Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

p27kip1 Modulates the Morphology and Phagocytic Activity of Microglia.

p27kip1 is a multifunctional protein that promotes cell cycle exit by blocking the activity of cyclin/cyclin-dependent kinase complexes as well as migration and motility via signaling pathways that converge on the actin and microtubule cytoskeleton. Despite the broad characterization of p27kip1 function in neural cells, little is known about its relevance in microglia. Here, we studied the role of p27kip1 in microglia using a combination of in vitro and in situ approaches. While the loss of p27kip1 did not affect microglial density in the cerebral cortex, it altered their morphological complexity in situ. However, despite the presence of p27kip1 in microglial processes, as shown by immunofluorescence in cultured cells, loss of p27kip1 did not change microglial process motility and extension after applying laser-induced brain damage in cortical brain slices. Primary microglia lacking p27kip1 showed increased phagocytic uptake of synaptosomes, while a cell cycle dead variant negatively affected phagocytosis. These findings indicate that p27kip1 plays specific roles in microglia.

Controversies and prospects about microglia in maternal immune activation models for neurodevelopmental disorders.

Activation of the maternal immune system during pregnancy is a well-established risk factor for neuropsychiatric disease in the offspring, yet, the underlying mechanisms leading to altered brain function remain largely undefined. Microglia, the resident immune cells of the brain, are key to adequate development of the central nervous system (CNS), and are prime candidates to mediate maternal immune activation (MIA)-induced brain abnormalities. As such, the effects of MIA on the immunological phenotype of microglia has been widely investigated. However, contradicting results due to differences in read-out and methodological approaches impede final conclusions on MIA-induced microglial alterations. The aim of this review is to critically discuss the evidence for an activated microglial phenotype upon MIA.

Cerebral Cortical Circuitry Formation Requires Functional Glycine Receptors.

The development of the cerebral cortex is a complex process that requires the generation, migration, and differentiation of neurons. Interfering with any of these steps can impair the establishment of connectivity and, hence, function of the adult brain. Neurotransmitter receptors have emerged as critical players to regulate these biological steps during brain maturation. Among them, α2 subunit-containing glycine receptors (GlyRs) regulate cortical neurogenesis and the present work demonstrates the long-term consequences of their genetic disruption on neuronal connectivity in the postnatal cerebral cortex. Our data indicate that somatosensory cortical neurons of Glra2 knockout mice (Glra2KO) have more dendritic branches with an overall increase in total spine number. These morphological defects correlate with a disruption of the excitation/inhibition balance, thereby increasing network excitability and enhancing susceptibility to epileptic seizures after pentylenetetrazol tail infusion. Taken together, our findings show that the loss of embryonic GlyRα2 ultimately impairs the formation of cortical circuits in the mature brain.

Age-specific function of α5ß1 integrin in microglial migration during early colonization of the developing mouse cortex.

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5ß1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5ß1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5ß1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5ß1 integrin in microglial migration during colonization of the embryonic brain.

Brivaracetam does not modulate ionotropic channels activated by glutamate, γ-aminobutyric acid, and glycine in hippocampal neurons.

Brivaracetam (BRV) is a selective, high-affinity ligand for synaptic vesicle protein 2A (SV2A), recently approved as adjunctive treatment for drug-refractory partial-onset seizures in adults. BRV binds SV2A with higher affinity than levetiracetam (LEV), and was shown to have a differential interaction with SV2A. Because LEV was reported to interact with multiple excitatory and inhibitory ligand-gated ion channels and that may impact its pharmacological profile, we were interested in determining whether BRV directly modulates inhibitory and excitatory ionotropic receptors in central neurons. Voltage-clamp experiments were performed in primary cultures of mouse hippocampal neurons. At a supratherapeutic concentration of 100 µm, BRV was devoid of any direct effect on currents gated by γ-aminobutyric acidergic type A, glycine, kainate, N-methyl-d-aspartate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Similarly to LEV, BRV reveals a potent ability to oppose the action of negative modulators on the inhibitory receptors. In conclusion, these results show that BRV contrasts with LEV by not displaying any direct action on inhibitory or excitatory postsynaptic ligand-gated receptors at therapeutic concentrations and thereby support BRV's role as a selective SV2A ligand. These findings add further evidence to the validity of SV2A as a relevant antiepileptic drug target and emphasize the potential for exploring further presynaptic mechanisms as a novel approach to antiepileptic drug discovery.

