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Skin reactions are well described complications of tattooing, usually provoked by red inks. Chemical characterizations of these inks are usually based on limited subjects and techniques. This study aimed to determine the organic and inorganic composition of inks using X-ray fluorescence spectroscopy (XRF), X-ray absorption spectroscopy (XANES) and Raman spectroscopy, in a cohort of patients with cutaneous hypersensitivity reactions to tattoo. A retrospective multicenter study was performed, including 15 patients diagnosed with skin reactions to tattoos. Almost half of these patients developed skin reactions on black inks. XRF identified known allergenic metals – titanium, chromium, manganese, nickel and copper – in almost all cases. XANES spectroscopy distinguished zinc and iron present in ink from these elements in endogenous biomolecules. Raman spectroscopy showed the presence of both reported (azo pigments, quinacridone) and unreported (carbon black, phtalocyanine) putative organic sensitizer compounds, and also defined the phase in which Ti was engaged. To the best of the authors' knowledge, this paper reports the largest cohort of skin hypersensitivity reactions analyzed by multiple complementary techniques. With almost half the patients presenting skin reaction on black tattoo, the study suggests that black modern inks should also be considered to provoke skin reactions, probably because of the common association of carbon black with potential allergenic metals within these inks. Analysis of more skin reactions to tattoos is needed to identify the relevant chemical compounds and help render tattoo ink composition safer.

Infrared (IR) spectromicroscopy allows chemical mapping of a kidney biopsy. It is particularly interesting for chemical speciation of abnormal tubular deposits and calcification. In 2017, using IR spectromicroscopy, we described a new entity called vancomycin cast nephropathy. However, despite recent progresses, the IR microspectrometer spatial resolution is intrinsically limited by diffraction (a few micrometers). Combining atomic force microscopy and IR lasers (AFMIR) allows acquisition of infrared absorption spectra with a resolution and sensitivity in between 10 and 100 nm. Here we show that AFMIR can be used on standard paraffin embedded kidney biopsies. Vancomycin cast could be identified in a damaged tubule. Interestingly unlike standard IR spectromicroscopy, AFMIR revealed heterogeneity of the deposits and established that vancomycin coprecipitated with phosphate containing molecules. These findings highlight the high potential of this approach with nanometric spatial resolution which opens new perspectives for studies on drug-induced nephritis, nanocrystals, and local lipid or carbohydrates alterations.

Synchrotron deep ultraviolet imaging highlighting pathological calcium oxalate detection in kidney is presented., Renal oxalosis is a rare cause of renal failure whose diagnosis can be challenging. Synchrotron deep ultraviolet (UV) fluorescence was assayed to improve oxalosis detection on kidney biopsies spatial resolution and sensitivity compared with the Fourier transform infrared microspectroscopy gold standard. The fluorescence spectrum of synthetic mono-, di- and tri-hydrated calcium oxalate was investigated using a microspectrometer coupled to the synchrotron UV beamline DISCO, Synchrotron SOLEIL, France. The obtained spectra were used to detect oxalocalcic crystals in a case control study of 42 human kidney biopsies including 19 renal oxalosis due to primary (PHO, n = 11) and secondary hyperoxaluria (SHO, n = 8), seven samples from PHO patients who received combined kidney and liver transplants, and 16 controls. For all oxalocalcic hydrates samples, a fluorescence signal is detected at 420 nm. These spectra were used to identify standard oxalocalcic crystals in patients with PHO or SHO. They also revealed micrometric crystallites as well as non-aggregated oxalate accumulation in tubular cells. A nine-points histological score was established for the diagnosis of renal oxalosis with 100% specificity (76–100) and a 73% sensitivity (43–90). Oxalate tubular accumulation and higher histological score were correlated to lower estimated glomerular filtration rate and higher urinary oxalate over creatinine ratio.

Graft microvasculature is a major target of donor-specific antibodies (DSA) and endothelial damage is direct evidence of antibody-mediated rejection (ABMR). Using immunohistochemistry, we analyzed the expression of three microvascular endothelial activation markers (fascin, vimentin, and hsp47), suggestive of endothelial-to-mesenchymal transition (EndMT) in 351 graft biopsies from 248 kidney recipients, with concomitant screening of circulating antihuman leukocyte antigen (HLA) DSA at the time of the biopsy. The factors associated with EndMT marker expression were DSA and the presence of microvascular inflammation (MI). EndMT expressing grafts had significantly more allograft loss compared to EndMT negative grafts (P < .0001). The expression of EndMT markers positively correlated with anti-HLA DSA class II mean fluorescence intensity (MFI) levels and especially identified DQ and DR antibodies as being more closely associated with microvascular injury. Moreover, only DSA linked to positive EndMT score affected allograft survival, regardless of DSA MFI levels or presence of C4d deposition. Thus, EndMT markers could represent a clinically relevant tool for early identification of ongoing endothelial injury, harmful DSA, and patients at high risk for allograft failure.

International audience; Carcinogenic risk of tattoo inks remains unclear, and their composition is poorly regulated. Various types of carcinogenic compounds can be found in tattoo inks, including primary aromatic amines (PAA), cleavage products of organic azo colourants, found in coloured inks.1 Many cases of skin cancers occurring within tattoos have been reported, including cases of keratoacanthoma (KA) developed on red ink.2 Pathogenesis of KA on tattoos remains unclear and is probably due to multiple factors: trauma induced by the needle during the tattoo itself, inflammatory reactions, ultraviolet radiation, direct carcinogenicity of the tattoo inks and individual background

International audience; Thyroid calcification is frequent in thyroid nodules. The aim of our study was to evaluate the prevalence of calcifications in thyroid tissue samples of patients with various thyroid diseases , and to identify their composition according to their localization. Among 50 thyroid samples included, 56% were malignant (papillary carcinoma) and 44% were benign (ade-noma, multinodular goiter, Graves' disease, sarcoidosis). Calcifications were found in 95% of samples using polarised light microscopy, whereas only 12% were described in initial pathological reports. Three types were individualised and analyzed by infrared spectrometry (μFTIR): colloid calcifications composed of calcium oxalate, capsular calcifications and psammoma bodies, both composed of calcium phosphate. Of notice, psammoma bodies characterized by FE-SEM were composed of concentric structure suggesting a slow process for crystal deposition. Calcium phosphates were found only in malignant samples whereas calcium oxalate was not associated with a define pathology. Proliferation assessed by KI67 staining was high (33% of positive follicles), and RUNX2, OPN, and CD44 positive staining were detected in thyrocytes with a broad variation between samples. However, thyr-ocyte proliferation and differentiation markers were not associated with the number of crystals. TRPV5 and CaSR expression was also detected in thyrocytes. mRNA transcripts expression was confirmed in a subgroup of 10 patients, altogether with other calcium transporters such as PMCA1 or Cav1.3. Interestingly, TRPV5 mRNA expression was significantly associated with number of colloid calcifications (rho =-0.72; p = 0.02). The high prevalence of calcium oxalate crystals within colloid gel raises intriguing issues upon follicle physiology for calcium and oxalate transport.

International audience; BACKGROUND: Sarcoidosis, characterized by epithelioid granulomas, is considered to be caused by a complex interplay between genetics and environmental agents. It has been hypothesized that exogenous inorganic particles as crystalline silica could be a causal or adjuvant agent in sarcoidosis onset.OBJECTIVES: To investigate the location, frequency and physicochemical characteristics of foreign materials and mineral tissue deposits in the granulomatous area of cutaneous sarcoidosis.METHODS: Skin biopsies (n = 14) from patients diagnosed with cutaneous sarcoidosis (mean age 43 years; 11 patients with extracutaneous involvement) were investigated using polarized light examination (PLE), μFourier Transform Infra-Red (μFT-IR) spectroscopy and Field Emission Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectroscopy (FE-SEM/EDX).RESULTS: Combined PLE, μFT-IR, FE-SEM/EDX analysis allowed to characterize mineral deposits in 7/14 biopsies (50%). It identified crystalline silica (SiO2 ) inside granulomas in three biopsies and calcite (CaCO3 ) at their periphery in 4.CONCLUSION: This study emphasizes the need of using combined methods for assessment of mineral deposits in granulomatous diseases. According to the location and characteristics of deposits, we can hypothesize that SiO2 particles contribute to the granuloma formation, whereas CaCO3 deposits are related to the granuloma biology. However, the significance of the association between SiO2 deposits and sarcoidosis is still disputed.

Nephrotoxicity is a specific and serious adverse effect of cisplatin which limits its utilization. Here we present an experimental approach to decipher its mechanisms focusing on the renal distribution of Pt and Zn in cisplatin exposed mice kidneys. We combined Synchrotron Radiation (S.R.) μX-ray fluorescence with statistical treatments. μX-ray fluorescence data were collected on a set of mice biopsies after cisplatin, oxaliplatin and carboplatin injections. Even if this investigation is based on a limited number of samples, these preliminary data seem to reveal a stronger correlation between the spatial repartition of Pt and Zn for cisplatin than for the other Pt containing drugs. The cisplatin injection induced a redistribution of medullary Zn across the corticomedullary junction where histological lesions develop. These results were confirmed by evaluation of Pearson's and Manders' co-localization coefficients. These data suggest that Zn is involved in the nephrotoxicity of cisplatin. This could lead to new diagnosis, physiopathological and even therapeutical approaches.

