François Jouret

Vacuolar H+-ATPase d2 subunit: molecular characterization, developmental regulation, and localization to specialized proton pumps in kidney and bone.

The ubiquitous multisubunit vacuolar-type proton pump (H+- or V-ATPase) is essential for acidification of diverse intracellular compartments. It is also present in specialized forms at the plasma membrane of intercalated cells in the distal nephron, where it is required for urine acidification, and in osteoclasts, playing an important role in bone resorption by acid secretion across the ruffled border membrane. It was reported previously that, in human, several of the renal pumps constituent subunits are encoded by genes that are different from those that are ubiquitously expressed. These paralogous proteins may be important in differential functions, targeting or regulation of H+-ATPases. They include the d subunit, where d1 is ubiquitous whereas d2 has a limited tissue expression. This article reports on an investigation of d2. It was first confirmed that in mouse, as in human, kidney and bone are two of the main sites of d2 mRNA expression. d2 mRNA and protein appear later during nephrogenesis than does the ubiquitously expressed E1 subunit. Mouse nephron-segment reverse transcription-PCR revealed detectable mRNA in all segments except thin limb of Henles loop and distal convoluted tubule. However, with the use of a novel d2-specific antibody, high-intensity d2 staining was observed only in intercalated cells of the collecting duct in fresh-frozen human kidney, where it co-localized with the a4 subunit in the characteristic plasma membrane-enhanced pattern. In human bone, d2 co-localized with the a3 subunit in osteoclasts. This different subunit association in different tissues emphasizes the possibility of the H+-ATPase as a future therapeutic target.

Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

Diagnosis of cyst infection in patients with autosomal dominant polycystic kidney disease: attributes and limitations of the current modalities

Cyst infection is a diagnostic challenge in patients with autosomal dominant polycystic kidney disease (ADPKD) because of the lack of specific manifestations and limitations of conventional imaging procedures. Still, recent clinical observations and series have highlighted common criteria for this condition. Cyst infection is diagnosed if confirmed by cyst fluid analysis showing bacteria and neutrophils, and as a probable diagnosis if all four of the following criteria are concomitantly met: temperature of >38°C for >3 days, loin or liver tenderness, C-reactive protein plasma level of >5 mg/dL and no evidence for intracystic bleeding on computed tomography (CT). In addition, the elevation of serum carbohydrate antigen 19-9 (CA19-9) has been proposed as a biomarker for hepatic cyst infection. Positron-emission tomography after intravenous injection of 18-fluorodeoxyglucose, combined with CT, proved superior to radiological imaging techniques for the identification and localization of kidney and liver pyocyst. This review summarizes the attributes and limitations of these recent clinical, biological and imaging advances in the diagnosis of cyst infection in patients with ADPKD.

Chloride Channels and Endocytosis: New Insights from Dent's Disease and ClC-5 Knockout Mice

Dent’s disease is a hereditary renal tubular disorder characterized by low-molecular weight (LMW) proteinuria, hypercalciuria and nephrolithiasis. The disease is due to mutations of ClC-5, a member of the family of voltage-gated CLC chloride channels. ClC-5 is expressed in part in cells lining the proximal tubule (PT) of the kidney, where it colocalizes with albumin-containing endocytic vesicles belonging to the receptor-mediated endocytic pathway that ensures efficient reabsorption of ultrafiltrated LMW proteins. Since progression along the endocytic apparatus requires endosomal acidification, it has been suggested that dysfunction of ClC-5 in endosomes may lead to inefficient reabsorption of LMW proteins and dysfunction of PT cells. Analysis of a ClC-5 knockout (KO) mouse model, displaying all the characteristic renal tubular defects of Dent’s disease, showed evidence of a severe LMW proteinuria. Cytochemical studies with the endocytic tracer, peroxidase, showed poor transfer into early endocytic vesicles, suggesting that impairment of receptor-mediated endocytosis in PT cells is the basis for the defective uptake of LMW proteins in patients with Dent’s disease. Endocytosis and processing of LMW proteins involve the multiligand tandem receptors, megalin and cubilin, that are abundantly expressed at the brush border of PT cells. Characterization of the endocytic defect in ClC-5 KO mice revealed that ligands of both megalin and cubilin were affected. The total kidney content of megalin and especially cubilin at the protein level was decreased but, more importantly, using analytical subcellular fractionation and quantitative immunogold labelling we demonstrated a selective disappearance of megalin and cubilin at the brush border of PT cells. These observations allowed us to conclude that defective protein endocytosis linked to ClC-5 inactivation is due at least in part to a major and selective loss of megalin and cubilin at the brush border, reflecting a trafficking defect in renal PT cells. These results improve our understanding of Dent’s disease, taken as a paradigm for renal Fanconi syndrome and nephrolithiasis, and demonstrate multiple roles for ClC-5 in the kidney. These studies also provided insights into important functions such as apical endocytosis, handling of proteins by renal tubular cells, calcium metabolism, and urinary acidification.

