Aims This joint document of the Italian Diabetes Society and the Italian Society of Nephrology reviews the natural history of diabetic kidney disease (DKD) in the light of the recent epidemiological literature and provides updated recommendations on anti-hyperglycemic treatment with non-insulin agents. Data Synthesis Recent epidemiological studies have disclosed a wide heterogeneity of DKD. In addition to the classical albuminuric phenotype, two new albuminuria-independent phenotypes have emerged, i.e., “nonalbuminuric renal impairment” and “progressive renal decline”, suggesting that DKD progression toward end-stage kidney disease (ESKD) may occur through two distinct pathways, albuminuric and nonalbuminuric. Several biomarkers have been associated with decline of estimated glomerular filtration rate (eGFR) independent of albuminuria and other clinical variables, thus possibly improving ESKD prediction. However, the pathogenesis and anatomical correlates of these phenotypes are still unclear. Also the management of hyperglycemia in patients with type 2 diabetes and impaired renal function has profoundly changed during the last two decades. New anti-hyperglycemic drugs, which do not cause hypoglycemia and weight gain and, in some cases, seem to provide cardiorenal protection, have become available for treatment of these individuals. In addition, the lowest eGFR safety thresholds for some of the old agents, particularly metformin and insulin secretagogues, have been reconsidered. Conclusions The heterogeneity in the clinical presentation and course of DKD has important implications for the diagnosis, prognosis, and possibly treatment of this complication. The therapeutic options for patients with type 2 diabetes and impaired renal function have substantially increased, thus allowing a better management of these individuals.

AIMS:This joint document of the Italian Society of Nephrology and the Italian Diabetes Society reviews the main indications to perform a renal biopsy in diabetic patients, according to the recommendations of a panel of experts based on all available scientific evidence.DATA SYNTHESIS:Renal biopsy has a pivotal role in assessing the nature and severity of renal injury in patients with diabetic kidney disease (DKD). The procedure is mandatory in the presence of one of more of the following features: rapid onset or progression of albuminuria or sudden onset of nephrotic syndrome, rapid GFR decline with or without albuminuria, hematuria, active urine sediment, clinical and/or laboratory suspicion of other systemic diseases, and, in patients with type 1 diabetes, short diabetes duration and absence of retinopathy. Indeed, ~40% of diabetic individuals with kidney injury undergoing renal biopsy are affected by a non-diabetic renal disease (NDRD). Furthermore, the histological evaluation of patients with suspected classical diabetic nephropathy allows to define the extent of glomerular, tubulo-interstitial and vascular lesions, thus providing important prognostic (and potentially therapeutic) data. In the future, the indications for renal biopsy might be extended to the definition of the histological lesions underlying the "nonalbuminuric" DKD phenotypes, as well as to the evaluation of the response to treatment with the new anti-hyperglycemic drugs that provide cardiorenal protection.CONCLUSIONS:In view of the heterogeneous clinical presentation and course of DKD and of the related heterogeneous histopathological patterns, a more extensive use of renal biopsy may be crucial to provide valuable information with important pathogenic, diagnostic, prognostic, and therapeutic implications.

Background. Organ donation refusal from relatives of potential donors with brain death significantly reduces organ availability. The need for organ donation has increased over time, but the shortage of available donors is the major limiting factor in transplantation. We analyzed the impact of a new systematic communication approach between medical staff and patients' relatives on the rate of consent to organ donation.Methods. The study was conducted as a single-center, non-randomized, controlled, before-and-after study at an 18-bed intensive care unit (ICU) of a university hospital. We compared the rate of consent for organ donation before and after the introduction of the new communication approach.Results. A total of 291 brain-dead patients were studied. The consent rate increased from 71% in the pre-intervention period (2007-2012) to 78.4% in the post-intervention period (2013-2015), with an 82.75% increase in the 2014 to 2015 period. During these periods, no significant variation of consent to organ donation was recorded at the national and regional levels.Conclusions. The introduction of a new communication approach between medical staff and relatives of brain-dead patients was associated with a significant increase in the rate of consent to donation. Our results highlight the importance of empathy with relatives in the ICU.

