischemia and home specifically to injured regions of the renal tubule. There they differentiate into renal tubular epithelial cells and appear to constitute the majority of the cells present in the previously necrotic tubules. Loss of stem cells following bone marrow ablation results in a greater rise in blood urea nitrogen after renal ischemia, while stem cell infusion after bone marrow ablation reverses this effect. Thus, therapies aimed at enhancing the mobilization, propagation, and/or delivery of bone marrow stem cells to the kidney hold potential as entirely new approaches for the treatment of acute tubular necrosis.

Distinct Macrophage Phenotypes Contribute to Kidney Injury and Repair

The ischemically injured kidney undergoes tubular cell necrosis and apoptosis, accompanied by an interstitial inflammatory cell infiltrate. In this study, we show that iNos-positive proinflammatory (M1) macrophages are recruited into the kidney in the first 48 hours after ischemia/reperfusion injury, whereas arginase 1- and mannose receptor-positive, noninflammatory (M2) macrophages predominate at later time points. Furthermore, depletion of macrophages before ischemia/reperfusion diminishes kidney injury, whereas depletion at 3 to 5 days after injury slows tubular cell proliferation and repair. Infusion of Ifnγ-stimulated, bone marrow-derived macrophages into macrophage-depleted mice at the time of kidney reperfusion restored injury to the level seen without macrophage depletion, suggesting that proinflammatory macrophages worsen kidney damage. In contrast, the appearance of macrophages with the M2 phenotype correlated with the proliferative phase of kidney repair. In vitro studies showed that IFNγ-stimulated, proinflammatory macrophages begin to express markers of M2 macrophages when cocultured with renal tubular cells. Moreover, IL-4-stimulated macrophages with an M2 phenotype, but not IFNγ-stimulated proinflammatory macrophages, promoted renal tubular cell proliferation. Finally, tracking fluorescently labeled, IFNγ-stimulated macrophages that were injected after injury showed that inflammatory macrophages can switch to an M2 phenotype in the kidney at the onset of kidney repair. Taken together, these studies show that macrophages undergo a switch from a proinflammatory to a trophic phenotype that supports the transition from tubule injury to tubule repair.

Stromal Cells Protect against Acute Tubular Injury via an Endocrine Effect

Emerging evidence suggests that the intravenous injection of bone marrow-derived stromal cells (BMSC) improves renal function after acute tubular injury, but the mechanism of this effect is controversial. In this article, we confirm that intravenous infusion of male BMSC reduced the severity of cisplatin-induced acute renal failure in adult female mice. This effect was also seen when BMSC (or adipocyte-derived stromal cells (AdSC)), were given by intraperitoneal injection. Infusion of BMSC enhanced tubular cell proliferation after injury and decreased tubular cell apoptosis. Using the Y chromosome as a marker of donor stromal cells, examination of multiple kidney sections at one or four days after cell infusion failed to reveal any examples of stromal cells within the tubules, and only rare examples of stromal cells within the renal interstitium. Furthermore, conditioned media from cultured stromal cells induced migration and proliferation of kidney-derived epithelial cells and significantly diminished cisplatin-induced proximal tubule cell death in vitro. Intraperitoneal administration of this conditioned medium to mice injected with cisplatin diminished tubular cell apoptosis, increased survival, and limited renal injury. Thus, marrow stromal cells protect the kidney from toxic injury by secreting factors that limit apoptosis and enhance proliferation of the endogenous tubular cells, suggesting that transplantation of the cells themselves is not necessary. Identification of the stromal cell-derived protective factors may provide new therapeutic options to explore in humans with acute kidney injury.

