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Since its first use several decades ago, scanning electron microscopy has been used in numerous investigations dedicated to biological systems. This contribution focuses on observations on pathological calcifications in order to review several major applications of primary importance to the clinician. Among these, we highlight such observations as medical diagnostic tools in pathologies arising from primary hyperoxaluria and urinary infections.

International audience; It is assumed that genetic diseases affecting the metabolism of cysteine and the kidney function lead to two different kinds of pathologies, namely cystinuria and cystinosis whereby generate L-cystine crystals. Recently, the presence of L-cysteine crystal has been underlined in the case of cystinosis. Interestingly, it can be strikingly seen that cystine ([-S-CH2-CH-(NH2)-COOH]2) consists of two cysteine (C3H7NO2S) molecules connected by a disulfide (S-S) bond. Therefore, the study of cystine and cysteine is important for providing a better understanding of cystinuria and cystinosis. In this paper, we elucidate the discrepancy between L-cystine and L-cysteine by investigating the theoretical and experimental infrared spectra (IR), X-ray diffraction (XRD) as well as Raman spectra aiming to obtain a better characterization of abnormal deposits related to these two genetic pathologies.

BACKGROUND AND AIMS Sickle cell disease (SCD) induces a decline in urine concentration and progressive deterioration of kidney (K) function. Hyperfiltration is often observed before this decline, and plasma urea is low. Oral urea or hydroxyurea improves the condition of these patients (pts). These observations draw the attention to renal urea handling in SCD. Studies suggest that addition of urea into the loop of Henle may indirectly modify NaCl concentration at the macula densa and induce a rise in glomerular filtration rate (GFR) by depressing the tubule-glomerular feedback (TGF) [1]. The urine-to-plasma urea concentration ratio (U/P urea) is a novel marker associated with GFR and the progression of disease in ADPKD [2]. The present study addresses the relationship of U/P urea with GFR in SCD pts. METHOD A total of 184 homozygous SCD pts referred to our department were included. They underwent extensive evaluation including measured GFR (mGFR) (51Cr EDTA clearance) and plasma and 24-h urine chemistries. The association between mGFR and U/P urea was tested using univariate, and multivariate stepwise regression analyses. RESULTS Mean age was 35.6 ± 2.8 years (mean ± SD) and body mass index (BMI) 22.2 ± 0.3 kg/m2. Mean mGFR was 107 ± 3 ml/min x 1.73m2. Hyperfiltration was observed in 37% of pts. Urine volume (UV) 1.71 ± 0.75 L/24h. Urea excretion 267 ± 151 mmol/24h. In 13% of pts fractional excretion of urea (FEurea) exceeded 50%. U/P urea (42 ± 20) varied from 6 to 112. It was not associated with 24-h urea excretion but showed a negative association with UV (P < 0.007). mGFR was strongly and positively associated with U/P urea (r = 0.58, P < 0.0001) but not with U/P osmolarity (see Figure and Table). mGFR was also associated with U/P creatinine although more weakly (r = 0.266, p < 0.0001) and with BMI and negatively associated with age, MAP and serum creatinine (creat). After adjustment for U/P creat, the association between mGFR and U/P urea remained highly significant, whereas, after adjustment for U/P urea, the association between mGFR and U/P creat disappeared. In multivariate regression, mGFR remained strongly associated with U/P urea. CONCLUSION Our data show a strong association between mGFR and U/P urea that remains highly significant after multivariate analysis. i) This suggests that a specific estimated GFR equation for SCD patients including the U/P urea ratio could be clinically more relevant than CKD-EPI equations. ii) A specific mechanism may link GFR and intrarenal urea handling, as U/P urea but not U/P osmolarity appears to be involved. Urea could play a role, indirectly, in the regulation of GFR [1]. In SCD, the longer transit time of the blood in vasa recta may favour countercurrent exchanges and lead to a greater addition of urea in loops of Henle (increasing the U/P urea ratio) and thus increasing GFR by reducing TGF. Sickled red cells and resulting in slower blood flow in the medulla may selectively influence urea handling, participating in high FEurea and GFR often observed in SCD pts. iii) As is the case in ADPKD, more attention should be given to longitudinal studies to the relationship between U/P urea and kidney disease progression in SCD pts.