The purpose of our study was to evaluate howhyperglycemia (HG) influences Lys63 protein ubiquitination and its involvement in tubular damage and fibrosis in diabetic nephropathy (DN). Gene and protein expression of UBE2v1, a ubiquitin-conjugating E2-enzyme variant that mediates Lys63-linked ubiquitination, and Lys63-ubiquitinated proteins increased in HK2 tubular cells under HG. Matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry identified 30 Lys63-ubiquitinated proteins, mainly involved in cellular organization, such as beta-actin, whose Lys63 ubiquitination increased under HG, leading to cytoskeleton disorganization. This effect was reversed by the inhibitor of the Ubc13/UBE2v1 complexNSC697923. Western blot analysis confirmed that UBE2v1 silencing in HK2 under HG, restored Lys63-beta-actin ubiquitination levels to the basal condition. Immunohistochemistry on patients with type 2 diabetic (T2D) revealed an increase in UBE2v1- and Lys63-ubiquitinated proteins, particularly in kidneys of patients with DN compared with control kidneys and other nondiabetic renal diseases, such as membranous nephropathy. Increased Lys63 ubiquitination both in vivo in patients with DN and in vitro, correlated with alpha-SMA expression, whereas UBE2v1 silencing reduced HG-induced alpha-SMA protein levels, returning themto basal expression. In conclusion, UBE2v1- and Lys63-ubiquitinated proteins increase in vitro under HG, as well as in vivo in T2D, is augmented in patients with DN, and may affect cytoskeleton organization and influence epithelial-to-mesenchymal transition. This process may drive the progression of tubular damage and interstitial fibrosis in patients with DN.Pontrelli, P., Conserva, F., Papale, M., Oranger, A., Barozzino, M., Vocino, G., Rochetti, M. T., Gigante, M., Castellano, G., Rossini, M., Simone, S., Laviola, L., Giorgino, F., Grandaliano, G., DiPaolo, S., Gesualdo, L. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

Three hundred sixty-six million people worldwide will be living with diabetes mellitus (DM) by 2030 ([1, 2]; http://www.idf.org/ global-diabetes-plan-2011-2021). Prospectively, 75–150 million of these patients will develop a diabetic nephropathy (DN) or a non-diabetic renal disease (NDRD), either isolated or superimposed on DN [3, 4]. To date, the differential diagnosis between ND and NDRD remains a challenge that nephrologists are trying to win [5]. DN is a widespread microangiopathic complication of DM eventually leading to end-stage renal disease over a variable number of years [6]. The natural history of DN, as originally depicted in patients with type 1 DM and later extrapolated to type 2 DM (T2DM), would comprise an early phase of microalbuminuria, which precedes overt nephropathy, characterized by macroalbuminuria and a progressive decline of glomerular filtration rate (GFR). Indeed, T2DM patients may present with overt proteinuria (and hypertension) already at the clinical onset of diabetic disease, or remain microalbuminuric without any progression to overt nephropathy, or even display renal insufficiency with minimal or absent proteinuria, this latter phenotype being increasingly recognized worldwide [7, 8]. In fact, the Renal Insufficiency and Cardiovascular Events (RIACE) Italian Multicenter Study (pts number = 15 773), published in 2011 [7], found a 56.6% of non-albuminuric chronic renal failure among patients with T2DM. It is likely that treatments such as the use of drugs blocking the renin angiotensin system (RAS) system or modulating dyslipidaemia and an aggressive glucose monitoring have changed the natural history of renal damage in DM shifting the microangiopathic forms of the disease towards macroangiopathic forms [9–11]. As a matter of fact, non-albuminuric renal impairment in the RIACE study was correlated with a higher prevalence of cardiovascular disease (CVD) and female sex [7, 8]. These data suggest that T2DM patients with non-albuminuric renal impairment have a macroangiopathic phenotype and a higher CVD burden. Consequently, some questions would arise: do these patients suffer from heart disease or kidney disease? By definition, DN recognizes a microangiopathic damage characterized by the clinical appearance of microalbuminuria. Is the reduction of GFR without an increase of albuminuria sufficient to rule out the diagnosis of DN? Is the reduction of GFR a haemodynamic effect due to RAS inhibition? The large variability in the clinical presentation of renal involvement in patients with T2DM, along with the possibility of glomerulonephritides independent from DM, make kidney biopsy a prerequisite for a correct and thorough diagnosis. However, not all nephrologists agree on the usefulness of renal biopsy, first of all because they believe that biopsy, in spite of the variability of clinical presentations, cannot but diagnose DN in the vast majority of patients with T2DM, which would render it useless and unethical, second because of the risk of major complications (haematuria, perirenal haematoma, arterial embolization and very rarely nephrectomy) [12–14]. Therefore, DN is commonly identified only on the basis of confusing clinical and laboratory data, and such diagnosis may in several cases result in misleading. A diagnostic model to correctly identify a pure DN has been recently developed by Zhou et al. [15] as well as by Liang et al. [5]. In the first report, several clinical and laboratory parameters such as years with diabetes, systolic blood pressure values, glycosylated haemoglobin levels, the absence of haematuria and diabetic retinopathy were significantly correlated in a logistic regression analysis with pure DN with a sensitivity and specificity higher than 90%. In the second report, the authors evaluated the predictive role of clinical and laboratory data to