Phosphoinositide 3-Kinase Regulates Phospholipase Cγ-mediated Calcium Signaling

It has been demonstrated that the lipid products of the phosphoinositide 3-kinase (PI3K) can associate with the Src homology 2 (SH2) domains of specific signaling molecules and modify their actions. In the current experiments, phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) was found to bind to the C-terminal SH2 domain of phospholipase Cγ (PLCγ) with an apparent K d of 2.4 μm and to displace the C-terminal SH2 domain from the activated platelet-derived growth factor receptor (PDGFR). To investigate the in vivorelevance of this observation, intracellular inositol trisphosphate (IP3) generation and calcium release were examined in HepG2 cells expressing a series of PDGFR mutants that activate PLCγ with or without receptor association with PI3K. Coactivation of PLCγ and PI3K resulted in an ∼40% increase in both intracellular IP3generation and intracellular calcium release as compared with selective activation of PLCγ. Similarly, the addition of wortmannin or LY294002 to cells expressing the wild-type PDGFR inhibited the release of intracellular calcium. Thus, generation of PtdIns-3,4,5-P3by receptor-associated PI3K causes an increase in IP3production and intracellular calcium release, potentially via enhanced PtdIns-4,5-P2 substrate availability due to PtdIns-3,4,5-P3-mediated recruitment of PLCγ to the lipid bilayer.

Phosphorylation-Dependent Paxillin-ERK Association Mediates Hepatocyte Growth Factor-Stimulated Epithelial Morphogenesis

Activation of the hepatocyte growth factor (HGF) receptor c-met results in the regulation of cell-matrix interactions, including the MAPK-dependent stimulation of epithelial cell morphogenesis. In the present study we demonstrate that HGF stimulates the localization of ERK to sites of cell-matrix interactions and that this is mediated by the tyrosine phosphorylation-dependent association of inactive ERK and the focal adhesion complex protein paxillin. In addition, paxillin was found to associate with the upstream MAP kinases Raf and MEK, resulting in a complex that can mediate localized ERK activation. Mutation of the ERK binding site in paxillin prevented HGF-stimulated ERK-paxillin association and eliminated HGF-induced cell spreading and branching process formation. These experiments reveal that paxillin-dependent ERK activation at sites of cell-matrix interaction is critical for HGF-stimulated epithelial morphogenesis.

Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1

Polycystic kidney disease (ADPKD) results from failure of the kidney to properly maintain three-dimensional structure after loss of either polycystin-1 or -2. Mice with kidney selective inactivation of Pkd1 during embryogenesis develop profound renal cystic disease and die from renal failure within 3 weeks of birth. In this model, cysts form exclusively from cells in which Cre recombinase is active, but the apparent pace of cyst expansion varies by segment and cell type. Intercalated cells do not participate in cyst expansion despite the presence of cilia up to at least postnatal day 21. Cystic segments show a persistent increase in proliferation as determined by bromodeoxyuridine (BrdU) incorporation; however, the absolute proliferative index is dependent on the underlying proliferative potential of kidney tubule cells. Components of the extracellular regulated kinase (MAPK/ERK) pathway from Ras through MEK1/2 and ERK1/2 to the effector P90(RSK) are activated in both perinatal Pkd1 and adult Pkd2 ortholgous gene disease models. The pattern of MAPK/ERK activation is focal and does not correlate with the pattern of active proliferation identified by BrdU uptake. The possibility of a causal relationship between ERK1/2 activation and cyst cell proliferation was assessed in vivo in the acute perinatal Pkd1 model of ADPKD using MEK1/2 inhibitor U0126. U0126 treatment had no effect on progression of cyst formation in this model at doses sufficient to reduce phospho-ERK1/2 in cystic kidneys. Cysts in ADPKD exhibit both increased proliferation and activation of MAPK/ERK, but cyst growth is not prevented by inhibition of ERK1/2 activation.

Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.