Experimental febrile seizures increase dendritic complexity of newborn dentate granule cells.

OBJECTIVE: Febrile seizures (FS) are fever-associated convulsions, being the most common seizure disorder in early childhood. A subgroup of these children later develops epilepsy characterized by a hyperexcitable neuronal network in the hippocampus. Hippocampal excitability is regulated by the hippocampal dentate gyrus (DG) where postnatal neurogenesis occurs. Experimental FS increase the survival of newborn hippocampal dentate granule cells (DGCs), yet the significance of this neuronal subpopulation to the hippocampal network remains unclear. In the current study, we characterized the temporal maturation and structural integration of these post-FS born DGCs in the DG. METHODS: Experimental FS were induced in 10-day-old rat pups. The next day, retroviral particles coding for enhanced green fluorescent protein (eGFP) were stereotactically injected in the DG to label newborn cells. Histochemical analyses of eGFP expressing DGCs were performed one, 4, and 8 weeks later and consisted of the following: (1) colocalization with neurodevelopmental markers doublecortin, calretinin, and the mature neuronal marker NeuN; (2) quantification of dendritic complexity; and (3) quantification of spine density and morphology. RESULTS: At neither time point were neurodevelopmental markers differently expressed between FS animals and normothermia (NT) controls. One week after treatment, DGCs from FS animals showed dendrites that were 66% longer than those from NT controls. At 4 and 8 weeks, Sholl analysis of the outer 83% of the molecular layer showed 20-25% more intersections in FS animals than in NT controls (p < 0.01). Although overall spine density was not affected, an increase in mushroom-type spines was observed after 8 weeks. SIGNIFICANCE: Experimental FS increase dendritic complexity and the number of mushroom-type spines in post-FS born DGCs, demonstrating a more mature phenotype and suggesting increased incoming excitatory information. The consequences of this hyperconnectivity to signal processing in the DG and the output of the hippocampus remain to be studied.

Analysis of alpha3 GlyR single particle tracking in the cell membrane.

Single particle tracking (SPT) of transmembrane receptors in the plasma membrane often reveals heterogeneous diffusion. A thorough interpretation of the displacements requires an extensive analysis suited for discrimination of different motion types present in the data. Here the diffusion pattern of the homomeric alpha3-containing glycine receptor (GlyR) is analyzed in the membrane of HEK 293 cells. More specifically, the influence of the alpha3 RNA splice variants alpha3K and alpha3L on lateral membrane diffusion of the receptor is revealed in detail. Using a combination of ensemble and local SPT analysis, free and anomalous diffusion parameters are determined. The GlyR alpha3 free diffusion coefficient is found to be 0.13 +/- 0.01 microm2/s and both receptor variants display confined motion. The confinement probability level and residence time are significantly elevated for the alpha3L variant compared to the alpha3K variant. Furthermore, for the alpha3L GlyR, the presence of directed motion was also established, with a velocity matching that of saltatory vesicular transport. These findings reveal that alpha3 GlyRs are prone to different types of anomalous diffusion and reinforce the role of RNA splicing in determining lateral membrane trafficking.

Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy.

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 µm(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.

Microglia proliferation is controlled by P2X7 receptors in a Pannexin-1-independent manner during early embryonic spinal cord invasion.