BACKGROUND AND AIMS Vancomycin stands out both as being a potent cause of AKI (V-AKI) and as one of the most universally prescribed antimicrobial drugs. From a pathophysiological standpoint, it was hitherto believed that V-AKI stemmed from the intracellular accumulation of vancomycin in the proximal tubule, which elicits oxidative stress and ultimately translates in cell apoptosis. Recently, a study has shown that vancomycin in the setting of overt drug overdose, can co-precipitate with uromodulin causing microspheric obstructive aggregates in the tubular lumen both in experimental models and in patients [1]. Further documentation of this novel mechanism has been hindered by the need to procure kidney tissue so as to perform ad hoc immunostaining. Furthermore, it remains to be proven that the presence of vancomycin microspheres represents a reliable marker for V-AKI. Urine based vancomycin-immunostaining may represent a valuable non-invasive approach. METHOD The urine of patients presenting with vancomycin overdose determined by plasma trough levels >15 mg/L along with acute kidney injury (according to KDIGO guidelines) were subjected to light microscopic urinalysis. Whenever microscopic urinalysis yielded the presence of casts, immunostaining was performed. Briefly, a pellet of urine was spread on a slide and immunostaining performed with a specific anti-vancomycin antibody (Abbot 6E-4421, 1/1000) prior to repeat microscopic examination. Infrared spectrophotometry was performed to ascertain the nature of the casts by comparing the spectrum to a reference vancomycin spectrum. Patients were deemed to have V-AKI whenever at least one vancomycin positive cast was observed following immunostaining. The urine of control patients was subjected to the same procedure: they were defined as patients exposed to vancomycin with or without vancomycin overdose but with no AKI. RESULTS A total of 5 patients with vancomycin overdose KDIGO stage 3 AKI were found to exhibit casts with positive vancomycin immunostaining (Fig. 1). On light microscopic urinalysis, vitreous casts devoid of refringence after polarization could be observed in each case. Upon infrared spectrophotometry, the casts displayed a Fourier transform spectrum consistent with vancomycin. In three of the cases, V-AKI was considered the sole or primary cause of AKI. The patient displayed abundant and markedly stained casts upon anti-vancomycin immunostaining (Fig. 1A). Overt and massive vancomycin overdosing was evidenced in all cases. In one case, the patient concurrently received piperacillin-tazobactam and in another case the patient was exposed to iodinated contrast media. The renal function course was favourable with full recovery in all three instances. In two cases, competing causes of AKI were either evidenced or not. In one case, the patient displayed septic shock which indicated vancomycin therapy. In another case, the patient was suspected to have immunoallergic intersititial nephritis. The patients exhibited rare casts with only faint staining (Fig. 1B). In both cases, vancomycin overdose could not be documented. V-AKI was deemed a possible concurrent cause of AKI. None of the 5 control patients yielded casts after light microscopic urinalysis. CONCLUSION Vancomycin cast detection on urine microscopy is feasible and can be enhanced by a straightforward immunostaining technique and represents a unique opportunity for a non-invasive diagnosis of V-AKI. Further studies are required to refine diagnostic criteria-based urine immunostaining detection. They also needed to determine whether detection of V-AKI merely reflects impaired vancomycin urinary excretion in the setting of AKI or can be regarded as a sensitive and specific marker of vancomycin-induced tubular obstruction and injury.

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Understanding the physico-chemistry related to cristalline pathologies constitutes a challenge in several medical specialities such as nephrology, dermatology or oncology. Regarding nephrology, the chemical diversity of concretions such as kidney stones calls for characterization techniques to determine the chemical composition of concretions. The starting point of this contribution is given by Fourier Transform InfraRed (FTIR) spectroscopy which is routinely used at the hospital to deter-mine the chemical composition of kidney stones as well as ectopic calcifications present in kidney biopsy. For kidney stones, the quantity of sample is sufficient to perform a significant analysis through classical FTIR. For ectopic calcifications, mu FTIR can be inefficient in the case of mu calcification in the tissue when their size is less than 10 mu m. For such samples, Optical PhotoThermal IR (OPT-IR) spectroscopy may constitute a way to overcome this experimental difficulty through the acquisition of IR spectrum with a spatial resolution close to 500 nm.To illustrate such opportunity, we first compare the IR spectrum acquired with a classical experimental set-up related to classical IR spectroscopy to IR spectrum collected with a OPT-IR one for different compounds namely calcium oxalate monohydrate, calcium oxalate dehydrate, calcium phosphate apatite and magnesium ammonium phosphate hexahydrate. Such comparison helps us to assess specificity of OPT-IR. Then, we consider several pathological calcifications associated to hyperoxaluria, adenine phosphoribosyltransferase (APRT) deficiency or the presence of Randall's plaque. We will see that the nanometer spatial resolution constitutes a major advantage versus a micrometre one. Also, in the case of Randall's plaque, we show that OPT-IR can determine the chemical composition of microscopic concretion without any kind of preparation. Such experimental fact is clearly a major advantage. Finally, we also extended this first investigation in nephrology by considering breast calcifications. In that case, if the number of chemical phases is quite low compared to the number of chemical phases identified in ectopic calcifications present in kidney (four instead of 24), the challenge is related to the possibility to distinguish between the different calcium phosphate namely amorphous carbonated calcium phosphate, CA and whitlockite.The complete set of data indicates the limitations and the advantages of OPT-IR spectroscopy.