International audience; The new rapid scan method, Flyscan mode, implemented on the DiffAbs beamline at Synchrotron SOLEIL, allows fast micro-X-ray fluorescence data acquisition. It paves the way for applications in the biomedical field where a large amount of data is needed to generate meaningful information for the clinician. This study presents a complete set of data acquired after injection of gold-cluster-enriched mesoporous silica nanospheres, used as potential theranostic vectors, into rats. While classical X-ray fluorescence investigations (using step-by-step acquisitions) are based on a limited number of samples (approximately one per day at the DiffAbs beamline), the Flyscan mode has enabled gathering information on the interaction of nanometer-scale vectors in different organs such as liver, spleen and kidney at the micrometer scale, for five rats, in only a single five-day synchrotron shift. Moreover, numerous X-ray absorption near-edge structure spectra, which are beam-time-consuming taking into account the low concentration of these theranostic vectors, were collected

International audience; Vancomycin is a widely prescribed antibiotic, but the exact nature of vancomycin-associated nephrotoxicity is unclear, in particular when considering the frequent coadministration of aminoglycosides. We describe here the initial case of a 56-year-old woman with normal renal function developing unexplained ARF without hypovolemia after administration of vancomycin without coadministration of aminoglycosides. Studying the patient’s renal biopsy specimen, we ascertained that obstructive tubular casts composed of noncrystal nanospheric vancomycin aggregates entangled with uromodulin explained the vancomycin-associated ARF. We developed in parallel a new immunohistologic staining technique to detect vancomycin in renal tissue and confirmed retrospectively that deleterious vancomycin-associated casts existed in eight additional patients with acute tubular necrosis in the absence of hypovolemia. Concomitant high vancomycin trough plasma levels had been observed in each patient. We also reproduced experimentally the toxic and obstructive nature of vancomycin-associated cast nephropathy in mice, which we detected using different in vivo imaging techniques. In conclusion, the interaction of uromodulin with nanospheric vancomycin aggregates represents a new mode of tubular cast formation, revealing the hitherto unsuspected mechanism of vancomycin-associated renal injury.

Because genetic background plays a pivotal role in humans and in various experimental models, we carefully monitored its impact on glomerular pathological characteristics during experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM-GN), using two leading mouse strains, 129S2/SvPas (129Sv) and C57bl/6J (B6J). These mice exhibited different severities of renal failure, hypertension, and glomerular lesions, according to their genetic background. In addition to the classic glomerular proliferative lesions, glomerular thrombotic microangiopathy (TMA) was found as a common genetic background-dependent histopathological hallmark of anti-GBM-GN, combined with hemolytic anemia and thrombocytopenia. Glomerular expression profiling, using microarrays and Western blot analysis in B6J TMA-resistant and 129Sv TMA-prone mice, demonstrated major differences in vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) 2 pathways, despite similar Vegfa expression levels. Further analysis revealed a lower basal glomerular endothelial Vegfr2 expression level in 129Sv TMA-prone mice compared with B6J TMA-resistant mice. This difference was even more pronounced during anti-GBM-GN, explaining why an exogenous VEGFA supply failed to rescue any 129Sv TMA lesions. Conversely, the systemic blocking of Vegfr2 amplified TMA lesions only in B6J mice. Herein, we specified the role that genetic background plays in determining, in particular, the level of Vegfr2 expression. We also demonstrated that glomerular Vegfr2-dependent TMA lesions are an underevaluated common hallmark of anti-GBM-GN in mice.

Introduction Les tatouages sont largement repandus dans la population (17 % des Francais de > 15 ans). Ils sont appliques en injectant des encres colorees dans le derme et destines a demeurer a vie, entrainant une exposition a long terme aux produits chimiques injectes. Les encres contiennent des agents potentiellement toxiques tels que le dioxyde de titane. La question de la carcinogenicite des encres reste ouverte. A ce jour, seuls les keratoacanthomes sur le rouge des tatouages semblent associes de facon non fortuite. L’objectif de notre etude etait de detecter et de caracteriser les composes chimiques presents au sein de biopsies de complications sur tatouage. Patients et methodes Nous avons realise une etude retrospective multicentrique sur 11 patients ayant presente une complication sur tatouage biopsiee. Les donnees demographiques, la presentation clinique de la reaction cutanee et la couleur de l’encre etaient collectees. Les biopsies cutanees etaient ensuite analysees en microscopie optique, puis par fluorescence X classique et induite par rayonnement synchrotron. Observations Parmi les 11 patients etudies, 9 presentaient une eruption papuleuse sur tatouage et deux une lesion tumorale. L’encre etait de couleur rouge chez 6 patients, noir chez 4 autres et turquoise chez un. L’examen en microscopie optique trouvait 4 infiltrats lymphohistiocytaires peri-vasculaire, 3 reactions lichenoides ( Fig. 1 ), 2 reactions granulomateuses, et 2 keratoacanthomes ( Fig. 2 ). L’analyse par fluorescence X revelait la presence de fer ( Fig. 1d et 2d ) et de zinc ( Fig. 1f et 2f ) dans l’ensemble des echantillons. Dans 6 cas la presence de titane ( Fig. 1e et 2e ) etait egalement trouvee, de localisation dermique ou a la racine des follicules pileux. Une correlation topographique directe pouvait systematiquement etre faite entre la localisation de ces metaux lourds et l’infiltrat inflammatoire ou tumoral. Dans 3 biopsies, une analyse complementaire par spectroscopie d’absorption X a permis de preciser l’origine exogene (lie a l’encre de tatouage) du zinc, se distinguant du zinc endogene associe a l’infiltrat inflammatoire dermique. Discussion Nous rapportons la plus grande analyse en fluorescence X de reactions cutanees sur tatouage. La presence, dans plus de la moitie des cas de titane, de localisation dermique souligne le role toxique de cet agent au cours des reactions cutanees sur tatouage. Ces resultats posent egalement la question de la toxicite du titane contenu dans les produits cosmetiques au cours de dermatose inflammatoire. Conclusion Notre etude a permis de determiner la nature des metaux lourds presents au cours des reactions cutanees sur tatouage, ainsi que leur topographie. D’autres etudes, utilisant ces techniques physico-chimiques pointues, sont necessaires pour une meilleure caracterisation des agents toxiques dans les encres et de leur imputabilite dans les reactions cutanees.

Introduction La maladie de Fabry (MF) est une maladie hereditaire liee a l’X due au deficit en une enzyme lysosomale, l’alpha-galactosidase A, responsable d’une accumulation lysosomale de globotriaosylceramide (Gb3) dans l’organisme resultant en une affection systemique. L’atteinte renale histologique est caracterisee par la presence de vacuoles intracytoplasmiques dans les podocytes en microscopie optique et par des inclusions multi-lamellaires denses aux electrons en microscopie electronique (ME). Ces lesions histologiques ne sont cependant pas specifiques de la MF. Observation Une patiente de 36 ans d’origine senegalaise, suivie pour un lupus systemique est adressee pour une proteinurie glomerulaire isolee. L’histoire lupique est marquee par des poussees cutaneo-articulaires iteratives depuis 2008 et une poussee renale en 2010 sous la forme d’une glomerulonephrite lupique de classe V traitee par rituximab et hydroxychloroquine (dose cumulee 1400 g). En 2017, apparition d’une proteinurie glomerulaire a 0,5 g/j sans hematurie ni insuffisance renale dans un contexte de poussee cutaneo-articulaire lupique. L’examen clinique etait sans particularite. Le bilan immunologique montrait une serologie lupique positive et une activation de la voie classique du complement. La biopsie renale revelait une glomerulonephrite extramembraneuse et la presence de vacuolisations intracytoplasmiques podocytaires. En ME, des inclusions intracytoplasmiques podocytaires en bulbe d’oignon formant des « corps myeliniques » etaient observees (Fig. 1). Il n’y avait pas d’argument clinique ou biologique (activite enzymatique de l’alpha-galactosidase normale) pour une MF. L’etude genetique est en cours. Discussion L’atteinte nephropathologique de la MF est typique mais n’est pas specifique. Des lesions identiques peuvent apparaitre au cours de certains traitements, notamment l’hydroxychloroquine et l’amiodarone. Nous rapportons ici un cas de podocytopathie renale induite par l’hydroxychloroquine. Il s’agit d’une complication rare de l’hydroxychloroquine, dose-dependante et generalement reversible a l’arret du traitement. Les mecanismes physiopathologiques impliquent une accumulation intralysosomale d’hydroxychloroquine qui entrainerait une inhibition des enzymes lysosomales par modification du pH lysosomal et donc une accumulation intralysosomale de glycosphingolipides comme dans la MF. L’hydroxychloroquine est egalement un puissant inhibiteur de l’autophagie, processus cellulaire particulierement important pour le fonctionnement podocytaire. Conclusion Mise en evidence d’une toxicite renale glomerulaire du plaquenil, peu decrite jusque la, mimant les lesions anatomopathologiques de la MF.