Microglia are known to invade the mammalian spinal cord (SC) at an early embryonic stage. While the mechanisms underlying this early colonization of the nervous system are still unknown, we recently found that it is associated, at least partially, with the ability of microglia to proliferate at the onset of motoneuron developmental cell death and of synaptogenesis in mouse embryo (E13.5). In vitro studies have shown that the proliferation and activation of adult microglia can be influenced by the purinergic ionotropic receptor P2X7 via a coupling with Pannexin-1. By performing patch-clamp recordings in situ using a whole-mouse embryonic SC preparation, we show here that embryonic microglia already express functional P2X7R. P2X7R activation evoked a biphasic current in embryonic microglia, which is supposed to reflect large plasma membrane pore opening. However, although embryonic microglia express pannexin-1, this biphasic current was still recorded in microglia of pannexin-1 knock-out embryos, indicating that it rather reflected P2X7R intrinsic pore dilatation. More important, we found that proliferation of embryonic SC microglia, but not their activation state, depends almost entirely on P2X7R by comparing wild-type and P2X7R-/- embryos. Absence of P2X7R led also to a decrease in microglia density. Pannexin-1-/- embryos did not exhibit any difference in microglial proliferation, showing that the control of embryonic microglial proliferation by P2X7R does not depend on pannexin-1 expression. These results reveal a developmental role of P2X7R by controlling embryonic SC microglia proliferation at a critical developmental state in the SC of mouse embryos.

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane.

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time-lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time-lapse analysis shows that embryonic microglia are highly dynamic cells.

Establishment of reference values for novel urinary biomarkers for renal damage in the healthy population: are age and gender an issue?

BACKGROUND: Recently, a lot of research has focused on the discovery of novel renal biomarkers. Among others, the urinary kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) have been proven to be promising biomarkers in a wide variety of renal pathologies. However, little is known about the normal concentrations in urine of healthy subjects. Therefore, the goal of our study is to establish reference values for urinary KIM-1, NGAL, N-acetyl-ß-D-glucosamidase (NAG), and cystatin C in a healthy population, taking into account possible effects of age and gender. METHODS: We collected urine samples from 338 healthy, nonsmoking subjects between 0 and 95 years old. Subjects with elevated α1-microglobulin values were excluded. Next to the urinary concentrations of KIM-1, NGAL, NAG, and cystatin C, we measured urinary creatinine and specific gravity to correct for urinary dilution. The possible effect of age and gender on the four urinary biomarkers was investigated, and the reference values were established. RESULTS: For the absolute urinary concentrations of the biomarkers, age had a significant effect on all the biomarkers, except for cystatin C, whereas gender significantly affected all four of them, except for NAG. The normalization of biomarkers for creatinine and specific gravity had an effect on the correlation between the biomarkers on one hand and age and gender on the other. CONCLUSIONS: In conclusion, age and gender had different effects on KIM-1, NGAL, NAG, and cystatin C. Based on this knowledge, age- and gender-specific reference values for KIM-1, NGAL, NAG, and cystatin C were established.

Dopamine-mediated striatal activity and function is enhanced in GlyRα2 knockout animals.

The glycine receptor alpha 2 (GlyRα2) is a ligand-gated ion channel which upon activation induces a chloride conductance. Here, we investigated the role of GlyRα2 in dopamine-stimulated striatal cell activity and behavior. We show that depletion of GlyRα2 enhances dopamine-induced increases in the activity of putative dopamine D1 receptor-expressing striatal projection neurons, but does not alter midbrain dopamine neuron activity. We next show that the locomotor response to d-amphetamine is enhanced in GlyRα2 knockout animals, and that this increase correlates with c-fos expression in the dorsal striatum. 3-D modeling revealed an increase in the neuronal ensemble size in the striatum in response to D-amphetamine in GlyRα2 KO mice. Finally, we show enhanced appetitive conditioning in GlyRα2 KO animals that is likely due to increased motivation, but not changes in associative learning or hedonic response. Taken together, we show that GlyRα2 is an important regulator of dopamine-stimulated striatal activity and function.

Experimental early-life febrile seizures induce changes in GABA(A) R-mediated neurotransmission in the dentate gyrus.