Although numerous pathologies are associated with abnormal skin deposits, these remain poorly described, as accurate characterization continues to present a challenge for dermatologists. Their submicrometer size as well as their diverse chemistry require various characterization tools. We aim to exemplify characterization of endogenous and exogenous skin deposits in some selected skin diseases using different physico-chemical techniques. We begin with a presentation of selected dis-eases associated with skin deposits. We then present those of our results which show their variety of structure, location and chemical composition, obtained with various tools: Field Emission Scanning Electron Microscopy coupled with Energy Dispersive X-ray Spectroscopy, X-ray fluorescence, vibra-tional spectroscopies, as well as techniques specific to synchrotron radiation. Our results constitute a real opportunity to improve diagnosis, and to understand the pathogenesis of many skin diseases, and opportunities for therapeutic intervention.

Most papers on kidney stones arising from infection concentrate on the mineral struvite. In this contribution, we would like to call attention to other mineral phases such as highly carbonated calcium phosphate apatite, ammonium urate, and whitlockite, by presenting clinical and chemical data. We start with epidemiological data which emphasize the increase in the prevalence of kidney stones related to infection. Then we present a statistical analysis of more than 85,000 stones which have been analysed at the Laboratoire des Lithiases of Assistance Publique-Hôpitaux de Paris which gives insights regarding the link between urinary tract infection and struvite, carbonated calcium phosphate apatite (carbapatite), and also surprisingly whitlockite. Some information regarding the pathogenesis of kidney stones linked to infection, the nature of the bacteria which have been identified, and the approach to precisely analyse infrared spectra to identify struvite, carbapatite, and whitlockite, conclude this first part. To complete this clinical description, we describe the crystallographic structure and the chemistry of three relevant compounds namely carbonated calcium phosphate, struvite, and whitlockite. To conclude this second part, the dependence of crystallite morphology of struvite on pH and on the presence, or absence, of bacteria, is described. Based on clinical and chemical data, it is becoming clear that struvite is not the only mineral intimately related to renal infectious processes, but that whitlockite and carbapatite with a high carbonation rate are strongly associated with urinary tract infection as well.

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Primary hyperparathyroidism (pHPT) has been reported to have a higher prevalence in sickle cell disease (SCD) patients, including a high rate of recurrence following surgery. However, most patients are asymptomatic at the time of diagnosis, with surprisingly infrequent hypercalciuria, raising the issue of renal calcium handling in SCD patients. We conducted a retrospective study including (1) 64 hypercalcemic pHPT non-SCD patients, (2) 177 SCD patients, divided into two groups of 12 hypercalcemic pHPT and 165 non-pHPT, (3) eight patients with a diagnosis of familial hypocalciuric hypercalcemia (FHH). Demographic and biological parameters at the time of diagnosis were collected and compared between the different groups. Determinants of fasting fractional excretion of calcium (FeCa2+) were also analyzed in non-pHPT SCD patients. Compared to non-SCD pHPT patients, our data show a similar ionized calcium and PTH concentration, with a lower plasmatic calcitriol concentration and a lower daily urinary calcium excretion in pHPT SCD patients (p &lt, 0.0001 in both cases). Fasting FeCa2+ is also surprisingly low in pHPT SCD patients, and thus inadequate to be considered hypercalcemia, recalling the FHH phenotype. FeCa2+ is also low in the non-pHPT SCD control group, and negatively associated with PTH and hemolytic biomarkers such as LDH and low hemoglobin. Our data suggest that the pHPT biochemical phenotype in SCD patients resembles the FHH phenotype, and the fasting FeCa2+ association with chronic hemolysis biomarkers strengthens the view of a potential pharmacological link between hemolytic by-products and calcium reabsorption, potentially through a decreased calcium-sensing receptor (CaSR) activity.

