Environmental exposure to arsenic and chromium in children is associated with kidney injury molecule-1.

Environmental hazards from natural or anthropological sources are widespread, especially in the north-central region of Mexico. Children represent a susceptible population due to their unique routes of exposure and special vulnerabilities. In this study we evaluated the association of exposure to environmental kidney toxicants with kidney injury biomarkers in children living in San Luis Potosi (SLP), Mexico. A cross-sectional study was conducted with 83 children (5-12 years of age) residents of Villa de Reyes, SLP. Exposure to arsenic, cadmium, chromium, fluoride and lead was assessed in urine, blood and drinking water samples. Almost all tap and well water samples had levels of arsenic (81.5%) and fluoride (100%) above the permissible levels recommended by the World Health Organization. Mean urine arsenic (45.6ppb) and chromium (61.7ppb) were higher than the biological exposure index, a reference value in occupational settings. Using multivariate adjusted models, we found a dose-dependent association between kidney injury molecule-1 (KIM-1) across chromium exposure tertiles [(T1: reference, T2: 467pg/mL; T3: 615pg/mL) (p-trend=0.001)]. Chromium upper tertile was also associated with higher urinary miR-200c (500 copies/µl) and miR-423 (189 copies/µL). Arsenic upper tertile was also associated with higher urinary KIM-1 (372pg/mL). Other kidney injury/functional biomarkers such as serum creatinine, glomerular filtration rate, albuminuria, neutrophil gelatinase-associated lipocalin and miR-21 did not show any association with arsenic, chromium or any of the other toxicants evaluated. We conclude that KIM-1 might serve as a sensitive biomarker to screen children for kidney damage induced by environmental toxic agents.

Age-related differences in susceptibility to cisplatin-induced renal toxicity.

Limited experimental models exist to assess drug toxicity in pediatric populations. We recently reported how a multi-age rat model could be used for pre-clinical studies of comparative drug toxicity in pediatric populations. The objective of this study was to expand the utility of this animal model, which previously demonstrated an age-dependent sensitivity to the classic nephrotoxic compound, gentamicin, to another nephrotoxicant, namely cisplatin (Cis). Sprague-Dawley rats (10, 25, 40 and 80 days old) were injected with a single dose of Cis (0, 1, 3 or 6 mg kg(-1) i.p.). Urine samples were collected prior and up to 72 h after treatment in animals that were >or= 25 days old. Several serum, urinary and 'omic' injury biomarkers as well as renal histopathology lesions were evaluated. Statistically significant changes were noted with different injury biomarkers in different age groups. The order of age-related Cis-induced nephrotoxicity was different than our previous study with gentamicin: 80 > 40 > 10 > 25 day-old vs 10 >or= 80 > 40 > 25-day-old rats, respectively. The increased levels of kidney injury molecule-1 (Kim-1: urinary protein/tissue mRNA) provided evidence of early Cis-induced nephrotoxicity in the most sensitive age group (80 days old). Levels of Kim-1 tissue mRNA and urinary protein were significantly correlated to each other and to the severity of renal histopathology lesions. These data indicate that the multi-age rat model can be used to demonstrate different age-related sensitivities to renal injury using mechanistically distinct nephrotoxicants, which is reflected in measurements of a variety of metabolite, gene transcript and protein biomarkers.

Kidney injury molecule-1 is an early noninvasive indicator for donor brain death-induced injury prior to kidney transplantation.

With more marginal deceased donors affecting graft viability, there is a need for specific parameters to assess kidney graft quality at the time of organ procurement in the deceased donor. Recently, kidney injury molecule-1 (Kim-1) was described as an early biomarker of renal proximal tubular damage. We assessed Kim-1 in a small animal brain death model as an early and noninvasive marker for donor-derived injury related to brain death and its sequelae, with subsequent confirmation in human donors. In rat kidney, real-time PCR revealed a 46-fold Kim-1 gene upregulation after 4 h of brain death. In situ hybridization showed proximal tubular Kim-1 localization, which was confirmed by immunohistochemistry. Also, Luminex assay showed a 6.6-fold Kim-1 rise in urine after 4 h of brain death. In human donors, 2.5-fold kidney injury molecule-1 (KIM-1) gene upregulation and 2-fold higher urine levels were found in donation after brain death (DBD) donors compared to living kidney donors. Multiple regression analysis showed that urinary KIM-1 at brain death diagnosis was a positive predictor of recipient serum creatinine, 14 days (p < 0.001) and 1 year (p < 0.05) after kidney transplantation. In conclusion, we think that Kim-1 is a promising novel marker for the early, organ specific and noninvasive detection of brain death-induced donor kidney damage.