Some recently published works contest the epithelial origin of myofibroblasts, which are the major extracellular matrix producers. However, our previous studies showed that, in tubular cells, some phenotypic changes reminiscent of epithelial-to-mesenchymal transition constitute an interesting early marker that predicts the progression of fibrosis in renal grafts. We hypothesized that activated epithelial cells could directly contribute to fibrogenesis, although they remain within the tubules. Using immunohistochemistry, we studied the association between epithelial phenotypic changes (de novo expression of vimentin and intracellular translocation of β-catenin) and the production of profibrotic molecules (connective tissue growth factor, HSP47, and laminin), in tubular epithelial cells from 93 renal grafts biopsied of 77 patients. We observed the de novo production of connective tissue growth factor, HSP47, and laminin in the tubular epithelial cells displaying epithelial phenotypic changes. The score of vimentin was significantly correlated with those of connective tissue growth factor (r = 0.785, P < .0001), HSP47 (r = 0.887, P < .0001), and laminin (r = 0.836, P < .0001). The level of tubular expression of mesenchymal cell markers and profibrogenic molecules, but not graft histologic lesions according to Banff acute or chronic scores, was correlated with graft dysfunction and proteinuria at the time of biopsy (r = -0.611, P < .0001 for vimentin with estimated glomerular filtration rate) (r = 0.42, P = .0006 for vimentin with proteinuria). Our results demonstrate that the epithelial phenotypic switch is associated with an active fibrogenic process in tubular epithelial cells and with graft injury indicators. Perpetuation of this tissue injury-repair response may drive fibrogenesis in renal grafts. This "repair response" represents an interesting marker for renal graft surveillance.

Biopsies of native or transplanted kidneys in patients suffering chronic or acute renal failure are commonly stained for tissue examination and search for possible crystal deposits which are then identified by polarizing microscopy and staining by von Kossa's method revealing mainly calcium deposits. Renal biopsies presumably containing crystal deposits were analyzed with a Spotlight 400 μFTIR (Fourier Transform Infra Red) imaging system in the mid-infrared spectral range to obtain infrared maps of tissue slides at high spatial resolution, down to 10 microns. When required, an optional ATR imaging accessory was used, improving the spatial resolution by a factor four, down to 3 microns at 1000 cm−1. Among the 685 renal biopsies, 72% contained abnormal non-proteic material. Among them, 2.16% contained drug crystals (triamterene, N-acetylsulfadiazine, ciprofloxacine, indinavir, atazanavir, foscarnet, and vancomycine). We focused on foscarnet and atazanavir deposits. In the case of foscarnet-induced renal failure, two types of crystals were found in one patient. They were located in different parts of the nephron: sodium and/or calcium phosphonoformate crystals within glomerules and carbapatite in the proximal tubular cells. By contrast, atazanavir was only found in the tubular lumen of the nephron.A precise identification of crystal deposits is essential for the diagnosis of an unexplained renal failure. Common histological procedures clearly fail to identify crystals deposits accurately. In most cases, light and polarizing microscopic examination should be completed by FTIR analysis.

International audience; Severe hypertension can lead to malignant hypertension (MH) with renal thrombotic microangiopathy and hemolysis. The role of plasma heme release in this setting is unknown. We aimed at evaluating the effect of a mild plasma heme increase by hemin administration in angiotensin II (AngII)-mediated hypertensive rats. Prevalence of MH and blood pressure values were similar in AngII and AngII + hemin groups. MH rats displayed a decreased renal blood flow (RBF), increased renal vascular resistances (RVR), and increased aorta and interlobar arteries remodeling with a severe renal microcirculation assessed by peritubular capillaries (PTC) rarefaction. Hemin-treated rats with or without AngII displayed also a decreased RBF and increased RVR explained only by PCT rarefaction. In AngII rats, RBF was similar to controls (with increased RVR). PTC density appeared strongly correlated to tubular damage score (rho = −0.65, p

International audience; Calcium oxalate nephrolithiasis is a common ailment. Frequent risk factors associated with are low diuresis, dietary imbalance, inherited or acquired metabolic disorders. Another important factor, namely the presence of a mineral deposit made of apatite at the surface of the papilla, named Randall's plaque (RP), has been recently underlined. In most cases, RP which serves as a nidus for kidney stone formation is made of calcium phosphate apatite (CA). However, RP does not seem to be composed exclusively of CA. We would like to assess the case of RP where sodium hydrogen urate monohydrate (NaUr) is also present in its chemical composition. To attain this goal, a set of experiments including Environmental Scanning Electron Microscopy (ESEM) and Synchrotron Radiation Fourier Transform InfraRed (SR-FTIR) has been performed to analyze papillae of six kidneys randomly selected after they were surgically removed for cancer. NaUr crystals were found in two samples. We show through ESEM an usual morphology of RP present at the surface of kidney stones in the presence of NaUr. Moreover, we discuss the presence of NaUr in the renal parenchyma and its spatial repartition with CA. The complete set of data indicates that different biochemical mechanisms are probably involved in the pathogenesis of RP. The next step will be to establish a significant relationship between these physicochemical data and the clinical and biochemical data of the patients.

International audience; Crystal formation in kidney tissue is increasingly recognized as a major cause of severe or acute renal failure. Kidney biopsies are currently performed and analyzed using different staining procedures. Unfortunately, none of these techniques are able to distinguish the different Ca phosphates (e.g., amorphous or nanostructured Ca phosphate apatite, octacalcium phosphate, brushite…) or Ca oxalates (whewellite and weddellite). Moreover, the crystal’s morphology, a structural parameter proven as a major information to the clinician regarding kidney stones, is not taken into account. Such major limitations call for a different research approach, based on physicochemical techniques. Here we propose classical observations through field-emission scanning electron microscopy experiments combined with energy dispersive spectroscopy as well as measurements through Raman and μFourier transform Infra-Red Spectroscopy. If necessary, in the case of microcrystals, observations using cutting edge technology such as Synchrotron Radiation (SR) – FTIR or SR-UV visible spectroscopy can be subsequently performed on the same sample. Taken together this set of diagnostic tools will help clinicians gather information regarding the nature and the spatial distribution at the subcellular scale of different chemical phases present in kidney biopsies as well as on the crystal morphology and therefore obtain more precise diagnosis.

International audience; Acute tubular damage is a major cause of renal failure, especially at the early phase of kidney transplant when ischemia-reperfusion injury and cyclosporin A toxicity may coexist. The mechanisms of the latter are largely unknown. Using an mRNA microarray on microdissected tubules from a rat model of cyclosporin A toxicity to describe the related epithelial-specific transcriptional signature in vivo, we found that cyclosporin A induces pathways dependent on the transcription factor ATF4 and identified nuclear protein transcriptional regulator 1 (Nupr1), a stress response gene induced by ATF4, as the gene most strongly upregulated. Upon cyclosporin A treatment, Nupr1-deficient mice exhibited worse renal tubular lesions than wild-type mice. In primary cultures treated with cyclosporin A, renal tubular cells isolated from Nupr1-deficient mice exhibited more apoptosis and ATP depletion than cells from wild-type mice. Furthermore, cyclosporin A decreased protein synthesis and abolished proliferation in wild-type tubular cells, but only reduced proliferation in Nupr1-deficient cells. Compared with controls, mouse models of ischemia-reperfusion injury, urinary obstruction, and hypertension exhibited upregulated expression of renal NUPR1, and cyclosporin A induced Nupr1 expression in cultured human tubular epithelial cells. Finally, immunohistochemical analysis revealed strong expression of NUPR1 in the nuclei of renal proximal tubules of injured human kidney allografts, but not in those of stable allografts. Taken together, these results suggest that epithelial expression of NUPR1 has a protective role in response to injury after renal transplant and, presumably, in other forms of acute tubular damage.

International audience; In this contribution, an approach developed previously for mice is used for human biopsy. In the case of patient 1, Pt detection is performed 6 days after the last oxaliplatin infusion, while for patient 2, the biopsy was performed more than 15 days after his first platin infusion and several dialysis. Even for these biological samples, experiments show that synchrotron mediated mXRF is a suitable tool to detect Pt in kidney biopsy, and thus probably for any organ exposed to Pt. Therefore, mXRF could also be of major interest to decipher the mechanism beyond Pt induced neurotoxicity, ototoxicity on human biopsy. Pharmacoavailability of chemotherapies is a major concern because some treatment failures are explained by poor tumor penetration of the active molecule. mXRF could be an elegant way to map the distribution of Pt inside cancerous cells at the micrometer scale. Pt and Zn are only two of the numerous trace elements that mXRF can detect; heavy metal intoxication diagnosis and the toxicity mechanism probably could also benefit from this innovative technique.