Objective Gitelman syndrome (GS) is the most frequent salt-wasting genetic tubulopathy and a source of hypokalaemia and hypomagnesemia. Chondrocalcinosis (CC) is a frequent feature of GS. The aim of our study was to determine the prevalence, distribution patterns, clinical phenotypes and risk factors for CC in GS. Methods This prospective study of a cohort of 57 patients with GS included a systematic screening for CC by peripheral joint radiography, cervical spine CT and joint US. The prevalence of cervical C1–C2 CC by CT was compared between 33 GS patients and sex- and age-matched controls. Clinical and biochemical features were analysed to identify factors associated with CC. Results Mean (s.d.) age of patients was 46.5 (12.4) years, 66.7% were women and 93.0% carried SLC12A3 mutations. Mean serum magnesium level was 0.60 (0.30) mmol/l. CC was observed in 79% of patients, with the highest prevalence at the cervical spine (81.8%) followed by the knee (52.6%), wrist (50.9%), ankle (38.6%), TM joint (36.4%), shoulder (33.3%), hip (22.8%), elbow (14.0%) and sclerochoroid (12.1%). Prevalence of CC at the C1–C2 level was higher in the GS cohort than control group (72.7% vs 9.1%) (adjusted odds ratio 21.0, 95% CI 2.8, 156.1, P = 0.003). Independent factors associated with CC were low serum magnesium level and age. Conclusion GS was associated with widespread CC, favoured by aging and hypomagnesemia. The C1–C2 level was the most affected site. Follow-up of this unique cohort will help understanding the clinical consequences of CC, especially the precise characterization of pyrophosphate arthropathy.

Chronic wounds, including leg ulcers, constitute an important medical problem among older patients. Dystrophic calcifications (DC) are associated with a variety of disorders, including leg ulcers. The aim of this study was to report the clinical and biological characteristics of older patients with DC in leg ulcers and to determine the morphology and chemical composition of these calcifications. We conducted a prospective monocentric study in our Geriatric-Wound and Healing ward, Rothschild Hospital, Paris, from January 2018 to December 2019. Patients with leg ulcers were screened for DC by palpation. Patients’ clinical, biological, and radiological findings were collected. DC morphology was analyzed using field-emission scanning electron microscopy and chemical composition was analyzed using µFourier transform infra-red spectroscopy and X-ray Fluorescence. Ten (7%) of the 143 patients hospitalized for leg ulcers presented DC. Older patients with DC were more likely to have leg ulcers with venous insufficiency (p = .015), colonized by Pseudomonas aeruginosa (p = .026), with a longer healing evolution (p = .0072) and hypercalcemia (p = .041). Five DC were extracted from ulcers: 2 presented 500 nm lacunar spheres and intermingled fibrils of about 10 nm in diameter, consistent with bacterial and biofilm imprints. DC were always composed of calcium–phosphate apatite and associated to the presence of zinc. Our analyses were consistent with the involvement of microorganisms and inflammatory process in DC formation. Early management of venous insufficiency, treatment of chronic bacterial colonization and use of calcium-solubilizing drugs seem to be rational strategies for calcified leg ulcer management in older patients.

Glomerular hyperfiltration alone or associated with albuminuria is a well-known feature of sickle cell associated nephropathy. Though, glomerular hyperfiltration is currently considered to be related to a high renal plasma flow and chronic hemolysis, cardiac output influence on measured glomerular filtration rate (mGFR) have not been investigated so far. Thirty seven homozygous sickle cell patients (SCA) from the RAND study investigated before and under angiotensin converting enzyme inhibitor (ACEI) were included. Both mGFR and cardiac index (CI) were high (> 110 ml/min/1.73 m2 and > 3.5 l/m2 in 81% and 97% of cases) with low systemic vascular resistance (SVR) (−5) in 38% of cases. mGFR association with CI and SVR were significant at baseline (respectively ρ: 0.44, p = 0.008 and ρ: − 0.37, p = 0.02) and under ACEI (p = 0.007 and 0.01 respectively), in accordance with previous data showing that hyperfiltration was linked to an increased glomerular perfusion and a glomerulomegaly rather than increased capillary hydrostatic pressure. Of notice, after adjustment on CI, mGFR remained associated with reticulocyte count and albuminuria under ACEI (p = 0.006 and 0.02 respectively). Our results suggest that hyperfiltration is tightly linked to an increased cardiac output which may account for an increased renal blood flow. Chronic hemolysis could be a relevant factor accounting for hyperfiltration potentially acting on glomerular enlargement which appears as a key factor. Our data suggest that cardiac output assessment is a relevant tool in the routine management and monitoring of SCA nephropathy.

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International audience; The SFBC working group aimed to deal with biological tests outside the french nomenclature that may be useful in the context of urinary exploration of metabolism. This section will be divides into three parts: 1) nutritional assessment using urinary urea; 2) metabolic assessment of urolithiasis; 3) exploration of tubulopathies. National and international recommendations support the evaluation of nutritional status from urea measurements in urine and dialysate with the following indications: primary metabolic evaluation of urolithiasis patients, monitoring of protein intake in chronic renal failure stage 3 or stage 5D with residual diuresis. For the management of the urolithiasis disease, biomedical tests recommended by the national and international guidelines are the measurement of the urinary density using refractometry in the primary metabolic evaluation as well as the determination of oxalemia in the diagnosis (patients with GFR< 30 mL/min/1.73 m2) and follow-up (patients with GFR< 60 mL/min/1.73 m2) of primary hyperoxaluria. The determination of the bicarbonaturia is retained for the in depth exploration of urolithiasis and tubular acidosis. The measure of chlore in urine is used to evaluate the volume status during metabolic alkalosis and to calculate the urinary anionic gap during metabolic acidosis.