Kidney injury molecule-1 expression in transplant biopsies is a sensitive measure of cell injury.

Kidney injury molecule-1 (KIM-1) is a specific histological biomarker for diagnosing early tubular injury on renal biopsies. In this study, KIM-1 expression was quantitated in renal transplant biopsies by immunohistochemistry and correlated with renal function. None of the 25 protocol biopsies showed detectable tubular injury on histologic examination, yet 28% had focal positive KIM-1 expression. Proximal tubule KIM-1 expression was present in all biopsies from patients with histological changes showing acute tubular damage and deterioration of kidney function. In this group, higher KIM-1 staining predicted a better outcome with improved blood urea nitrogen (BUN), serum creatinine, and estimated glomerular filtration rate (eGFR) over an ensuing 18 months. KIM-1 was expressed focally in affected tubules in 92% of kidney biopsies from patients with acute cellular rejection. By contrast, there was little positive staining for Ki-67, a cell proliferation marker, in any of the groups. KIM-1 expression significantly correlated with serum creatinine and BUN, and inversely with the eGFR on the biopsy day. Our study shows that KIM-1 staining sensitively and specifically identified proximal tubular injury and correlated with the degree of renal dysfunction. KIM-1 expression is more sensitive than histology for detecting early tubular injury, and its level of expression in transplant biopsies may indicate the potential for recovery of kidney function.

Urinary biomarkers in the early diagnosis of acute kidney injury.

A change in the serum creatinine is not sensitive for an early diagnosis of acute kidney injury. We evaluated urinary levels of matrix metalloproteinase-9 (MMP-9), N-acetyl-beta-D-glucosaminidase (NAG), and kidney injury molecule-1 (KIM-1) as biomarkers for the detection of acute kidney injury. Urine samples were collected from 44 patients with various acute and chronic kidney diseases, and from 30 normal subjects in a cross-sectional study. A case-control study of children undergoing cardio-pulmonary bypass surgery included urine specimens from each of 20 patients without and with acute kidney injury. Injury was defined as a greater than 50% increase in the serum creatinine within the first 48 h after surgery. The biomarkers were normalized to the urinary creatinine concentration at 12, 24, and 36 h after surgery with the areas under the receiver-operating characteristic curve compared for performance. In the cross-sectional study, the area under the curve for MMP-9 was least sensitive followed by KIM-1 and NAG. Combining all three biomarkers achieved a perfect score diagnosing acute kidney injury. In the case-control study, KIM-1 was better than NAG at all time points, but combining both was no better than KIM-1 alone. Urinary MMP-9 was not a sensitive marker in the case-control study. Our results suggest that urinary biomarkers allow diagnosis of acute kidney injury earlier than a rise in serum creatinine.

Kidney injury molecule-1 is an early biomarker of cadmium nephrotoxicity.

Cadmium (Cd) exposure results in injury to the proximal tubule characterized by polyuria and proteinuria. Kidney injury molecule-1 (Kim-1) is a transmembrane glycoprotein not normally detected in the mature kidney, but is upregulated and shed into the urine following nephrotoxic injury. In this study, we determine if Kim-1 might be a useful early biomarker of Cd nephrotoxicity. Male Sprague-Dawley rats were given daily injections of Cd for up to 12 weeks. Weekly urine samples were analyzed for Kim-1, protein, creatinine, metallothionein, and Clara cell protein CC-16. Significant levels of Kim-1 were detected in the urine by 6 weeks and continued to increase throughout the treatment period. This appearance of Kim-1 occurred 4-5 weeks before the onset of proteinuria, and 1-3 weeks before the appearance of metallothionein and CC-16. Higher doses of Cd gave rise to higher Kim-1 excretion. Reverse transcriptase-polymerase chain reaction (RT-PCR) expression analysis showed that Kim-1 transcript levels were increased after 6 weeks at the low dose of Cd. Immunohistochemical analysis showed that Kim-1 was present in proximal tubule cells of the Cd-treated rats. Our results suggest that Kim-1 may be a useful biomarker of early stages of Cd-induced proximal tubule injury.