Introduction La calciphylaxie uremique est rare chez les patients dialyses mais engage le pronostic vital. L’histologie cutanee montre des calcifications arteriolaires profondes et une panniculite necrotique. L’objectif de notre etude etait de caracteriser sur le plan chimique les depots cutanes observes au cours de la calciphylaxie, d’etudier leur localisation subcellulaire et leurs interactions tissulaires. Materiel et methodes Nous avons inclus dans une etude multicentrique retrospective (2006–2017), les patients ayant presente une calciphylaxie uremique typique sur le plan clinique et histologique. Les biopsies cutanees des patients et de 5 temoins presentant une arteriosclerose cutanee etaient analysees en microscopie optique (MO) avant d’etre etudiees par spectroscopie infrarouge (IR) et microscopie electronique a balayage (MEB) couplee a la micro-analyse X (EDX). Resultats Trente-six patients (26 F/10 H) ont ete inclus, d’âge moyen 64 ans . Trente (80 %) etaient dialyses, 33 (92 %) hypertendus, 23 (64 %) diabetiques et 16 (44 %) traites par anti-vitamines K. Le delai median entre le debut de la dialyse et la survenue de la calciphylaxie etait de 24 mois. Les manifestations dermatologiques etaient majoritairement la necrose (72 %) et l’ulceration (67 %), sur les membres inferieurs (92 %). Vingt neuf biopsies cutanees ont ete analysees. En IR, les depots etaient constamment composes de carbapatite (phosphate de calcium carbonate cristallise). L’etude en MEB montrait des calcifications arteriolaires constantes, massives, circonferentielles ( Fig. 1 A) des vaisseaux dermiques profonds et hypodermiques, differentes des calcifications localisees a la media de l’arteriosclerose ( Fig. 1 B). Elles etaient associees a des calcifications hypodermiques, en plaques ou spheriques disposees en cadre autour des adipocytes ( Fig. 2 ). Discussion Nous presentons la 1ere etude chimique et morphologique des depots cutanes observes dans la calciphylaxie uremique : il s’agit constamment de carbapatite, de localisation vasculaire et tissulaire, dans le derme profond et l’hypoderme. L’atteinte constante de l’ensemble de la paroi vasculaire est probablement responsable de la thrombose arteriolaire, entrainant une necrose adipocytaire. La liberation de phospholipides qui s’en suivrait pourrait servir de noyau de nucleation des depots calciques peri-adipocytaire, a confirmer par des experiences complementaires. La caracterisation precise de la nature chimique et de la localisation des depots calciques cutanes justifie l’approche therapeutique par le thiosulfate de sodium, explique l’absence d’efficacite des vasodilatateurs et incite a developper des traitements axes sur l’inhibition de la formation de cristaux phosphocalciques. D’autres etudes utilisant ces techniques physico-chimiques seront necessaires pour une meilleure comprehension de la physiopathologie de la calciphylaxie et de potentielles nouvelles pistes therapeutiques.

Introduction Apres une transplantation renale, 4 a 78 % des patients presentent des calcifications du greffon. L’abondance de celles-ci est correlee au pronostic du greffon, avec une moins bonne survie renale en cas de nombreuses calcifications. Nous avons mis au point un modele murin de nephropathie cristalline ischemique afin d’etudier la cinetique d’apparition des cristaux, leurs compositions, localisations et le role d’une proliferation tubulaire precoce ou retardee apres insuffisance renale aigue ischemique. Materiels et methodes Souris femelles C57BL6 J ayant un regime comprenant glucose phosphate disodique et gavage par ergocalciferol soumises a une ischemie renale de 45 minutes avec nephrectomie controlaterale (IRA). En fonction des groupes, induction (ou non) d’une proliferation tubulaire a j2 par l’administration intraperitoneale de FGF7 en fin de chirurgie. Analyse histologique, infrarouge, en microscopie electronique a balayage et immunohistochimique des reins a differents temps pour les groupes IRA avec et sans FGF7, IRA avec et sans diete phosphatee et mesure des clairances de l’inuline a j30. Resultats On observe a j2 des depots de lipoproteines et de carbapatite a la jonction corticomedullaire (JCM) et une plus faible proportion de cristaux medullaires (CM). Leur nombre est majore dans le groupe IRA + FGF7. A j6, ces depots sont tres abondants. A j30, on observe une clairance presque complete des cristaux corticaux et de JCM. Les CM persistent, mais sont moins abondants dans le groupe FGF7. On n’observe pas de cristaux dans les groupes sans diete enrichie en phosphates (PO4). Nos resultats preliminaires suggerent une diminution de la clairance de l’inuline a j30 dans le groupe IRA + diete PO4 par rapport aux groupes IRA + diete PO4 + FGF7 et IRA sans diete PO4. Discussion La presence d’une diete PO4 lors d’une IRA est responsable a court terme de calcifications de JCM reversibles, mais de CM tubulaires persistantes a j30 qui semblent deleteres sur la fonction renale. L’administration de FGF7 au moment de l’IRA, responsable d’une proliferation tubulaire precoce, semble benefique sur les calcifications et la fonction renale a long terme en conditions de sursaturation urinaire. Conclusion Ce travail ouvre la perspective d’une utilisation de FGF7 au decours d’une greffe renale afin d’ameliorer le pronostic renal a long terme.

During human glomerulonephritis, the severity of injuries correlates with glomerular fibrin deposits, which are tightly regulated by the intraglomerular fibrinolytic system. Here, we evaluated the role of vitronectin (VTN; also known as complement S protein), the principal cofactor of the plasminogen activator inhibitor-1 (PAI-1), in a mouse model of acute glomerulonephritis. We found that in mice subjected to nephrotoxic serum, the absence of VTN resulted in a lower glomerular PAI-1 activity and a higher glomerular fibrinolytic activity. Challenged VTN(-/-) mice displayed significantly less fibrin deposits, proteinuria, and renal failure than their wild-type counterparts. Notably, this protective effect afforded by VTN deficiency was still observed after a C3 depletion. Finally, the injection of VTN(+/+) serum in VTN(-/-) mice induced the glomerular deposition of VTN, increased PAI-1 deposition, decreased glomerular fibrinolytic activity, and aggravated glomerular injury. As in mice, abundant glomerular VTN deposits were also observed in patients with severe glomerulonephritis. Here, we show that plasma-exchange therapy, admittedly beneficial in this clinical context, induces a significant depletion in circulating VTN, which might modulate PAI-1 activity locally and accelerate the clearance of fibrin deposits in the glomeruli. Collectively, these results demonstrate that VTN exerts a deleterious role independently from complement, by directing PAI-dependent fibrinolysis in the glomerular compartment.

Introduction A partir d’une etude transcriptomique differentielle de rein de rats hypertendus et nephrotiques, nous avons identifie et etudie, pour la premiere fois, l’Isthmine-1 (ISM-1), une molecule impliquee dans le syndrome nephrotique (SN). Resultats L’immunohistochimie montre que l’ISM-1 est exprimee physiologiquement dans le rein chez le rat, la souris et chez l’homme. Son expression est podocytaire en microscopie electronique, confirmee par une co-localisation avec la nephrine. Puisque ses deux recepteurs : l’integrine alphaV beta5 et GRP78 (Bip) sont connus pour etre impliques respectivement dans la permeabilite vasculaire et dans des glomerulopathies, nous avons etudie l’ISM-1 au cours de 2 modeles experimentaux de SN chez le rat par l’injection de puromycine d’aminonucleoside (PAN) et d’adriamycine (ADR). Dans ces 2 modeles, nous avons observe une augmentation du taux d’ARNm d’ISM-1 ainsi que de ses recepteurs dans les glomerules isoles, contrastant avec la diminution de l’expression de la nephrine. Afin de demontrer l’implication de l’ISM-1 dans le SN, nous avons inhibe son expression par l’administration d’antisens anti-ISM-1 a des rats nephrotiques PAN et ADR. Chez les rats PAN, l’inhibition de l’ISM-1 ne modifie pas l’evolution de la maladie probablement par une severite importante du modele. Nous poursuivons l’investigation des rats ADR dont le developpement de la maladie est plus progressif. En parallele, nous avons incube des podocytes humains en culture avec des doses croissantes d’ISM-1 recombinante et observe une diminution de la viabilite cellulaire. Cette baisse de la viabilite des podocytes s’accompagne d’une inhibition de l’autophagie visible par l’accumulation de la proteine P62 et d’une induction de la caspase 8 active, proteine impliquee dans l’apoptose. L’immuno-precipitation de GRP78 et de l’integrine avb5 a partir des glomerules nous a permis de confirmer l’interaction de l’ISM-1 et avec ses recepteurs dans le rein. Discussion Nous developpons actuellement des vecteurs viraux qui seront injectes a la souris pour induire une forte expression d’ISM-1. Nous attendons que ces souris developpent une maladie glomerulaire. Conclusion Nos resultats montrent pour la premiere fois que l’ISM-1 a une expression physiologique podocytaire. Son expression et celle de ses recepteurs sont augmentees au cours des syndromes nephrotiques experimentaux. In vitro, L’ISM-1 semble avoir un role deletere sur les podocytes par inhibition de l’autophagie.

