Identificazione del primo biomarcatore diagnostico urinario per il rene a spugna midollare (MSK): risultato di uno studio di proteomica mediante spettrometria di massa

Il rene a spugna midollare (o medullary sponge kidney, MSK) \ue8 una malformazione renale, caratterizzata da modificazioni ectasiche e cistiche dei dotti collettori della porzione midollare delle piramidi renali, che assumono una forma simile a quella delle spugne dopo essere state strizzate e da cui deriva la sua denominazione. Sebbene la sua prevalenza nella popolazione generale non sia nota con esattezza (\uf07e 5% dei casi su 10.000-100.000), MSK \ue8 stato diagnosticato nel 20% dei pazienti con calcoli renali ricorrenti. La sua patogenesi non \ue8 del tutto chiara e negli ultimi anni si \ue8 fatta strada l\u2019idea che si tratti di una patologia ereditaria di tipo autosomico dominante a penetranza incompleta. La diagnosi di MSK \ue8 radiografica e l\u2019urografia con mezzo di contrasto \ue8 l\u2019indagine di riferimento. Negli ultimi anni per\uf2 si \ue8 diffusa una tendenza a ridurre l\u2019impiego dell\u2019urografia a seguito dell\u2019introduzione di indagini che richiedono basse concentrazioni o assenza di mezzo di contrasto quali: ecografia, tomografia computerizzata (TAC spirale), risonanza magnetica. Questo si \ue8 tradotto in una riduzione del numero di casi di MSK diagnosticati. Infatti l\u2019ecografia renale, cos\uec come la TAC, non consentono di mettere in evidenza i reperti tipici e patognomonici di questa condizione. Il rischio che l\u2019MSK possa essere sempre meno diagnosticata genera la necessit\ue0 di introdurre specifici biomarker diagnostici e clinici. Pertanto nel nostro studio abbiamo utilizzato l\u2019analisi proteomica, mediante spettrometria di massa, al fine di individuare nuovi elementi biologici coinvolti nella fisiopatologia dell\u2019MSK e per identificare potenziali biomarker diagnostici da introdurre nella pratica clinica. Sono stati arruolati 22 pazienti affetti da MSK e 22 pazienti con nefrolitiasi calcica idiopatica (ICN). Due dei pazienti con ICN (ICN con cisti), sebbene non avessero segni clinici e radiologici di MSK, mostravano cisti su entrambi i reni. Le urine di 10 pazienti con MSK e 11 pazienti con ICN, scelti in maniera random, sono state sottoposte a spettrometria di massa mediante lo spettrometro linear trap Quadrupole Orbitrap Velos Pro. Dalle analisi di spettrometria di massa sono state identificate 1529 proteine. Di queste 884 (58%) erano in comune tra i due gruppi di pazienti mentre 249 (16%) e 396 (26%) erano associate rispettivamente a ICN e MSK. Tramite Volcano Plot e curva ROC \ue8 stato possibile restringere, rispettivamente, a 328 e 44 il numero di proteine specifiche per MSK. Inoltre, 119 proteine differenziavano i pazienti con cisti dai pazienti con nefrolitiasi che non presentavano cisti. In particolare, un totale di 22 proteine risultava up-regolato nel confronto tra pazienti MSK e ICN mentre 15 proteine risultavano sotto-espresse nel gruppo di pazienti con cisti rispetto al gruppo di pazienti senza cisti. La sovrapposizione tra i 3 metodi statistici e l\u2019applicazione dell\u2019algoritmo Support Vector Machine ha portato all\u2019identifcazione di cinque proteine: glypican-1 (GPC-1), plexin domain\u2013containing protein 1 (PLXDC-1), beta-hexosaminidase (HEXA), epididymis-specific alpha-mannosidase (MAN2B2) e laminin subunit alpha 2 (LAMA-2). Quest\u2019ultima proteina, \ue8 risultata il miglior biomarcatore per MSK, poich\ue9 era statisticamente up-regolata in entrambe le analisi univariate, aveva un\u2019elevata area sotto la curva e mostrava il pi\uf9 basso ranking, ottenuto con algoritmo di support vector machine, dei 16 biomarcatori selezionati. Diversi autori hanno suggerito che la laminina potrebbe avere un ruolo fondamentale nella formazione e crescita delle cisti. La validazione con metodica ELISA effettuata sulle urine di tutti i pazienti inclusi nello studio confermava i dati di proteomica. In conclusione, LAMA-2 potrebbe rappresentare un ottimo candidato come biomarcatore per la diagnosi di MSK e potrebbe aiutare i clinici ad identificare precocemente i pazienti con questa patologia e ad iniziare terapie atte a ridurre le complicanze renali e sistemiche

Everolimus-associated interstitial pneumonia in a renal transplant patient: a case report

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Proteomics Insights into Medullary Sponge Kidney Disease: Review of the Recent Results of an Italian Research Collaborative Network