Increased susceptibility of aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction.

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism underlying the increased susceptibility to injury remains undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic injury and to identify candidate genes that may mediate this response. Renal slices prepared from young (5 mo), aged ad libitum (aged-AL; 24 mo), and aged caloric-restricted (aged-CR; 24 mo) male Fischer 344 rats were subjected to ischemic stress (100% N(2)) for 0-60 min. As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts. Importantly, caloric restriction attenuated the increased susceptibility to injury. In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the viability experiments. RNA was isolated and the corresponding cDNA was hybridized to CodeLink Rat Whole Genome Bioarray slides. Subsequent gene expression analysis was performed using GeneSpring software. Using two-sample t-tests and a twofold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction, including claudin-7, kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7). Claudin-7 gene expression peaked at 18 mo; however, increased protein expression in certain tubular epithelial cells was seen at 24 mo. Kim-1 gene expression was not elevated at 8 or 12 mo but was at 18 and 24 mo. However, changes in Kim-1 protein expression were only seen at 24 mo and corresponded to increased urinary levels. Importantly, these changes were attenuated by caloric restriction. MMP-7 gene expression was decreased at 8 mo, but an age-dependent increase was seen at 24 mo. Increased MMP-7 protein expression in tubular epithelial cells at 24 mo was correlated with the gene expression pattern. In summary, we identified genes changed by aging and changes attenuated by caloric restriction. This will facilitate investigation into the molecular mechanism mediating the age-related increase in susceptibility to injury.

Renal stem cells in recovery from acute kidney injury.

The kidney has a dramatic capacity to regenerate after injury. Whether stem cells are the source of the epithelial progenitors replacing injured and dying tubular epithelium is currently an area of intense investigation. Studies from our laboratory and others have supported a model whereby many surviving renal epithelial cells after injury become dedifferentiated and take on mesenchymal characteristics. These cells proliferate to restore the integrity of the denuded basement membrane, and subsequently redifferentiate into a functional epithelium. An alternative possibility is that a minority of surviving intratubular cells possess stem cell properties and selectively proliferate after damage to neighboring cells. Some evidence exists to support this hypothesis but it has not yet been rigorously evaluated. A third hypothesis is that extratubular cells contribute to repair of damaged epithelium. Bone marrow-derived stem cells have been proposed to contribute to this process but our work and work of others indicates that the vast majority of tubular cells derive from an intrarenal source. Recent evidence suggests that interstitial cells may represent another extratubular stem cell niche. The fundamental unanswered questions in this field include whether renal stem cells exist in the adult, and if they do where are they located (interstitium, tubule, cortex, medulla) and what markers can be relied upon for the isolation and purification of these putative renal stem cells. In this review we focus on our current understanding of the potential role of renal and extrarenal stem cells in repair of the adult kidney and highlight some of the controversies in this field.

Renal stem cells in recovery from acute kidney injury.

The kidney has a dramatic capacity to regenerate after injury. Whether stem cells are the source of the epithelial progenitors replacing injured and dying tubular epithelium is currently an area of intense investigation. Studies from our laboratory and others have supported a model whereby many surviving renal epithelial cells after injury become dedifferentiated and take on mesenchymal characteristics. These cells proliferate to restore the integrity of the denuded basement membrane, and subsequently redifferentiate into a functional epithelium. An alternative possibility is that a minority of surviving intratubular cells possess stem cell properties and selectively proliferate after damage to neighboring cells. Some evidence exists to support this hypothesis but it has not yet been rigorously evaluated. A third hypothesis is that extratubular cells contribute to repair of damaged epithelium. Bone marrow-derived stem cells have been proposed to contribute to this process but our work and work of others indicates that the vast majority of tubular cells derive from an intrarenal source. Recent evidence suggests that interstitial cells may represent another extratubular stem cell niche. The fundamental unanswered questions in this field include whether renal stem cells exist in the adult, and if they do where are they located (interstitium, tubule, cortex, medulla) and what markers can be relied upon for the isolation and purification of these putative renal stem cells. In this review we focus on our current understanding of the potential role of renal and extrarenal stem cells in repair of the adult kidney and highlight some of the controversies in this field.

Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol.

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.

Differential role of cytosolic phospholipase A2 in the invasion of brain microvascular endothelial cells by Escherichia coli and Listeria monocytogenes.