The main manifestation of acute interstitial nephritis (AIN) due to immune checkpoint inhibitors is acute kidney injury. We report here a biopsy-proven AIN revealed by tubular acidosis. This case highlights that immune checkpoint inhibitor prescribers must be aware of electrolytic disorders since tubular dysfunction can precede serum creatinine increase and reveal renal toxicity.

Background Despite new therapeutics, the prognosis for pancreatic cancer remains poor. Pancreatic surgery is a therapeutic option in non-metastatic forms. The consequences for renal function are poorly described. Methods Patients who underwent surgery for pancreatic cancer between 1 January 2010 and 1 January 2017 and who experienced kidney biopsy in the Pitié-Salpêtrière Hospital were analysed. Results Two hundred and ninety-four patients had pancreatic surgery during the period of analysis and five of them had a kidney biopsy (mean ± SD 20 months ±13.6 months after surgery) during the post-operative follow-up. Among these patients, three exhibited oxalate nephropathy (ON), indicating that the prevalence of ON in patients with pancreatectomy is at least 1%. ON may be insidious, with chronic renal failure without urinary abnormalities. All patients had a high oxalate-to-creatinine ratio in urine sample. Renal function improved after specific management of ON in two patients. Pancreaticoduodenectomy may represent a higher risk of ON than left pancreatectomy. Conclusion Although rare and underestimated, ON appears to be a real risk after pancreatic resection. Early detection may preserve renal function.

Resume La cystinurie est une maladie monogenique induisant une perte de fonction d’un transporteur d’acides amines dibasiques implique dans la reabsorption de cystine au niveau des tubules renaux proximaux. L’excretion urinaire de cystine chez ces patients est augmentee d’un rapport 10 a 60 par rapport au sujet sain, responsable d’une sursaturation avec precipitation de cristaux de cystine dans les reins et de calculs dans les cavites excretrices. La physiopathologie de la cystinurie est particuliere : la concentration plasmatique de cystine est souvent normale alors que l’absorption intestinale est diminuee. Ce paradoxe est explique par une absorption intestinale preservee de cysteine et de methionine qui sont deux acides amines soufres metabolises notamment en cystine. Le principe du traitement consiste a empecher la formation de cristaux de cystine dans les reins afin de prevenir la survenue d’une maladie lithiasique, mais aussi d’une authentique nephropathie cristalline responsable d’une insuffisance renale chez plus de la moitie des patients. Le traitement medical des patients cystinuriques repose (1) sur une diminution de l’excretion urinaire quotidienne de cystine qui est le reflet de l’absorption intestinale et donc des apports en methionine ; (2) sur une augmentation du seuil de solubilite urinaire de la cystine afin de prevenir une precipitation cristalline intratubulaire et intracavitaire par une dilution et une alcalinisation des urines avec des valeurs cibles de 3 L et un pH urinaire aux alentour de 7,5. Le traitement par sulfhydryle dont le but est de dissocier les ponts disulfures des molecules de cystine avec un effet modeste sur la dissolution des calculs de cystine constitues ne devrait en toute logique etre utilise en dernier recours une fois les mesures dietetiques et la cible de pH urinaire obtenue.

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OBJECTIVE The coexistence of calcium pyrophosphate dihydrate (CPPD) and monosodium urate monohydrate crystals in gouty tophi has rarely been reported. We undertook this study to investigate CPPD crystal deposits in a series of surgically removed gouty tophi and to identify factors associated with these deposits. METHODS Twenty-five tophi from 22 gout patients were analyzed using polarized light microscopy, field emission scanning electron microscopy (FESEM), and μ Fourier transform infrared (μFTIR) spectroscopy. RESULTS Tophi consisted of multiple lobules separated by fibrous septa and surrounded by a foreign-body giant cell reaction. CPPD crystal aggregates were identified in 9 of 25 tophi from 6 patients. CPPD crystals were dispersed or highly compacted, localized at the edge or inside the tophus lobules, with some lobules completely filled with crystals. Both monoclinic and triclinic CPPD crystal phases were identified using FESEM and μFTIR. Compared to patients without CPPD, those with CPPD-containing tophi were older (mean 60.5 years versus 47.2 years; P = 0.009), and had longer-term gout duration (mean 17.0 years versus mean 9.0 years; P < 0.05) and tophi duration (mean 10.0 years versus mean 4.6 years; P < 0.01). None of the patients had radiographic chondrocalcinosis of the knee or wrist. CONCLUSION CPPD crystal formation seems to be a late and frequent event of tophus maturation, occurring more frequently with aging, and could contribute to the speed of tophus dissolution and the apparent persistence of tophus sometimes observed even after effective, long-lasting urate-lowering therapy.