Background: Medullary sponge kidney (MSK) disease is a rare and neglected congenital condition typically associated with nephrocalcinosis/nephrolithiasis, urinary concentration defects, and cystic anomalies in the precalyceal ducts that, although sporadic in the general population, is relatively frequent in renal stone formers. The physiopathologic mechanism associated with this disease is not fully understood, and omics technologies may help address this gap. Summary: The aim of this review was to provide an overview of the current state of the application of proteomics in the study of this rare disease. In particular, we focused on the results of our recent Italian collaborative studies that, analyzing the MSK whole and extracellular vesicle urinary content by mass spectrometry, have displayed the existence of a large and multifactorial MSK-associated biological machinery and identified some main regulatory biological elements able to discriminate patients affected by this rare disorder from those with idiopathic calcium nephrolithiasis and autosomal dominant polycystic kidney disease (including laminin subunit alpha 2, ficolin 1, mannan-binding lectin serine protease 2, complement component 4-binding protein β, sphingomyelin, ephrins). Key Messages: The application of omics technologies has provided new insights into the comprehension of the physiopathology of the MSK disease and identified novel potential diagnostic biomarkers that may replace in future expensive and invasive radiological tests (including CT) and select novel therapeutic targets potentially employable, whether validated in a large cohort of patients, in the daily clinical practice

Monoclonal antibody therapy and renal transplantation: focus on adverse effects.

A series of monoclonal antibodies (mAbs) are commonly utilized in renal transplantation as induction therapy (a period of intense immunosuppression immediately before and following the implant of the allograft), to treat steroid-resistant acute rejections, to decrease the incidence and mitigate effects of delayed graft function, and to allow immunosuppressive minimization. Additionally, in the last few years, their use has been proposed for the treatment of chronic antibody-mediated rejection, a major cause of late renal allograft loss. Although the exact mechanism of immunosuppression and allograft tolerance with any of the currently used induction agents is not completely defined, the majority of these medications are targeted against specific CD proteins on the T or B cells surface (e.g., CD3, CD25, CD52). Moreover, some of them have different mechanisms of action. In particular, eculizumab, interrupting the complement pathway, is a new promising treatment tool for acute graft complications and for post-transplant hemolytic uremic syndrome. While it is clear their utility in renal transplantation, it is also unquestionable that by using these highly potent immunosuppressive agents, the body loses much of its innate ability to mount an adequate immune response, thereby increasing the risk of severe adverse effects (e.g., infections, malignancies, haematological complications). Therefore, it is extremely important for clinicians involved in renal transplantation to know the potential side effects of monoclonal antibodies in order to plan a correct therapeutic strategy minimizing/avoiding the onset and development of severe clinical complications

Inhibition of heparanase protects against chronic kidney dysfunction following ischemia/reperfusion injury

Renal ischemia/reperfusion (I/R) injury occurs in patients undergoing renal transplantation and with acute kidney injury and is responsible for the development of chronic allograft dysfunction as characterized by parenchymal alteration and fibrosis. Heparanase (HPSE), an endoglycosidase that regulates EMT and macrophage polarization, is an active player in the biological response triggered by ischemia/reperfusion (I/R) injury. I/R was induced in vivo by clamping left renal artery for 30 min in wt C57BL/6J mice. Animals were daily treated and untreated with Roneparstat (an inhibitor of HPSE) and sacrificed after 8 weeks. HPSE, fibrosis, EMT-markers, inflammation and oxidative stress were evaluated by biomolecular and histological methodologies together with the evaluation of renal histology and measurement of renal function parameters. 8 weeks after I/R HPSE was upregulated both in renal parenchyma and plasma and tissue specimens showed clear evidence of renal injury and fibrosis. The inhibition of HPSE with Roneparstat-restored histology and fibrosis level comparable with that of control. I/R-injured mice showed a significant increase of EMT, inflammation and oxidative stress markers but they were significantly reduced by treatment with Roneparstat. Finally, the inhibition of HPSE in vivo almost restored renal function as measured by BUN, plasma creatinine and albuminuria. The present study points out that HPSE is actively involved in the mechanisms that regulate the development of renal fibrosis arising in the transplanted organ as a consequence of ischemia/reperfusion damage. HPSE inhibition would therefore constitute a new pharmacological strategy to reduce acute kidney injury and to prevent the chronic pro-fibrotic damage induced by I/R

Personalization of the Immunosuppressive Treatment in Renal Transplant Recipients: The Great Challenge in "Omics" Medicine.