Hepatocyte growth factor (HGF) modulates cell adhesion, migration, and branching morphogenesis in cultured epithelial cells, events that require regulation of cell-matrix interactions. Using mIMCD-3 epithelial cells, we studied the effect of HGF on the focal adhesion proteins, focal adhesion kinase (FAK) and paxillin and their association. HGF was found to increase the tyrosine phosphorylation of paxillin and to a lesser degree FAK. In addition, HGF induced association of paxillin and activated ERK, correlating with a gel retardation of paxillin that was prevented with the ERK inhibitor U0126. The ability of activated ERK to phosphorylate and induce gel retardation of paxillin was confirmed in vitro in both full-length and amino-terminal paxillin. Several potential ERK phosphorylation sites in paxillin flank the paxillin-FAK association domains, so the ability of HGF to regulate paxillin-FAK association was examined. HGF induced an increase in paxillin-FAK association that was inhibited by pretreatment with U0126 and reproduced by in vitro phosphorylation of paxillin with ERK. The prevention of the FAK-paxillin association with U0126 correlated with an inhibition of the HGF-mediated FAK tyrosine phosphorylation and inhibition of HGF-dependent cell spreading and adhesion. An examination of cellular localization of FAK and paxillin demonstrated that HGF caused a condensation of focal adhesion complexes at the leading edges of cell processes and FAK-paxillin co-localization in these large complexes. Thus, these data suggest that HGF can induce serine/threonine phosphorylation of paxillin most probably mediated directly by ERK, resulting in the recruitment and activation of FAK and subsequent enhancement of cell spreading and adhesion.

The Lipid Products of Phosphoinositide 3-Kinase Increase Cell Motility through Protein Kinase C

Phosphoinositide 3-kinase has been implicated as an activator of cell motility in a variety of recent studies, yet the role of its lipid product, phosphatidylinositol 1,4,5-trisphosphate (PtdIns-3,4,5-P3), has yet to be elucidated. In this study, three independent preparations of PtdIns-3,4,5-P3 were found to increase the motility of NIH 3T3 cells when examined utilizing a microchemotaxis chamber. Dipalmitoyl L-α-phosphatidyl-D-myo-inositol 3,4,5-triphosphate (Di-C16-PtdIns-3,4,5-P3) also produced actin reorganization and membrane ruffling. Cells pretreated with 12-O-tetradecanoylphorbol-13-acetate to cause down-regulation of protein kinase C (PKC) exhibited complete inhibition of cell motility induced by Di-C16-PtdIns-3,4,5-P3. These results are consistent with previous observations that PtdIns-3,4,5-P3 activates Ca2+-independent PKC isoforms in vitro and in vivo and provide the first demonstration of an in vivo role for the lipid products of the phosphoinositide 3-kinase. PtdIns-3,4,5-P3 appears to directly initiate cellular motility via activation of a PKC family member.

The impact of aging on kidney repair

The process of normal aging affects organ homeostasis as well as responses to acute and chronic injury. In view of the rapid growth in the elderly population, it is increasingly important for us to develop a mechanistic understanding of how these age-dependent changes can impact the susceptibility and response of the kidney to injurious stimuli. In this overview, we focus on the current understanding of those mechanisms by reviewing how cellular changes in the aging kidney might lead to a diminished proliferative reserve, an increased tendency for apoptosis, alterations in growth factor profiles, and changes in potential progenitor and immune cell functions. A better understanding of these processes may help us to define new targets for studying kidney repair and could ultimately lead to new therapeutic strategies that are specifically tailored for treatment of the elderly population.

Adult stem cells in the repair of the injured renal tubule

The capacity of the kidney to regenerate functional tubules following episodes of acute injury is an important determinant of patient morbidity and mortality in the hospital setting. After severe injury or repeated episodes of injury, kidney recovery can be significantly impaired or even fail completely. Although significant advances have been made in the clinical management of such cases, there is no specific therapy that can improve the rate or effectiveness of the repair process. Recent studies have indicated that adult stem cells, either in the kidney itself or derived from the bone marrow, could participate in this repair process and might therefore be utilized clinically to treat acute renal failure. This review will focus on our current understanding of these stem cells, the controversies surrounding their in vivo capacity to repopulate the renal tubule, and further investigations that will be required before stem cell therapy can be considered for use in the clinical setting.