International audience; The examination of the urinary sediment of a 64-year-old woman showed the presence of three different types of crystals, all with unusual morphology, which could not be identified with bright field microscopy, polarized light, and the knowledge of urine pH (7.5). The use of microscopic infrared spectroscopy, Raman spectroscopy and energy dispersive X-ray spectroscopy led to the identification of the three types of crystals as calcite, vaterite and aragonite, which are all variants of calcium carbonate crystals. This paper confirms the complex morphology and nature that urinary crystals may at times have and the utility of advanced infrared spectroscopy techniques for their identification.

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International audience; Dans cette contribution, nous étudions les calculs rénaux contenant la whitlockite (Wk). En premier lieu, la présence du fer dans la formule stœchiométrique de la Wk est discutée grâce à la fluorescence X. Puis, nous abordons la relation à l’infection à l’aide d’observations au Microscope électronique à balayage (MEB) aptes à mettre en évidence de possibles empreintes de bactéries. Elles montrent que le processus infectieux est établi pour des teneurs supérieures à 20%, ceux-ci ne pouvant toutefois pas être écartés en dessous de cette teneur. Finalement, l’étude de relation entre la taille des cristaux de Wk et l’infection par DRX ne permet pas de l’établir. Le message pour le clinicien est le suivant : pour un patient asymptomatique, si le calcul ne contient ni struvite, ni apatite carbonatée et moins de 20% de Wk, il convient d’observer par MEB les calculs rénaux afin de pouvoir écarter ou non un processus infectieux.

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The prevalence of crystalline pathologies including urolithiasis, gallstones, vascular calcifications and crystalline arthritis, is very high in the general population beyond 60 years old. Characterization of microcrystals in tissue at the micrometer and at the nanometer scale through physico-chemical techniques constitutes a new opportunity for the physician to decipher the early stage of the pathogenesis of these biological entities. In this review, such description indicates a wide variety of the chemical process associated to the nucleation process directly from supersaturated solution or from organic support such as DNA or elastin. We will also discuss the case of vesicles which play a major role in the case of ectopic calcification situated in kidney tissue, namely the Randall's plaque. All this research focused on the very first steps of the genesis of pathological calcifications constitute a major step to develop specific therapy able to avoid the formation of these abnormal deposits in tissues. As already underlined, crystals may be the consequence of various pathologies, but they are also involved in the dysfunction of the tissues.

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Introduction Dans la necrolyse epidermique toxique (NET), la cicatrisation commence vers le 8e jour et se poursuit sur 2 a 3 semaines. Nous decrivons trois patients admis en reanimation pour NET, et dont l’evolution etait caracterisee par des troubles de cicatrisation avec calcifications dermiques pour lesquels nous avons evoque la responsabilite de la caspofungine, utilisee dans les infections fongiques invasives. Materiel et methodes Les donnees des patients (1 femme, 2 hommes) a l’admission sont dans le Tableau. Alors que les patients cicatrisaient de maniere satisfaisante, l’evolution etait marquee par une re-aggravation cutanee avec survenue d’erosions fibrineuses rugueuses a la palpation. La biopsie cutanee a l’aggravation revelait dans les cas 1 et 2 une necrose epidermique entierement calcifiee confirmee par la coloration Von Kossa, et dans le 3e cas une ulceration epidermique avec microcalcifications dermiques en regard. La calcemie ionisee etait de 1,19 (0,97–1,25) mmol/L (N 1,15–1,33) chez les trois patients. Aucun patient ne recevait de supplementation calcique. La supplementation en phosphore et les soins locaux etaient habituels (chlorhexidine diluee et vaseline). Pour etudier la nature des calcifications, on realisait une analyse en spectroscopie infrarouge : ces depots etaient composes de carbapatite. La microscopie electronique a balayage montrait que ces depots etaient formes de volumineuses plaques dont l’analyse structurelle sub-micrometrique revelait de petites spherules semblant emaner du derme et fusionner pour former ces plaques. L’analyse en spectroscopie de rayon X a dispersion d’energie confirmait la nature calcique de ces spherules micrometriques. L’hypothese d’une secretion calcique par une differentiation osteoblastique des fibroblastes etait ecartee par la negativite des marqueurs immunohistochimiques d’expression osteocytaire. La reprise des dossiers medicaux revelait que les trois patients etaient traites par caspofungine au moment de la re-aggravation cutanee : patient 1 et 3 pour choc septique a Candida parapsilosis ; patient 2 pour choc septique non documente. Le role de la caspofungine dans cette re-aggravation etait evoque. Discussion La caspofungine modifie les flux calciques intracellulaires via un effet agoniste sur le recepteur de la ryanodine (RyR), exprime sur les keratinocytes. Un tel effet agoniste a montre dans un modele murin qu’il provoquait un retard de cicatrisation. Ainsi, la caspofungine a pu favoriser chez nos patients une necrose des keratinocytes en regeneration et l’apparition de calcifications chez nos patients. La caspofungine pourrait favoriser des troubles de cicatrisation au cours de la NET. Cette hypothese devra etre etayee par des investigations in vitro.