PURPOSE: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ-aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA(A) R function in dentate granule cells. METHODS: We applied an immature rat model of hyperthermia (HT)-induced FS. GABA(A) R-mediated neurotransmission was studied using whole-cell patch-clamp recordings from dentate granule neurons in hippocampal slices within 6-9 days post-HT. KEY FINDINGS: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole-cell GABA responses revealed a doubled GABA(A) R sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole-cell GABA responses showed similar kinetics in postsynaptic GABA(A) Rs of HT and NT rats. quantitative real-time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA(A) R subunit expression, which was most pronounced for the α3 subunit. SIGNIFICANCE: The data support the hypothesis that FS persistently alter neuronal excitability.

Experimental early-life febrile seizures cause a sustained increase in excitatory neurotransmission in newborn dentate granule cells.

Prolonged febrile seizures (FS) are a risk factor for the development of hippocampal-associated temporal lobe epilepsy. The dentate gyrus is the major gateway to the hippocampal network and one of the sites in the brain where neurogenesis continues postnatally. Previously, we found that experimental FS increase the survival rate and structural integration of newborn dentate granule cells (DGCs). In addition, mature post-FS born DGCs express an altered receptor panel. Here, we aimed to study if these molecular and structural changes are accompanied by an altered cellular functioning. Experimental FS were induced by hyperthermia in 10-days-old Sprague-Dawley rats. Proliferating progenitor cells were labeled the next day by injecting green fluorescent protein expressing retroviral particles bilaterally in the dentate gyri. Eight weeks later, spontaneous excitatory and inhibitory postsynaptic events (sEPSCs and sIPSCs, respectively) were recorded from labeled DGCs using the whole-cell patch-clamp technique. Experimental FS resulted in a robust decrease of the inter event interval (p < .0001) and a small decrease of the amplitude of sEPSCs (p < .001). Collectively the spontaneous excitatory charge transfer increased (p < .01). Experimental FS also slightly increased the frequency of sIPSCs (p < .05), while the amplitude of these events decreased strongly (p < .0001). The net inhibitory charge transfer remained unchanged. Experimental, early-life FS have a long-term effect on post-FS born DGCs, as they display an increased spontaneous excitatory input when matured. It remains to be established if this presents a mechanism for FS-induced epileptogenesis.

Glycine release from radial cells modulates the spontaneous activity and its propagation during early spinal cord development.

Rhythmic electrical activity is a hallmark of the developing embryonic CNS and is required for proper development in addition to genetic programs. Neurotransmitter release contributes to the genesis of this activity. In the mouse spinal cord, this rhythmic activity occurs after embryonic day 11.5 (E11.5) as waves spreading along the entire cord. At E12.5, blocking glycine receptors alters the propagation of the rhythmic activity, but the cellular source of the glycine receptor agonist, the release mechanisms, and its function remain obscure. At this early stage, the presence of synaptic activity even remains unexplored. Using isolated embryonic spinal cord preparations and whole-cell patch-clamp recordings of identified motoneurons, we find that the first synaptic activity develops at E12.5 and is mainly GABAergic. Using a multiple approach including direct measurement of neurotransmitter release (i.e., outside-out sniffer technique), we also show that, between E12.5 and E14.5, the main source of glycine in the embryonic spinal cord is radial cell progenitors, also known to be involved in neuronal migration. We then demonstrate that radial cells can release glycine during synaptogenesis. This spontaneous non-neuronal glycine release can also be evoked by mechanical stimuli and occurs through volume-sensitive chloride channels. Finally, we find that basal glycine release upregulates the propagating spontaneous rhythmic activity by depolarizing immature neurons and by increasing membrane potential fluctuations. Our data raise the question of a new role of radial cells as secretory cells involved in the modulation of the spontaneous electrical activity of embryonic neuronal networks.

Glycine enhances microglial intracellular calcium signaling. A role for sodium-coupled neutral amino acid transporters.