Invasion of brain microvascular endothelial cells (BMECs) is a key step in the pathogenesis of meningitis due to Escherichia coli and Listeria monocytogenes. Although host cell actin cytoskeletal rearrangements are essential in BMEC invasion by E. coli K1 and L. monocytogenes, the underlying signaling mechanisms remain unclear. This study demonstrates that host cell cytosolic phospholipase A2 (cPLA2) contributes to E. coli K1 invasion of BMECs but not to L. monocytogenes invasion of BMECs. This difference was observed with 4-bromophenacyl bromide, a nonselective PLA2 inhibitor, and arachidonyl trifluoromethyl ketone, a selective cPLA2 inhibitor, and was confirmed with BMEC derived from cPLA2 knockout mice. Activation of cPLA2 leads to generation of intracellular arachidonic acid, which is metabolized via cyclooxygenase (COX) and lipo-oxygenase (LOX) pathways into eicosanoids. COX and LOX inhibitors also significantly inhibit E. coli K1 invasion of BMECs.

Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia.

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100-200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.

Nuclear translocation of cytosolic phospholipase A2 is induced by ATP depletion.

Phospholipase A(2) (PLA(2)) enzymes may play a role in cellular injury due to ATP depletion. Renal Madin-Darby canine kidney cells were subjected to ATP depletion to assess the effects of cellular energy metabolism on cytosolic PLA(2) (cPLA(2)) regulation. ATP depletion results in a decrease in soluble cPLA(2) activity and an increase in membrane-associated activity, which is reversed upon restoration of ATP levels by addition of dextrose. In ATP-depleted cells cPLA(2) mass shifts from cytosol to nuclear fractions. GFP-cPLA(2) is localized at the nuclear membrane of stably transfected ATP-depleted LLC-PK(1) cells under conditions where [Ca(2+)](i) is known to increase. cPLA(2) translocation does not occur if the increase in [Ca(2+)](i) increase is inhibited. If [Ca(2+)](i) is allowed to increase when ATP is depleted and the cells are then lysed, cPLA(2) remains associated with nuclear fractions even if the homogenate [Ca(2+)] is markedly reduced. In contrast, cPLA(2), which becomes associated with the nucleus when [Ca(2+)](i) is increased using ionophore, readily dissociates from the nuclear fractions of ATP-replete cells upon reduction of homogenate [Ca(2+)]. Okadaic acid inhibits the ATP depletion-induced association of cPLA(2) with nuclear fractions. Thus energy deprivation results in [Ca(2+)]-induced nuclear translocation, which is partially prevented by a phosphatase inhibitor.

PLIP, a novel splice variant of Tip60, interacts with group IV cytosolic phospholipase A(2), induces apoptosis, and potentiates prostaglandin production.

The group IV cytosolic phospholipase A(2) (cPLA(2)) has been localized to the nucleus (M. R. Sierra-Honigmann, J. R. Bradley, and J. S. Pober, Lab. Investig. 74:684-695, 1996) and is known to translocate from the cytosolic compartment to the nuclear membrane (S. Glover, M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and M. H. Gelb, J. Biol. Chem. 270:15359-15367, 1995; A. R. Schievella, M. K. Regier, W. L. Smith, and L. L. Lin, J. Biol. Chem. 270:30749-30754, 1995). We hypothesized that nuclear proteins interact with cPLA(2) and participate in the functional effects of this translocation. We have identified a nuclear protein, cPLA(2)-interacting protein (PLIP), a splice variant of human Tip60, which interacts with the amino terminal region of cPLA(2). Like Tip60, PLIP cDNA includes the MYST domain containing a C2HC zinc finger and well-conserved similarities to acetyltransferases. Both PLIP and Tip60 coimmunoprecipitate and colocalize with cPLA(2) within the nuclei of transfected COS cells. A polyclonal antibody raised to PLIP recognizes both PLIP and Tip60. Endogenous Tip60 and/or PLIP in rat mesangial cells is localized to the nucleus in response to serum deprivation. Nuclear localization coincides temporally with apoptosis. PLIP expression, mediated by adenoviral gene transfer, potentiates serum deprivation-induced prostaglandin E(2) (PGE(2)) production and apoptosis in mouse mesangial cells from cPLA(2)(+/+) mice but not in mesangial cells derived from cPLA(2)(-/-) mice. Thus PLIP, a splice variant of Tip60, interacts with cPLA(2) and potentiates cPLA(2)-mediated PGE(2) production and apoptosis.