La cristallurie est une balance entre les promoteurs et les inhibiteurs ; les premiers favorisant la croissance cristalline et les seconds l’inhibant. Les dosages biologiques permettent de mettre en evidence un ou plusieurs facteurs lithogenes frequemment observes chez des patients lithiasiques. L’objectif de notre travail a ete d’etudier l’impact de l’acidification ou de l’alcalinisation des urines de patients lithiasiques lors d’un bilan de cristallurie : recueil de la totalite des urines du matin, apportees dans les 2 a 3 heures, a temperature ambiante au laboratoire pratiquant l’examen. Quatre-vingts patients lithiasiques (40 pour le calcium urinaire et 40 pour l’acide urique urinaire) ont ete selectionnes a partir des resultats de la cristallurie : plus de 20 cristaux calcium-dependants ou d’acide urique/mm3. Pour le calcium et l’acide urique, des differences de concentration apres respectivement acidification et alcalinisation sont observees avec un r2 = 0,43 (p < 0,01) et r2 = 0,36 (p < 0,01) respectivement. En conclusion, dans cette population lithiasique avec presence de cristaux, cette etape pre-analytique est indispensable pour le calcium urinaire et l’acide urique urinaire permettant d’etablir un diagnostic de certitude indispensable pour le patient.

Crystalluria is a marker of urine supersaturation with substances deriving from metabolic disorders, inherited diseases or drugs. The investigation of crystalluria must be done according to a protocol which includes the delivery to the laboratory of a proper urine sample, the use of a microscope equipped with polarized light, the accurate knowledge of urine pH, and a comprehensive examination of the crystals, which is based on their identification, quantification and size measurement. For unusual crystals, infrared spectroscopy may also be needed. If the formation of stones is always preceded by crystalluria, the reverse is not true. In addition to the crystalline composition, stone morphology provides valuable information on stone activity and, for some crystalline species, major information regarding the underlying pathology. Fourier transform infrared spectroscopy (FTIR) reliably identify specific forms of nephrolithiasis, as common-type stones made of calcium oxalate (CaOx) and/or calcium phosphate that is combined with morphology classification; using this method, stones may be classified into 6 types subdivided in 22 subtypes. The investigation of crystalluria is an inexpensive and valuable tool for the detection and the monitoring of inherited and acquired diseases associated with urinary stone formation or acute or chronic renal function impairment from intrarenal crystal precipitation. Selective FTIR identification of the composition of core (or the umbilication), middle part, and surface of every stone allows identification of the initiating lithogenic process (in the nucleus or in the Randall's plaque) and the factors which subsequently contributed to stone growth. In conclusion, the proposed morpho-constitutional method of urinary stone analysis, which moreover is rapid and low cost, provides clinically relevant orientations for targeted etiologic evaluation.

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; Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzyme deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria, promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome. It has been shown to inhibit neuronal lactate dehydrogenase 5 enzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, stiripentol decreased the synthesis of oxalate by hepatocytes in a dose-dependent manner. In vivo, oral administration of stiripentol significantly reduced urine oxalate excretion in rats. Stiripentol protected the kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, stiripentol significantly improved renal function. Patients affected by Dravet syndrome and treated with stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received stiripentol for several weeks, and urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.

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.