Mesenchymal Stromal Cell Therapy in Ischemia/Reperfusion Injury.

Ischemia/reperfusion injury (IRI) represents a worldwide public health issue of increasing incidence. IRI may virtually affect all organs and tissues and is associated with significant morbidity and mortality. Particularly, the duration of blood supply deprivation has been recognized as a critical factor in stroke, hemorrhagic shock, or myocardial infarction, as well as in solid organ transplantation (SOT). Pathophysiologically, IRI causes multiple cellular and tissular metabolic and architectural changes. Furthermore, the reperfusion of ischemic tissues induces both local and systemic inflammation. In the particular field of SOT, IRI is an unavoidable event, which conditions both short- and long-term outcomes of graft function and survival. Clinically, the treatment of patients with IRI mostly relies on supportive maneuvers since no specific target-oriented therapy has been validated thus far. In the present review, we summarize the current literature on mesenchymal stromal cells (MSC) and their potential use as cell therapy in IRI. MSC have demonstrated immunomodulatory, anti-inflammatory, and tissue repair properties in rodent studies and in preliminary clinical trials, which may open novel avenues in the management of IRI and SOT.

AMP-activated Protein Kinase (AMPK) Activation and Glycogen Synthase Kinase-3β (GSK-3β) Inhibition Induce Ca2+-independent Deposition of Tight Junction Components at the Plasma Membrane

Extracellular Ca2+ is essential for the development of stable epithelial tight junctions. We find that in the absence of extracellular Ca2+, AMP-activated protein kinase (AMPK) activation and glycogen synthase kinase (GSK)-3β inhibition independently induce the localization of epithelial tight junction components to the plasma membrane. The Ca2+-independent deposition of junctional proteins induced by AMPK activation and GSK-3β inhibition is independent of E-cadherin. Furthermore, the nectin-afadin system is required for the deposition of tight junction components induced by AMPK activation, but it is not required for that induced by GSK-3β inhibition. Phosphorylation studies demonstrate that afadin is a substrate for AMPK. These data demonstrate that two kinases involved in regulating cell growth and metabolism act through distinct pathways to influence the deposition of the components of epithelial tight junctions.

A novel renal carbonic anhydrase type III plays a role in proximal tubule dysfunction

Dysfunction of the proximal tubule (PT) is associated with variable degrees of solute wasting and low-molecular-weight proteinuria. We measured metabolic consequences and adaptation mechanisms in a model of inherited PT disorders using PT cells of ClC-5-deficient (Clcn5Y/-) mice, a well-established model of Dents disease. Compared to cells taken from control mice, those from the mutant mice had increased expression of markers of proliferation (Ki67, proliferative cell nuclear antigen (PCNA), and cyclin E) and oxidative scavengers (superoxide dismutase I and thioredoxin). Transcriptome and protein analyses showed fourfold induction of type III carbonic anhydrase in a kidney-specific manner in the knockout mice located in scattered PT cells. Kidney-specific carbonic anhydrase type III (CAIII) upregulation was confirmed in other mice lacking the multiligand receptor megalin and in a patient with Dents disease due to an inactivating CLCN5 mutation. The type III enzyme was specifically detected in the urine of mice lacking ClC-5 or megalin, patients with Dents disease, and in PT cell lines exposed to oxidative stress. Our study shows that lack of PT ClC-5 in mice and men is associated with CAIII induction, increased cell proliferation, and oxidative stress.