Renal transplantation represents the most favorable treatment for patients with advanced renal failure and it is followed, in most cases, by a significant enhancement in patients' quality of life. Significant improvements in one-year renal allograft and patients' survival rates have been achieved over the last 10 years primarily as a result of newer immunosuppressive regimens. Despite these notable achievements in the short-term outcome, long-term graft function and survival rates remain less than optimal. Death with a functioning graft and chronic allograft dysfunction result in an annual rate of 3%-5%. In this context, drug toxicity and long-term chronic adverse effects of immunosuppressive medications have a pivotal role. Unfortunately, at the moment, except for the evaluation of trough drug levels, no clinically useful tools are available to correctly manage immunosuppressive therapy. The proper use of these drugs could potentiate therapeutic effects minimizing adverse drug reactions. For this purpose, in the future, "omics" techniques could represent powerful tools that may be employed in clinical practice to routinely aid the personalization of drug treatment according to each patient's genetic makeup. However, it is unquestionable that additional studies and technological advances are needed to standardize and simplify these methodologies

How has peritoneal dialysis changed over the last 30 years: experience of the Verona dialysis center

The last decade has witnessed considerable improvement in dialysis technology and changes in clinical management of patients in peritoneal dialysis (PD) with a significant impact on long term clinical outcomes. However, the identification of factors involved in this process is still not complete.METHODS:Therefore, to assess this objective, we retrospectively analyzed clinical records of 260 adult patients who started PD treatment from 1983 to 2012 in our renal unit. For the analysis, we divided them into three groups according to the time of starting dialysis: GROUP A (n: 62, 1983-1992), GROUP B (n: 66, 1993-2002) and GROUP C (n: 132, 2003 to 2012).RESULTS:Statistical analysis revealed that patients included in the GROUP C showed a reduction in mean patients' age (p = 0.03), smoking habit (p = 0.001), mean systolic blood pressure (p < 0.0001) and an increment in hemoglobin levels (p < 0.0001) and residual diuresis (p = 0.016) compared to the other two study groups. Additionally, patients included in GROUP C, mainly treated with automated peritoneal dialysis, showed a reduced risk of all-causes mortality and a decreased risk to develop acute myocardial infarction and cerebrovascular disease. Patients' age, diabetes mellitus and smoking habit were all positively associated with a significant increased risk of mortality in our PD patients, while serum albumin levels and residual diuresis were negatively correlated.CONCLUSIONS:Therefore, the present study, revealed that in the last decade there has been a growth of our PD program with a concomitant modification of our patients' characteristics. These changes, together with the evident technical advances, have caused a significant improvement of patients' survival and a decrement of the rate of hospitalization. Moreover, it reveals that our pre-dialysis care, modifying the above-mentioned factors, has been a major cause of these clinical improvements

Systemic and Nonrenal Adverse Effects Occurring in Renal Transplant Patients Treated with mTOR Inhibitors

The mammalian target of rapamycin inhibitors (mTOR-I), sirolimus and everolimus, are immunosuppressive drugs largely used in renal transplantation. The main mechanism of action of these drugs is the inhibition of the mammalian target of rapamycin (mTOR), a regulatory protein kinase involved in lymphocyte proliferation. Additionally, the inhibition of the crosstalk among mTORC1, mTORC2, and PI3K confers the antineoplastic activities of these drugs. Because of their specific pharmacological characteristics and their relative lack of nephrotoxicity, these inhibitors are valid option to calcineurine inhibitors (CNIs) for maintenance immunosuppression in renal transplant recipients with chronic allograft nephropathy. However, as other immunosuppressive drugs, mTOR-I may induce the development of several adverse effects that need to be early recognized and treated to avoid severe illness in renal transplant patients. In particular, mTOR-I may induce systemic nonnephrological side effects including pulmonary toxicity, hematological disorders, dysmetabolism, lymphedema, stomatitis, cutaneous adverse effects, and fertility/gonadic toxicity. Although most of the adverse effects are dose related, it is extremely important for clinicians to early recognize them in order to reduce dosage or discontinue mTOR-I treatment avoiding the onset and development of severe clinical complications

Involvement of heparanase in the pathogenesis of acute kidney injury: Nephroprotective effect of PG545

Despite the high prevalence of acute kidney injury (AKI) and its association with increased morbidity and mortality, therapeutic approaches for AKI are disappointing. This is largely attributed to poor understanding of the pathogenesis of AKI. Heparanase, an endoglycosidase that cleaves heparan sulfate, is involved in extracellular matrix turnover, inflammation, kidney dysfunction, diabetes, fibrosis, angiogenesis and cancer progression. The current study examined the involvement of heparanase in the pathogenesis of ischemic reperfusion (I/R) AKI in a mouse model and the protective effect of PG545, a potent heparanase inhibitor. I/R induced tubular damage and elevation in serum creatinine and blood urea nitrogen to a higher extent in heparanase over-expressing transgenic mice vs. wild type mice. Moreover, TGF-\u3b2, vimentin, fibronectin and \u3b1-smooth muscle actin, biomarkers of fibrosis, and TNF\u3b1, IL6 and endothelin-1, biomarkers of inflammation, were upregulated in I/R induced AKI, primarily in heparanase transgenic mice, suggesting an adverse role of heparanase in the pathogenesis of AKI. Remarkably, pretreatment of mice with PG545 abolished kidney dysfunction and the up-regulation of heparanase, pro-inflammatory (i.e., IL-6) and pro-fibrotic (i.e., TGF-\u3b2) genes induced by I/R. The present study provides new insights into the involvement of heparanase in the pathogenesis of ischemic AKI.Our results demonstrate that heparanase plays a deleterious role in the development of renal injury and kidney dysfunction,attesting heparanase inhibition as a promising therapeutic approach for AKI