Introduction La sarcoidose est une maladie inflammatoire systemique de cause inconnue. Elle touche preferentiellement le systeme lymphatique et le poumon. L’atteinte renale est rare, responsable d’une nephropathie interstitielle granulomateuse. L’hypercalciurie et l’hypercalcemie sont frequentes et contribueraient a la formation des cristaux susceptibles d’induire des lithiases, des coliques nephretiques, voire une nephrocalcinose. La prevalence et la nature des calcifications au cours de la sarcoidose renale sont inconnues. De plus, les differentes techniques de depistage des cristaux dont la coloration a base de nitrate d’argent, n’ont jamais ete evaluees. Objectifs Nous avons etudie retrospectivement les biopsies renales des patients ayant presente une sarcoidose renale de l’hopital Tenon entre 2000 et 2017 avec pour objectifs de preciser la prevalence et la nature des cristaux renaux et determiner la technique la plus performante pour leur depistage. Patients/materiels et methodes Les fragments biopsiques renaux inclus en paraffine ont ete examines selon les modalites suivantes : examen en lumiere polarisee (LP) et directe (LD) d’une coupe non coloree, examen en LP et LD d’une coupe coloree par l’hematoxiline-eosine (HE), examen en lumiere directe d’une coupe coloree par le Yasue (specifique du phosphate de calcium) et etude en microspectroscopie infrarouge. Les fragments biopsiques renaux congeles ont ete examines selon les modalites suivantes : examen en LP et LD d’une coupe non coloree et microspectroscopie infrarouge. Resultats Sur les 54 patients biopsies pour une sarcoidose, nous disposons de tissu inclus en paraffine et de tissu congele pour, respectivement, 35 et 33 patients ; les 2 types de tissu etaient disponibles pour 21 patients. Sur les colorations HE et de Yasue, nous avons retrouve au moins une calcification renale en LD et/ou LP chez 10 (28,6 %) et 9 (28,1 %) patients respectivement. Des cristaux etaient visibles uniquement chez 4 patients a partir des coupes non colorees de tissu inclus en paraffine. L’examen des coupes non colorees de tissu congele a mis en evidence des cristaux chez 14 patients en LD (42 %) et 16 en LP (48 %). La nature des cristaux est en cours de determination grâce a une etude en microspectroscopie infrarouge avec transformation de Fourier. Discussion L’observation de coupes non colorees de tissu renale congele semble etre la technique la plus sensible pour la mise en evidence de calcifications renales. La prevalence des calcifications est elevee au cours de la sarcoidose renale. Nous disposerons pour la premiere fois de la nature precise de toutes ces calcifications.

Antibody-mediated rejection (ABMR) is a leading cause of allograft loss. Treatment efficacy depends on accurate diagnosis at an early stage. However, sensitive and reliable markers of antibody-endothelium interaction during ABMR are not available for routine use. Using immunohistochemistry, we retrospectively studied the diagnostic value of three markers of endothelial-to-mesenchymal transition (EndMT), fascin1, vimentin, and heat shock protein 47, for ABMR in 53 renal transplant biopsy specimens, including 20 ABMR specimens, 24 cell-mediated rejection specimens, and nine normal grafts. We validated our results in an independent set of 74 unselected biopsy specimens. Endothelial cells of the peritubular capillaries in grafts with ABMR expressed fascin1, vimentin, and heat shock protein 47 strongly, whereas those from normal renal grafts did not. The level of EndMT marker expression was significantly associated with current ABMR criteria, including capillaritis, glomerulitis, peritubular capillary C4d deposition, and donor-specific antibodies. These markers allowed us to identify C4d-negative ABMR and to predict late occurrence of disease. EndMT markers were more specific than capillaritis for the diagnosis and prognosis of ABMR and predicted late (up to 4 years after biopsy) renal graft dysfunction and proteinuria. In the independent set of 74 renal graft biopsy specimens, the EndMT markers for the diagnosis of ABMR had a sensitivity of 100% and a specificity of 85%. Fascin1 expression in peritubular capillaries was also induced in a rat model of ABMR. In conclusion, EndMT markers are a sensitive and reliable diagnostic tool for detecting endothelial activation during ABMR and predicting late loss of allograft function.

International audience; Noninfectious mixed cryoglobulinemic GN (MCGN) has been poorly investigated. We analyzed presentation and outcome of 80 patients with biopsy-proven MCGN, which were identified in the retrospective French CryoVas survey. MCGN was related to primary Sjögren's syndrome in 22.5% of patients and to lymphoproliferative disorders in 28.7% of patients, and was defined as essential in 48.8% of patients. At presentation, hematuria, proteinuria ≥1 g/d, hypertension, and renal failure were observed in 97.4%, 84.8%, 85.3%, and 82.3% of cases, respectively. Mean±eGFR was 39.5±20.4 ml/min per 1.73 m2. Membranoproliferative GN was the predominant histologic pattern, observed in 89.6% of cases. Renal interstitium inflammatory infiltrates were observed in 50% of cases. First-line treatment consisted of steroids alone (27.6%) or in association with rituximab (21.1%), alkylating agents (36.8%) or a combination of cyclophosphamide and rituximab (10.5%). After a mean follow-up of 49.9±45.5 months, 42.7% of patients relapsed with a renal flare in 75% of cases. At last follow-up, mean eGFR was 50.2±26.1 ml/min per 1.73 m2 with 9% of patients having reached ESRD; 59% and 50% of patients achieved complete clinical and renal remission, respectively. A rituximab+steroids regimen prevented relapses more effectively than steroids alone or a cyclophosphamide+steroids combination did, but was associated with a higher rate of early death when used as first-line therapy. Severe infections and new-onset B-cell lymphoma occurred in 29.1% and 8.9% of cases, respectively; 24% of patients died. In conclusion, noninfectious MCGN has a poor long-term outcome with severe infections as the main cause of death

Epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs) may participate in the pathogenesis of renal fibrosis. We performed a prospective study of EMT markers in protocol biopsies obtained 3 months after engraftment from 56 patients who received deceased donor kidneys and who had stable renal function. The presence of EMT was examined, and quantified by immunohistochemical staining for vimentin and translocation of beta-catenin to the cytoplasm. EMT status was defined as the presence of EMT markers inor = 10% of TECs. EMT features were virtually absent in implantation biopsies, whereas 41% of the grafts were EMT-positive in the absence of advanced chronic allograft nephropathy. Thirteen patients (23%) had borderline changes or acute rejection. EMT features were more frequent in these patients than in those with normal kidney grafts (vimentin expression, p = 0.003; beta-catenin translocation, p = 0.002). EMT in grafts corresponded with elevated serum creatinine of the donor before the recovery of kidney (p = 0.02) and longer cold ischemia time (p = 0.02). In contrast, the donor age had no influence on the expression of EMT markers. These results suggest that EMT is an early and frequent phenomenon in kidney transplants that could be triggered by immunological and/or ischemic tubular injury.

The myelin protein 0 (MPZ or P0) is a transmembrane glycoprotein that represents the most abundant myelin component. Mutations in the P0 gene are associated with one form of autosomal dominant demyelinating peripheral neuropathy, Charcot-Marie-Tooth disease type 1B (CMT1B). Because CMT1 may be associated with renal involvement, mostly focal segmental glomerulosclerosis, we hypothesized that P0 could be expressed in the kidney. P0 mRNA was detected by reverse transcriptase–PCR in the human and mouse renal cortex. P0 transcripts were identified by in situ hybridization at different stages of the mouse kidney development, especially in embryonic structures that give rise to the glomerulus. P0 protein was also detected by Western blot in human and rat glomerular extracts and in a human podocyte cell line using a monoclonal anti-P0 antibody. Immunofluorescence studies on human kidney sections showed that the podocytes were intensely labeled. Immunogold electron microscopy disclosed a predominant staining of the membranes of intracellular vesicles in podocytes. P0 was also detected in the podocyte cell membrane, including at the foot processes. P0 −/− mice exhibited mild growth retardation and demyelinating neuropathy similar to the one observed in patients with CMT1B. They also presented mild albuminuria, without significant ultrastructural change of the glomerular basement membrane or the podocytes. These results demonstrate that P0, the major myelin protein, is also expressed during nephrogenesis and in mature kidney, mostly in podocytes. They suggest that P0 gene mutations might be involved in renal diseases.