The inhibitory neurotransmitter glycine is known to enhance microglial nitric oxide production. However, up to now, the mechanism is undocumented. Since calcium is an important second messenger in both immune and glial cells, we studied the effects of glycine on intracellular calcium signaling. We found that millimolar concentrations of glycine enhance microglial intracellular calcium transients induced by 100 µM ATP or by 500 nM thapsigargin. This modulation was unaffected by the glycine receptor antagonist strychnine and could not be mimicked by glycine receptor agonists such as taurine or ß-alanine, indicating glycine receptor independency. The modulation of calcium responses could be mimicked by several structurally related amino acids (e.g., serine, alanine, or glutamine) and was inhibited in the presence of the neutral amino acid transporter substrate α-aminoisobutyric acid (AIB). We correlated these findings to immunofluorescence glycine uptake experiments which showed a clear glycine uptake which was inhibited by AIB. Furthermore, all amino acids that were shown to modulate calcium responses also evoked AIB-sensitive inward currents, mainly carried by sodium, as demonstrated by patch clamp experiments. Based on these findings, we propose that sodium-coupled neutral amino acid transporters are responsible for the observed glycine modulation of intracellular calcium responses.

Collection and storage requirements for urinary kidney injury molecule-1 (KIM-1) measurements in humans.

BACKGROUND: Urinary kidney injury molecule-1 (KIM-1) is a recently discovered biomarker for early renal damage. However, little is known about the collection and storage requirements prior to its measurement in human urine. METHODS: Samples of healthy volunteers were collected and aliquoted. The effect of pre-freezing time, thawing, addition of protease inhibitors, centrifugation, storage time (up to 1.5 years) and temperature (4°C, -20°C and -80°C) was tested. RESULTS: Addition of protease inhibitors and centrifugation prior to freezing did not affect the KIM-1 measurements. When samples were kept at room temperature for longer than 3 h before freezing or defrosted more than 1 h before measurement, mean KIM-1 values differed significantly compared to aliquots with minimal pre-freezing and thawing time. Samples frozen at -80°C were stable for up to 1.5 years; however an increasing number of freeze-thaw cycles adversely affected KIM-1 measurements. When stored at 4°C and -20°C, samples were less stable compared to those stored at -80°C. CONCLUSIONS: This study recommends that urine samples collected for KIM-1 measurements are frozen within 3 h after voiding and only be defrosted immediately prior to measurement. Addition of protease inhibitor and centrifugation prior to measurement is not necessary. Samples are preferably stored at -80°C and freeze-thaw cycles should be avoided.

The association between urinary kidney injury molecule 1 and urinary cadmium in elderly during long-term, low-dose cadmium exposure: a pilot study.

BACKGROUND: Urinary kidney injury molecule 1 is a recently discovered early biomarker for renal damage that has been proven to be correlated to urinary cadmium in rats. However, so far the association between urinary cadmium and kidney injury molecule 1 in humans after long-term, low-dose cadmium exposure has not been studied. METHODS: We collected urine and blood samples from 153 non-smoking men and women aged 60+, living in an area with moderate cadmium pollution from a non-ferrous metal plant for a significant period. Urinary cadmium and urinary kidney injury molecule 1 as well as other renal biomarkers (alpha1-microglobulin, beta2-microglobulin, blood urea nitrogen, urinary proteins and microalbumin) were assessed. RESULTS: Both before (r = 0.20; p = 0.01) and after (partial r = 0.32; p < 0.0001) adjustment for creatinine, age, sex, past smoking, socio-economic status and body mass index, urinary kidney injury molecule 1 correlated with urinary cadmium concentrations. No significant association was found between the other studied renal biomarkers and urinary cadmium. CONCLUSIONS: We showed that urinary kidney injury molecule 1 levels are positively correlated with urinary cadmium concentration in an elderly population after long-term, low-dose exposure to cadmium, while other classical markers do not show an association. Therefore, urinary kidney injury molecule 1 might be considered as a biomarker for early-stage metal-induced kidney injury by cadmium.