International audience; Importance: Calcific uremic arteriolopathy (CUA), a rare, potentially fatal, disease with calcium deposits in skin, mostly affects patients with end-stage renal disease who are receiving dialysis. Chemical composition and structure of CUA calcifications have been poorly described.Objectives: To describe the localization and morphologic features and determine the precise chemical composition of CUA-related calcium deposits in skin, and identify any mortality-associated factors.Design, Setting, and Participants: A retrospective, multicenter cohort study was conducted at 7 French hospitals including consecutive adults diagnosed with CUA between January 1, 2006, and January 1, 2017, confirmed according to Hayashi clinical and histologic criteria. Patients with normal renal function were excluded. For comparison, 5 skin samples from patients with arteriolosclerosis and 5 others from the negative margins of skin-carcinoma resection specimens were also analyzed.Main Outcomes and Measures: Localization and morphologic features of the CUA-related cutaneous calcium deposits were assessed with optical microscopy and field-emission-scanning electron microscopy, and the chemical compositions of those deposits were evaluated with μ Fourier transform infrared spectroscopy, Raman spectroscopy, and energy dispersive radiographs.Results: Thirty-six patients (median [range] age, 64 [33-89] years; 26 [72%] female) were included, and 29 cutaneous biopsies were analyzed. Calcific uremic arteriolopathy and arteriolosclerosis skin calcifications were composed of pure calcium-phosphate apatite. Calcific uremic arteriolopathy vascular calcifications were always circumferential, found in small to medium-sized vessels, with interstitial deposits in 22 (76%) of the samples. A thrombosis, most often in noncalcified capillary lumens in the superficial dermis, was seen in 5 samples from patients with CUA. Except for calcium deposits, the vessel structure of patients with CUA appeared normal, unlike thickened arteriolosclerotic vessel walls. Twelve (33%) patients died of CUA.Conclusions and Relevance: Calcific uremic arteriolopathy-related skin calcifications were exclusively composed of pure calcium-phosphate apatite, localized circumferentially in small to medium-sized vessels and often associated with interstitial deposits, suggesting its pathogenesis differs from that of arteriolosclerosis. Although the chemical compositions of CUA and arteriolosclerosis calcifications were similar, the vessels' appearances and deposit localizations differed, suggesting different pathogenetic mechanisms.

The search for crystalluria and morphologic analysis of urinary crystals is of interest in the diagnostic evaluation and assessment of efficacy of therapeutic strategies in stone formers. In common calcium oxalate (CaOx) nephrolithiasis, identification of the monohydrate or dihydrate form of CaOx orients toward hyperoxaluria or hypercalciuria as the main lithogenic mechanism, respectively. An unusual high abundance of crystals exclusively made of CaOx monohydrate is highly suggestive of primary hyperoxaluria, the most severe of all types of renal stone diseases. The identification of crystal species such as struvite, cystine, 2,8-dihydroxyadenine, which are not found in normal urine, is indicative of specific pathological conditions, namely, infection stones and hereditary diseases such as cystinuria and dihydroxyadeninuria. Such diseases expose to progressive loss of renal function unless they are prevented by specific therapeutic measures. The presence of crystals made of drugs may entail the risk of kidney dysfunction because of crystallization of the drug or its metabolites.Serial determination of crystalluria, which reflects the activity of stone disease and its response to therapeutic measures, is of major interest in the follow-up of patients suffering from nephrolithiasis. It should be more largely used in clinical practice in most stone formers, still more for the surveillance of patients affected by severe, hereditary renal stone diseases such as primary hyperoxaluria, cystinuria, and dihydroxyadeninuria, where determination of the global crystal volume allows assessing the efficacy of therapeutic measures and optimizing the medical management of the patients., L’étude de la cristallurie et de ses caractéristiques qualitatives et quantitatives est d'un intérêt clinique majeur pour le diagnostic de la maladie lithiasique et l’évaluation de l'efficacité des mesures thérapeutiques proposées aux patients. Dans la lithiase oxalocalcique commune, la forme cristalline, whewellite ou weddellite, oriente respectivement vers une hyperoxalurie ou une hypercalciurie. Une quantité élevée de cristaux de whewellite traduit une forte hyperoxalurie et doit faire rechercher une hyperoxalurie primaire. L'identification de cristaux particuliers tels que la struvite, la cystine, la dihydroxy-2,8-adénine permet le diagnostic de pathologies spécifiques qui exposent à une altération progressive de la fonction rénale lorsqu'elles ne sont pas traitées précocement par des mesures adaptées. La présence de cristaux médicamenteux peut expliquer l'origine d'une insuffisance rénale aiguë développée sous traitement.Au cours du suivi des patients souffrant de lithiase, la détermination de la cristallurie sur des prélèvements sériés est d'un grand intérêt pour la prévention du risque de récidive de calculscar elle reflète l'activité de la maladie lithiasique et sa réponse aux mesures diététiques et thérapeutiques. Elle peut donc bénéficier à la plupart des patients lithiasiques. Elle se doit d’être réalisée chez tout patient souffrant d'une forme héréditaire sévère de lithiase telle que cystinurie, hyperoxalurie primaire ou dihydroxyadéninurie pour en optimiser la prise en charge thérapeutique, la détermination du volume cristallin global dans ces pathologies cristallines sévères permettant d'adapter le traitement médical, d’évaluer l'efficacité des mesures thérapeutiques proposées et de modifier celles-ci en fonction des résultats obtenus.