Introduction La sarcoidose est caracterisee histologiquement par la presence de granulomes epithelioides sans necrose caseeuse. L’atteinte cutanee est frequente et la presentation clinique variee. Certaines etudes ont suspecte le role de particules minerales environnementales dans la formation des lesions sarcoidosiques. L’objectif de notre etude etait de detecter et de caracteriser d’eventuelles particules minerales au sein et en peripherie des granulomes. Patients et methodes Nous avons conduit une etude retrospective sur 10 patients ayant une sarcoidose avec atteinte cutanee, de janvier 2010 a decembre 2015. Les biopsies cutanees ont ete analysees en microscopie optique standard couplee a un examen en lumiere polarisee, avant d’etre etudiees par spectroscopie infrarouge (IR) et microscopie electronique a balayage (MEB) couplee a la micro-analyse X (EDX). Resultats Notre avons etudie les biopsies cutanees de 7 hommes et 3 femmes, d’âge moyen 43,5 ans. Les lesions cutanees etaient le plus souvent des papules (7 patients). La biopsie cutanee a ete realisee sur les membres pour 6 patients, le visage pour 3 et le dos pour un. L’etude en microscopie optique trouvait systematiquement des granulomes epithelioides gigantocellulaires sans necrose caseeuse. Ils etaient localises dans le derme chez 9 patients et dans l’hypoderme chez un. L’examen en lumiere polarisee detectait la presence d’elements birefringents au centre des granulomes dans 3 cas. L’etude par IR, puis par MEB couplee a l’EDX permettait d’affirmer que ces elements correspondaient a de la silice minerale cristalline SiO2. Il s’agissait pour deux patients, d’un corps etranger intragranulomateux unique d’environ 10 μm et pour un autre, de plusieurs elements d’1 μm en amas. Par ailleurs, il etait egalement identifie dans 4 cas, en peripherie des granulomes, la presence de depots de carbonate de calcium. Discussion L’association inconstante de silice avec les granulomes de sarcoidose peut etre expliquee par (1) un role inducteur ou adjuvant dans la survenue de la sarcoidose, (2) une reaction granulomateuse au contact de corps etrangers chez les sujets developpant une sarcoidose, (3) le recrutement dans les granulomes de macrophages ayant phagocyte une particule de SiO2. Les desordres phosphocalciques induits par les granulomes pourraient, par ailleurs, expliquer les depots calciques perigranulomateux. Conclusion Nous presentons la premiere etude des granulomes sarcoidosiques par IR, MEB et EDX. Elle a permis la detection de silice cristalline intragranulomateuse chez 3 patients, possible facteur precipitant la maladie, et la detection de carbonate de calcium chez 4 patients en peripherie des granulomes, probablement produit par les granulomes. Des etudes prospectives portant sur un plus grand nombre de patients sont necessaires pour une meilleure comprehension de la maladie.

Cyclosporine (CsA), a widely used immunosuppressive drug, is an effective treatment of sight-threatening posterior idiopathic uveitis. CsA's main side effect is nephrotoxicity. The aim of this single-center prospective cohort study (conducted in a tertiary care teaching hospital in Paris, France) was to assess the long-term renal tolerance of a low-dose CsA treatment in patients with previously healthy kidneys on clinical, biologic, and pathologic criteria. Forty-one patients treated with 4.3 +/- 1.6 mg/kg body wt per day CsA for 44.9 +/- 3.6 mo were included. Mean follow-up was 55.4 +/- 0.2 mo. BP, CsA trough level, and renal function were prospectively monitored together with blood urea, creatinine clearance, GFR, and effective renal plasma flow. Eleven patients underwent serial kidney biopsies before and after 2 yr of a 4 +/- 0.9 mg/kg daily CsA treatment. Sustained low-dose CsA treatment induced a significant increase in plasma creatinine (P < 0.0001), a significant decrease in creatinine clearance (P < 0.0001), and isotopic GFR (P < 0.0001) over time. The highest dose induced more severe alterations in any of the renal parameters than the lowest dose. Prevalence of hypertension was particularly high. Histopathologic data showed significant interstitial fibrosis (P < 0.003) and tubular atrophy (P < 0.003) after 2 yr. Low-dose long-term CsA treatment induces significant renal impairment and a high incidence of hypertension. Our study suggests that lowering daily dosage may prevent CsA-induced nephrotoxicity if a daily dose of < or =3 mg/kg is used. Whether once established it is reversible is still prospective, although the occurrence of interstitial fibrosis in the kidney would argue against reversibility.

Background. The epithelial response to injury is stereotypical and reminiscent of epithelial-to-mesenchymal transitions (EMTs), such as those observed during embryogenesis and tumour metastasis. In the context of solid organ transplantation, EMT-like features are often acquired by epithelial cells and are predictive of graft fibrosis. Here, we studied the possible involvement of several major transcriptional regulators, including snail1, phospho-Smad 2/3 and zeb1, in EMT induction in human renal grafts. Methods. We used immunohistochemistry to detect the presence of these EMT transcriptional regulators along with that of two validated EMT markers (intra-cytoplasmic translocation of β-catenin, de novo expression of vimentin), in 103 renal graft biopsy samples taken for routine surveillance or for a clinical indication. Results. We observed the nuclear accumulation of snail1 and phospho-smad2/3 in tubular cells displaying EMT. The level of snail1 was significantly correlated with the scores of EMT markers (β-catenin: ρ= 0.94, P < 0.0001; vimentin: ρ= 0.93, P < 0.0001) and with deteriorated graft function and proteinuria at the time of biopsy. Furthermore, intense staining for both snail1 and vimentin in tubular cells (≥10% of tubules) was predictive of graft dysfunction 21 months post-biopsy, independently of the other known risk factor for long-term graft dysfunction. In contrast, in both normal and diseased graft, zeb1 expression was detected exclusively in the endothelial cells of glomeruli and peritubular capillaries. Conclusion. This study suggests that snail1 is closely related to the fibrogenic, EMT-like response of the tubular epithelium in human renal grafts and predictive of graft function loss.

Background Erythropoietin (Epo) is a growth factor whose synthesis mainly takes place in the kidney. Epo has been shown to support the growth not only of erythroid progenitor cells but also of certain other cell types. We attempted to establish whether Epo enhances the recovery from acute renal failure induced by cisplatin. Methods Sprague-Dawley rats were randomized into three groups. In the cisplatin group, animals received one intraperitoneal injection of cisplatin (6 mg/kg) and a daily injection of placebo for 9 days. In the cisplatin+Epo group, animals received intrapertoneal cisplatin and a daily injection of Epo (100 IU/kg) for 9 days. In the control group, animals received both placebo preparations alone. Para-aminohippuric acid and inulin clearances were determined after 4 and 9 days to evaluate renal blood flow and glomerular filtration rate. In addition, light microscopy and immunohistochemistry examinations were performed, and in situ proliferating cell nuclear antigen (PCNA) staining was done to estimate the degree of renal tubular cell regenerative activity. The potential role of epithelial growth factor (EGF) was evaluated by semi-quantitative assessment of EGF immunostaining. Results Renal blood flow and glomerular filtration rate decreased significantly in cisplatin and cisplatin+Epo groups versus control group at day 4. However, at day 9, they both were significantly greater in cisplatin+Epo-treated animals than in rats that had received cisplatin alone. Tubular cell regeneration was significantly enhanced at day 4 in cisplatin+Epo group, compared with cisplatin and control groups respectively. EGF immunostaining was not significantly different between the three groups. Conclusion Epo significantly enhanced the rate of recovery from acute renal failure induced by cisplatin. PCNA staining indicated that Epo might act directly via stimulation of tubular cell regeneration.

The nephrotoxic potentials of a high-osmolar contrast medium, diatrizoate, and of a low-osmolar contrast medium, ioxaglate, were compared during early degenerative gentamicin-induced nephropathy in the rat. Male rats (13-22/group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery, and either diatrizoate or ioxaglate was administered (1 ml/min for 3 min) via an aortic puncture into the remaining kidney. Some of the rats received chronic treatment with gentamicin (50 mg/kg/day i.m., 4 days), starting 2 days before and ending 1 day after contrast medium administration. Two control groups, only one of which received gentamicin, were subjected to a 3-min renal ischemia. The creatinine clearance (CrCl) per 100 g body weight was determined before and 24 and 48 h after contrast medium injection. A second study (6 rats/group) evaluated urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion and the histologic appearance of the kidneys (blinded analysis) in the same experimental groups. Gentamicin induced a significant decrease in CrCl at baseline (0.35 +/- 0.19 vs. 0.41 +/- 0.19 ml/min; p0.01) and an increase in urinary NAG (128 +/- 92 vs. 39 +/- 57 mumol/h/mmol creatinine; p0.01). Taking into account these differences at baseline, univariate repeated-measures analysis showed that on day 1 diatrizoate caused a more marked decrease in CrCl than ioxaglate (p0.05), whether or not gentamicin was also administered. On day 2, the depressant effect of diatrizoate associated with gentamicin persisted (CrCl vs. day 0 = -0.19 +/- 0.10 ml/min), while that of diatrizoate alone returned to baseline (-0.05 +/- 0.24 ml/min).(ABSTRACT TRUNCATED AT 250 WORDS)