Feasibility of high resolution SPECT imaging on conscious mice: preliminary study in kidney pharmacokinetic

Since the initial report of the trigemino-cardiac reflex (TCR) during tumour surgery in the cerebellopontine angle [1], there has been growing and continuing debate about this reflex and increasing awareness of the phenomenon [2–4]. While a few clinical reports have underlined that the TCR is manifested by the sudden development of cardiac dysrhythmia up to asystole, arterial hypotension, apnoea and gastric hypermotility after central or peripheral stimulation of any division of the trigeminal nerve [3], the physiological function of this reflex is not yet fully understood. The current theoretical explanation for the physiological mechanism of the TCR is that sensory nerve endings of the trigeminal nerve send neuronal signals via the gasserian ganglion to the sensory nucleus of the trigeminal nerve, forming the afferent pathway of the reflex arc [3]. This afferent pathway continues along the short internuncial nerve fibres in the reticular formation to connect with the efferent pathways in the motor nucleus of the vagus nerve [3]. The vagus provides parasympathetic innervation to the heart, vascular smooth muscle and abdominal viscera. Vagal stimulation via these connections after trigeminal nerve activation likely accounts for the reflexive response. Since it is generally accepted that the diving reflex and ischaemic tolerance involve, at least in part, similar physiological mechanisms [3, 5, 6], the existence of such endogenous (neuro)protective strategies may stress the clinical importance of the TCR, a term that also subsumes the diving reflex. Even though no convincing experimental data exist, the TCR may be a specific example of a group of related responses generally defined by Wolf as “oxygen-conserving reflexes” [7]. Within seconds after the initiation of such a reflex, there is a powerful and differentiated activation of sympathetic nerves [7]. The subsequent elevation in cerebral blood flow is not associated with changes in the cerebral metabolic rate of oxygen (CMRO2) or the cerebral metabolic rate of glucose (CMRglc) and hence represents a primary cerebrovascular vasodilation [7]. It is generally accepted that various noxious stimuli can, when applied below the threshold of brain damage, induce tolerance in the brain against a subsequent deleterious stimulus of the same or even another modality; the latter is called “cross tolerance” [8] and probably involves separate systems of neurons of the central nervous system [3]. The one which mediates reflexive neurogenic protection emanates from oxygen-sensitive sympatho-excitatory reticulospinal neurons of the rostral ventrolateral medulla oblongata. These cells, excited within seconds by a reduction in cerebral blood flow or CMRO2, initiate the systemic vascular components [9]. They profoundly increase regional cerebral blood flow without changing CMRO2 or CMRglc and hence rapidly and efficiently provide the brain with oxygen [9]. The system mediating reflex protection projects via as yet undefined pathways from the rostral ventrolateral medulla oblongata to the upper brainstem and/or thalamus and finally engages a small population of neurons in the cortex which appear to be dedicated to reflexively transducing a neuronal signal into cerebrovascular vasodilatation and synchronisation of electrocortical activity [9]. Reticulospinal neurons of the rostral ventrolateral medulla oblongata are “premotor” neurons and, as such, are critical for detecting and initiating the vascular, cardiac and respiratory responses to brainstem hypoxia and ischaemia. The systemic response to excitation of rostral ventrolateral medulla oblongata neurons, however, results from activation of a network of effector neurons distributed elsewhere in the central nervous system. Thus, sympathetic excitation is mediated by an excitatory projection to spinal preganglionic sympathetic neurons and the bradycardia via projections to cardiovagal motor medullary neurons [3, 9]. The integrated response functions to redistribute blood from viscera to brain in response to a challenge to cerebral metabolism. Little is known about the potential to visualise these cascades in the different brain regions by means of current state-of-the-art imaging techniques. Direct imaging studies on the influence of TCR in structures of the central nervous system have not yet been described in either humans or animals. Functional magnetic resonance imaging studies (fMRI), however, allow repeated evaluation of activity changes during cardiovascular changes over the entire brain at relatively high resolution and with minimal invasiveness. From fMRI studies in neuronal structures after baroreceptor reflex activation [10]—that may present similarities to studies after TCR initiation—one can assume that the areas responsive to TCR activation may also include extramedullary regions not traditionally considered to be components of central cardiovascular reflex networks (e.g. cerebellarrelated structures), and many of these components may show both shortand long-term response patterns. In addition, intrinsic optical imaging has been used to examine the rostral ventrolateral medullary surface and to control site responses to pressor and depressor challenges during sleep that resulted in somatomotor, respiratory, heart rate or electroencephalographic indications of late-developing compensatory responses [11]. These studies demonstrate that transient cardiovascular changes elicit discrete cardiovascular changes over multiple brain regions and that these changes are often expressed unilaterally even in response to a bilateral challenge. These experimental findings are in accordance with other imaging studies of central nervous brain organisation in humans [12]. That the brain may have neuronal systems dedicated to protecting itself from (ischaemic) damage at first appears a novel concept, but it is, upon reflection, not surprising given that the brain is not injured in naturalistic behaviours characterised by very low levels of regional cerebral blood