Expression of fibronectin splice variants in the postischemic rat kidney.

Using an in vivo rat model of unilateral renal ischemia, we previously showed that the expression and distribution of fibronectin (FN), a major glycoprotein of plasma and the extracellular matrix, dramatically changes in response to ischemia-reperfusion. In the distal nephron in particular, FN accumulates in tubular lumens, where it may contribute to obstruction. In the present study, we examine whether the tubular FN is the plasma or cellular form, each of which is produced by alternative splicing of a single gene transcript. We demonstrate that FN in tubular lumens does not contain the extra type III A (EIIIA) and/or the extra type III B (EIIIB) region, both of which are unique to cellular FN. It does, however, contain the V95 region, which in the rat is a component of FNs in both plasma and the extracellular matrix. Expression of FN containing EIIIA increases dramatically in the renal interstitium after ischemic injury and continues to be produced at high levels 6 wk later. V95-containing FN also increases in the interstitial space, albeit more slowly and at lower levels than FN containing EIIIA; it also persists 6 wk later. FN containing the EIIIB region is not expressed in the injured kidney. The presence of V95 but not the EIIIA or EIIIB regions of FN in tubular lumens identifies the origin of FN in this location as the plasma; tubular FN is ultimately voided in the urine. The data indicate that both plasma and cellular FNs containing the V95 and/or EIIIA regions may contribute to the pathogenesis of acute renal failure and to the repair of the injured kidney.

TRIP-Br: a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1.

We report the isolation of TRIP-Br1, a transcriptional regulator that interacts with the PHD-bromodomain of co-repressors of Krüppel-associated box (KRAB)-mediated repression, KRIP-1(TIF1beta) and TIF1alpha, as well as the co-activator/adaptor p300/CBP. TRIP-Br1 and the related protein TRIP-Br2 possess transactivation domains. Like MDM2, which has a homologous transactivation domain, TRIP-Br proteins functionally contact DP-1, stimulating E2F-1/DP-1 transcriptional activity. KRIP-1 potentiates TRIP-Br protein co-activation of E2F-1/DP-1. TRIP-Br1 is a component of a multiprotein complex containing E2F-1 and DP-1. Co-expression of the retinoblastoma gene product (RB) abolishes baseline E2F-1/DP-1 transcriptional activity as well as TRIP-Br/KRIP-1 co-activation, both of which are restored by the adenovirus E1A oncoprotein. These features suggest that TRIP-Br proteins function at E2F-responsive promoters to integrate signals provided by PHD- and/or bromodomain- containing transcription factors. TRIP-Br1 is identical to the cyclin-dependent kinase 4 (cdk4)-binding protein p34(SEI-1), which renders the activity of cyclin D/cdk4 resistant to the inhibitory effect of p16(INK4a) during late G(1). TRIP-Br1(p34(SEI-1)) is differentially overexpressed during the G(1) and S phases of the cell cycle, consistent with a dual role for TRIP-Br1(p34(SEI-1)) in the regulation of cell cycle progression through sequential effects on the transcriptional activity of E2F-responsive promoters during G(1) and S phases.

Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity.

Inflammation causes the induction of cyclooxygenase-2 (Cox-2), leading to the release of prostanoids, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity. Peripheral inflammation also generates pain hypersensitivity in neighbouring uninjured tissue (secondary hyperalgesia), because of increased neuronal excitability in the spinal cord (central sensitization), and a syndrome comprising diffuse muscle and joint pain, fever, lethargy and anorexia. Here we show that Cox-2 may be involved in these central nervous system (CNS) responses, by finding a widespread induction of Cox-2 expression in spinal cord neurons and in other regions of the CNS, elevating prostaglandin E2 (PGE2) levels in the cerebrospinal fluid. The major inducer of central Cox-2 upregulation is interleukin-1beta in the CNS, and as basal phospholipase A2 activity in the CNS does not change with peripheral inflammation, Cox-2 levels must regulate central prostanoid production. Intraspinal administration of an interleukin-converting enzyme or Cox-2 inhibitor decreases inflammation-induced central PGE2 levels and mechanical hyperalgesia. Thus, preventing central prostanoid production by inhibiting the interleukin-1beta-mediated induction of Cox-2 in neurons or by inhibiting central Cox-2 activity reduces centrally generated inflammatory pain hypersensitivity.