International audience; This study investigated the role of discoidin domain receptor 1 (DDR1), a collagen receptor that displays tyrosine-kinase activity, in the development of glomerulonephritis. Crescentic glomerulonephritis was induced in DDR1-deficient mice and their wild-type (WT) littermates as controls, by injection of alloimmune sheep nephrotoxic serum (NTS). Histological, functional and transcriptomic studies were performed. Glomerulonephritis produced a 17-fold increase of DDR1 expression, predominantly in glomeruli. DDR1 deletion protected NTS-treated mice against glomerular disease (proteinuria/creatininuria 5.5±1.1 vs. 13.2±0.8 g/mmol in WT, crescents 12±2 vs. 24±2% of glomeruli, urea 16±2 vs. 28±5 mM), hypertension (123±11 vs. 157±8 mmHg), and premature death (70 vs. 10% survival) (all P

International audience; Thrombospondin-1 (TSP-1) is an endogenous activator of transforming growth factor-β (TGF-β), and an anti-angiogenic factor, which may prevent kidney repair. Here we investigated whether TSP-1 is involved in the development of chronic kidney disease using rats with unilateral ureteral obstruction, a well-known model to study renal fibrosis. Obstruction of 10 days duration induced inflammation, tubular cell atrophy, dilation, apoptosis, and proliferation, leading to interstitial fibrosis. TSP-1 expression was increased in parallel to that of collagen III and TGF-β. Relief of the obstruction at day 10 produced a gradual improvement in renal structure and function, the reappearance of peritubular capillaries, and restoration of renal VEGF content over a 7- to 15-day post-relief period. TSP-1 expression decreased in parallel with that of TGF-β1 and collagen III. Mice in which the TSP-1 gene was knocked out displayed less inflammation and had better preservation of renal tissue and the peritubular capillary network compared to wild-type mice. Additional studies showed that the inflammatory effect of TSP-1 was mediated, at least in part, by monocyte chemoattractant protein-1 and activation of the Th17 pathway. Thus, TSP-1 is an important profibrotic and inflammatory mediator of renal disease. Blockade of its action may be a treatment against the development of chronic kidney disease.

International audience; Recent findings in idiopathic membranous nephropathy (MN) suggest that in most patients, the disease is because of anti-phospholipase A(2) receptor (PLA(2) R1) autoantibodies. Our aim was to analyze the prevalence and significance of anti-PLA(2) R1 antibodies in recurrent and de novo MN after transplantation. We assessed circulating PLA(2) R1 autoantibodies by a direct immunofluorescence assay based on human embryonic kidney cells transfected with a PLA(2) R1 cDNA, and the presence of PLA(2) R1 antigen in immune deposits. We showed that PLA(2) R1 was involved in 5 of 10 patients with recurrent MN, but in none of the 9 patients with de novo MN. We also showed a marked heterogeneity in the kinetics and titers of anti-PLA(2) R1, which may relate to different pathogenic potential. We provide evidence that some patients with PLA(2) R1-related idiopathic MN and anti-PLA(2) R1 antibodies at the time of transplantation will not develop recurrence. Because PLA(2) R1 autoantibody was not always associated with recurrence, its predictive value should be carefully analyzed in prospective studies.

The transcription factor Snail is an important repressor of E-cadherin gene expression. It plays a key role in the induction of epithelial-mesenchymal transition, an essential process important not only in embryonic development and tumor progression but also in organ fibrogenesis. We studied the expression of Snail by immunohistochemistry, along with several epithelial phenotypic changes suggestive of epithelial-mesenchymal transition, in 14 patients with multiple myeloma cast nephropathy. This nephropathy is characterized by a rapid progression toward fibrosis. As controls, we used normal kidneys and kidneys from patients displaying an idiopathic nephrotic syndrome, a syndrome unassociated with renal fibrosis. We discovered that, in all patients with multiple myeloma nephropathy, a drastic accumulation of Snail is seen in the nuclei from tubular epithelial cells showing epithelial phenotypic changes. In contrast, normal and idiopathic nephrotic syndrome kidneys did not exhibit either of these markers. Snail, a major player in the process of epithelial-to-mesenchymal transition, is highly expressed by tubular epithelial cells during multiple myeloma nephropathy. It is, therefore, a potential target to prevent multiple myeloma kidneys from fibrosing. Intranuclear accumulation of Snail is a characteristic in phenotypically altered tubular cells from multiple myeloma kidneys. The epithelial-mesenchymal transition pathway could, therefore, be involved in the rapid renal fibrogenesis observed in this setting.

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is accumulated in plasma during chronic kidney disease (CKD). It is considered an independent mortality and cardiovascular risk factor in CKD patients. To test the involvement of ADMA in CKD progression, we investigated the effects of chronic ADMA administration on renal structure and compared these effects with NG-nitro-L-arginine methyl ester (L-NAME) treatment, a widely used exogenous inhibitor of NOS that induces CKD. Three groups of uninephrectomized mice were studied: ADMA (60 mg/kg per day), L-NAME (60 mg/kg per day), and isotonic saline (control) were infused through osmotic mini-pumps for 8 weeks. ADMA and L-NAME induced hypertension (PAS 167 ± 16 and 168 ± 10 versus 100 ± 4 mmHg, p < 0.01, respectively). High level of ADMA was associated with increased renal oxidative stress. ADMA treatment induced glomerular and vascular fibrosis as evidenced by the elevated deposits of collagen I, III, and fibronectin (p < 0.01). A similar profile was observed in the L-NAME group. Mice treated with ADMA had reduced peritubular capillaries versus controls (p < 0.01). Collagen I mRNA expression and renal TGF-β1 concentrations were higher in the ADMA and L-NAME groups. Increased level of TGF-β1 was associated with a significant rise of HIF-1α and endothelin-1 expression. These results demonstrate for the first time that elevated concentrations of ADMA are associated with the development of renal fibrosis. These data suggest that in pathophysiological conditions of endothelial dysfunction, the exaggerated endogenous synthesis of ADMA could contribute to CKD progression by favouring hypertension, extracellular matrix synthesis, and rarefaction of peritubular capillaries.

Renal fibrosis is defined by the exaggerated accumulation of extracellular matrix proteins. Tissue transglutaminase (TG2) modifies the stability of extracellular matrix proteins and renders the extracellular matrix resistant to degradation. In addition, TG2 also activates transforming growth factor-beta (TGF-beta). We investigated the involvement of TG2 in the development of renal fibrosis using mice with a knockout of the TG2 gene (KO). These mice were studied at baseline and 12 days after unilateral ureteral obstruction, which induced a significant increase in interstitial TG2 expression in wild-type mice (P < 0.001). Interstitial fibrosis was evident in both groups, but total and fibrillar collagen was considerably lower in KO mice as compared with wild-type (P < 0.001). Similarly, mRNA and protein expression of collagen I were significantly lower in KO animals (P < 0.05). A statistically significant reduction in renal inflammation and fewer myofibroblasts were observed in KO mice (P < 0.01). Free active TGF-beta was decreased in KO mice (P < 0.05), although total (active + latent) TFG-beta concentration did not differ between groups. These results show that mice deficient in TG2 are protected against the development of fibrotic lesions in obstructive nephropathy. This protection results from reduced macrophage and myofibroblast infiltration, as well as from a decreased rate of collagen I synthesis because of decreased TGF-beta activation. Our results suggest that inhibition of TG2 may provide a new and important therapeutic target against the progression of renal fibrosis.

Introduction La vancomycine est un antibiotique largement prescrit et dont la nephrotoxicite est toujours discutee. Les excipients historiques ou son association aux aminosides ont pu etre mis en cause. Observation Nous decrivons ici le cas d’une patiente de 56 ans diabetique, obese (IMC = 40 kg/m2), normorenale et atteinte de leucemie aigue myeloblastique de type 2. Elle a developpe une insuffisance renale aigue apres administration intraveineuse de vancomycine seule (3 grammes par jour pendant 3 jours) prescrite en raison d’une aplasie febrile sans defaillance hemodynamique, et dans les suites d’une chimiotherapie ne comportant aucun nephrotoxique. A j3, la concentration residuelle en vancomycine etait de 87 mg/L. La fonction renale s’etant degradee (creatininemie 372 μmol/L) le traitement fut interrompu. La proteinurie etait mesuree a 0,10 g/L, et le sediment urinaire etait inactif. Une biopsie renale fut finalement realisee a j18, montrant des lesions epitheliales tubulaires severes, avec un infiltrat inflammatoire interstitiel important et des depots intra-tubulaires atypiques et granuleux, peu polarisants et faiblement refringents. Une immuno-histochimie (IHC) et une analyse en spectrometrie infrarouge indiquent que ces formations sont constituees de vancomycine. Ces images, amorphes, etaient aussi retrouvees dans les urines a l’examen direct. Nous avons induit la meme toxicite dans un modele animal (deux injections de 25 mg de vancomycine par voie intraperitoneale a des souris adultes) avec une insuffisance renale aigue a j2, une necrose tubulaire d’aspect similaire avec des depots granuleux intra-tubulaires ayant eux-aussi le spectre infrarouge de la vancomycine. Conclusion Notre cas demontre une toxicite tubulaire aigue de la vancomycine, revelee par une necrose tubulaire avec precipitation intra-tubulaire de vancomycine, attestee par immuno-histochimie et par spectrometrie infrarouge. Ces lesions sont aussi confirmees experimentalement chez la souris. Pour nous, l’imputabilite est donc certaine.