Ubiquitous and kidney-specific subunits of vacuolar H+-ATPase are differentially expressed during nephrogenesis.

The vacuolar H(+)-ATPase (V-ATPase) is a ubiquitous multisubunit pump that is responsible for acidification of intracellular organelles. In the kidney, a particular form of V-ATPase, made of specific subunits isoforms, has been located at the plasma membrane of intercalated cells (IC). Mutations in genes encoding IC-specific subunits cause infant distal renal tubular acidosis (dRTA), suggesting that the segmental distribution of these subunits is acquired at birth or during early infancy. However, the comparative ontogeny of the IC-specific versus the ubiquitous subunits of V-ATPase and the mechanisms involved in their segmental expression remain unknown. Real-time reverse transcription-PCR, in situ hybridization, immunoblotting, immunostaining, and subcellular fractionation analyses characterized the expression and distribution of V-ATPase subunits, transcription factors, and differentiation markers during mouse nephrogenesis. Ubiquitous A, E1, B2, G1, and C1 subunits showed an early (embryonic day 13.5 [E13.5]) and stable expression throughout nephrogenesis, followed by a slight increase around birth. The developmental pattern of a1 was bimodal, with early induction, gradual decrease during organogenesis, and neonatal increase. These patterns contrasted with the later (from E15.5) and progressive expression of IC-specific a4, B1, G3, and C2 subunits, after the induction of the forkhead transcription factor Foxi1. From E15.5, Foxi1 mRNA was detected in IC, where it co-distributed with B1 in late nephrogenesis. Immunostaining showed that the distribution of ubiquitous E1 and B2 was acquired from E15.5, whereas a4 was located in IC during late nephrogenesis. Subcellular fractionation showed that in both fetal and mature (cortex and medulla) kidneys, E1 and a4 were located in endosomes. These data demonstrate a differential expression and a coordinate regulation of IC-specific versus ubiquitous V-ATPase subunits during nephrogenesis. They provide new insights into the complex regulation of V-ATPase subunits, the maturation of IC along the nephron, and the pathophysiology of hereditary dRTA.

Mesenchymal stromal cell therapy in conditions of renal ischaemia/reperfusion

Acute kidney injury (AKI) represents a worldwide public health issue of increasing incidence, with a significant morbi-mortality. AKI treatment mostly relies on supportive manoeuvres in the absence of specific target-oriented therapy. The pathophysiology of AKI commonly involves ischaemia/reperfusion (I/R) events, which cause both immune and metabolic consequences in renal tissue. Similarly, at the time of kidney transplantation (KT), I/R is an unavoidable event which contributes to early graft dysfunction and enhanced graft immunogenicity. Mesenchymal stromal cells (MSCs) represent a heterogeneous population of adult, fibroblast-like multi-potent cells characterized by their ability to differentiate into tissues of mesodermal lineages. Because MSC have demonstrated immunomodulatory, anti-inflammatory and tissue repair properties, MSC administration at the time of I/R and/or at later times has been hypothesized to attenuate AKI severity and to accelerate the regeneration process. Furthermore, MSC in KT could help prevent both I/R injury and acute rejection, thereby increasing graft function and survival. In this review, summarizing the encouraging observations in animal models and in pilot clinical trials, we outline the benefit of MSC therapy in AKI and KT, and envisage their putative role in renal ischaemic conditioning.