Introduction L’hyperoxalurie par la formation de depots d’oxalate de calcium (CaOx) dans le rein qu’elle entraine est une cause rare d’insuffisance renale chronique. Les techniques de reference pour detecter les cristaux dans les biopsies renales sont la microscopie a polarisation qui est peu specifique et la microspectrometrie infrarouge a transformee de Fourrier (IRTF) dont la resolution spatiale est limitee a la dizaine de micrometre en raison des longueurs d’ondes utilisees. Nous presentons ici une nouvelle approche basee sur la microspectroscopie l’UV. Patients et methodes Le spectre de fluorescence de l’oxalate de calcium synthetique sous forme mono-, bi- et trihydratee apres excitation a 275 nm a ete acquis sur un microspectrometre directement couple au faisceau synchrotron (ligne DISCO, SOLEIL). Le meme spectre a ete ensuite recherche a l’aide d’un microscope a fluorescence inverse sur les biopsies de deux patients. Le patient 1 avait 77 ans au moment de la biopsie realisee pour une insuffisance renale aigue de profil tubulo-interstitiel. L’etude histologique a montre des lesions de necrose tubulaire aigue associees a de nombreux cristaux intra-tubulaires et interstitiels de CaOx identifies par microscopie IRTF et microscopie a lumiere polarisee. La patiente 2 avait 7 mois au moment de la biopsie pour insuffisance renale aigue. L’etude histologique a montre quelques lesions tubulaires associees a de rares cristaux refringents en lumiere polarisee trop petits pour etre analysables en IRTF. Resultats L’oxalate de calcium quel que soit son etat d’hydratation presente un pic d’emission de fluorescence a 440 nm apres excitation a 275 nm. On peut separer ce pic de celui emis par les acides amines aromatiques. Nous avons retrouve ce pic et pu l’utiliser pour confirmer la presence de CaOx sur la biopsie du patient 1 et la reveler sur celle du patient 2 avec une resolution spatiale submicrometrique. Discussion La fluorescence UV pourrait devenir la methode la plus sensible pour la detection du CaOx dans les tissus biologiques. Conclusion Cette nouvelle technique d’imagerie pourrait permettre de mieux comprendre et diagnostiquer les nephropathies tubulo-interstitielles dependantes de l’oxalate.

Renal hyperplasia and hypertrophy are early events after nephron reduction which precede progressive destruction of the remnant kidney. Restriction of dietary sodium content was shown to reduce renal lesions following nephron reduction. AP-1 is a transcription factor, resulting from heterodimerization of fos and jun proteins, which mediates the effects of mitogenic growth factors. To elucidate the role of AP-1 in growth processes involved in renal deterioration, we evaluated whether restriction of dietary sodium content (0.25 vs. 0.50% sodium w/w) affected AP-1-DNA binding and hyperplasia in the remnant kidney after nephron reduction (70% nephrectomy). Cell proliferation, evaluated by PCNA immunostaining, increased progressively from day 7 to day 60 in glomeruli, proximal and distal tubules and loops of Henle of nephrectomized (Nx) rats compared to control sham-operated (C) animals. AP-1-DNA binding activity increased 7 and 14 days after surgery, but it was reduced below C values at day 60. c-fos and c-jun expression were also reduced in Nx rats at day 60. Sodium restriction significantly reduced the number of PCNA-stained cells in glomeruli and tubules at days 14 and 60, but not at day 7, whereas it decreased AP-1 activation at all times of the study. This effect was associated to a marked reduction of renal lesions in Nx rats. In conclusion, we showed that, after nephron reduction, the beneficial effect of sodium restriction was associated with a reduction of hyperplasia and AP-1 activation, but that the latter did not parallel delayed cell proliferation rate in remaining nephrons. Thus, we propose that different transduction pathways are involved in cell proliferation after nephron reduction, according to the time of evolution of renal lesions.

Between November 1983 and December 1991, we undertook a systematic study of kidney in deceased AIDS patients using light and immunofluorescence microscopy. Among the 116 examined kidneys (from 106 men and 10 women), nine (7.75%) showed diffuse mesangial IgA deposits. By light microscopy, glomerular lesions were absent or moderate (mesangial hypertrophy or some mesangial deposits). Urinary abnormalities were mild in all cases. Our study shows that the association between IgA nephropathy and HIV infection is not rare, and a review of the literature disclosed 18 reported cases. Some reported immunopathogenic data support the possibility of a link between HIV infection and IgA nephropathy.

A possible involvement of endothelium derived relaxing nitric oxide (NO) in the pathogenesis of iodinated contrast media (CM)-induced nephrotoxicity was investigated in the rat.Male rats (6 to 12 per group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery and a low-osmolar contrast medium (CM), ioxaglate, was injected (1 mL/min; 3 minutes) via an aortic puncture in the single remaining kidney. Contrast medium was injected with or without the NO-synthase inhibitor L-NAME (100 mg/kg intravenously [i.v.] 5 minutes before CM). One group received L-Arginine, the physiological precursor of NO (100 mg/kg i.v.), 5 minutes before L-NAME. Phenylephrine (300 micrograms/kg; 30 min) was used as a vasoconstrictive NO-independent control. The effects of iohexol, another low-osmolar CM, on creatinine clearance (CrCl) were also studied with and without pretreatment with L-NAME. A control group was subjected to a 3-minute renal ischemia only. Creatinine clearance and urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion were determined before, and 24 and 48 hours after CM administration. Blinded histologic analysis was carried out after completion of the study.When administered alone, neither L-NAME nor L-arginine modified CrCl. Ioxaglate mildly but significantly decreased CrCl at 24 hours (-26.5% of preinjection value). This was similar to the effect observed in the control group subjected to ischemia only. When associated with L-NAME, ioxaglate markedly decreased CrCl (-58 + 11% at 24 hours, P.05 vs. ioxaglate alone). A similar interaction was noted in the case of iohexol. L-NAME also markedly increased ioxaglate-induced urinary NAG excretion. Phenylephrine had a similar impact on renal function. L-arginine pretreatment reduced the increase in serum creatinine induced by L-NAME+ioxaglate (68 + 17 mumol/L vs. 175 + 59 mumol/L for L-NAME+ioxaglate; P.05) and urinary NAG excretion. Ioxaglate alone induced only tubular epithelial vacuolization. When associated with L-NAME, this CM induced tubular and vascular lesions, as well as necrosis in the outer medulla. Such histologic effects were clearly inhibited by L-arginine.These data indicate that L-NAME, a specific inhibitor of NO-synthase, and phenylephrine, accentuate the nephrotoxicity of CM in the rat. This is consistent with results from the literature showing that CM-toxicity is enhanced by renal ischemia.

The long-term renal effects of cisplatin have been very poorly studied. Therefore we investigated the chronic renal effects of various doses of cisplatin in three groups of male Sprague-Dawley rats. Group I received two injections of 5 mg/kg body weight (bw) at 4-week intervals, group II four injections of 2.5 mg/kg bw at 4-weeks intervals, and group III one injection of 5 mg/kg bw and four injections of 2.5 mg/kg bw at 4-weeks intervals. Controls received an equivalent amount of isotonic saline. Each group was evaluated 1, 3, or 6 months after the last injection of cisplatin. One, 3 and 6 months after the last injection, cisplatin induced a marked decrease in glomerular filtration rate (GFR) evaluated as clearance of [99mTc]DTPA and creatinine clearance in all treated rats. Urinary NAG excretion remained unaltered. At 3 months post-cisplatin treatment GFR was significantly less (P < 0.05) in group III (0.18 +/- 0.02 ml/min/100 g bw) when compared with group I (0.23 +/- 0.02 ml/min/100 g bw) or II (0.23 +/- 0.04 ml/min/100 g bw). In group I GFR was similar 1 month (0.24 +/- 0.02), 3 months (0.23 +/- 0.02) and 6 months (0.23 +/- 0.03 ml/min/100 g bw) after cisplatin treatment. Cisplatin induced atrophy and dilatation of tubules with mononuclear cell infiltration associated with cyst formation. The glomerular and tubulointerstitial lesions were significantly enhanced in group III when compared with groups I and II. This study indicates that repeated administration of cisplatin may induce a chronic tubulointerstitial nephropathy associated with a marked decrease in GFR, which is stable over time. The incidence and severity of chronic cisplatin toxicity is dose-related and is not modified by dividing the dose.