Diabetic nephropathy (DN) has become the most frequent cause of chronic kidney disease worldwide due to the constant increase of the incidence of type 2 diabetes mellitus in developed and developing countries. The understanding of the pathophysiological mechanisms of human diseases through a large-scale characterization of the protein content of a biological sample is the key feature of the proteomics approach to the study of human disease. We discuss the main results of over 10 years of tissue and urine proteomics studies applied to DN in order to understand how far we have come and how far we still have to go before obtaining a full comprehension of the molecular mechanisms involved in the pathogenesis of DN and identifying reliable biomarkers for accurate management of patients.

Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system. The analysis of PBMCs phosphoproteome might help elucidate the signaling pathways essential to their biological role in health, immunological diseases and cancer. Enrichment of phosphoproteins becomes a prerequisite for phosphoproteome analysis and conventionally requires a multi-step procedure and sophisticated equipments. In this study, we standardized 2D-PAGE phosphoproteome analysis of PBMCs and compared two phosphoprotein enrichment methods, lanthanum chloride precipitation and affinity micro-column. Further, the different specificity for PBMCs phosphorylated proteins of each method was investigated.

retrograde into Bowman's space without mixing between compartments.Vacuum-assisted seeding improved cell delivery into vasculature and collecting systems: For the former, by demonstrating appropriate cells in the glomerular and peritubular capillary vasculature (e.g.GFP+).For the latter, by showing pancreatic islet cells in Bowman's space and tubules (e.g.insulin+).Conclusions: We have successfully developed protocols to transition from our rat model to pig kidneys for decellularization, sterilization and cell seeding.We selectively deployed cells antegrade through the vasculature into glomerular tufts, and retrograde up tubules into Bowman's space.Pancreatic islet cells successfully grew in the collecting system and demonstrated insulin production.This approach is very promising for tissue engineering a pig kidney trans-organ using a patient's iPSCs, and thereby crafting a vascularized pancreas xenotransplant.

Summary Background and objectives IgA nephropathy has variable clinical presentation and progression. Its definitive diagnosis and prognosis require renal biopsy. The identification of new biomarkers allowing noninvasive diagnosis and monitoring of disease activity would be advantageous. This study analyzed the urine proteome of IgA nephropathy patients at an early stage of disease. Design, setting, participants, & measurements Urine from 49 IgA nephropathy patients, 42 CKD patients, and 40 healthy individuals was analyzed by surface-enhanced laser desorption/ionization time of flight/mass spectrometry. Differentially excreted proteins were identified by matrix-enhanced laser desorption/ionization time of flight/mass spectrometry, confirmed by immunologic methods, and validated in an independent set of patients (14 IgA nephropathy and 24 CKD). All patients were recruited at the Division of Nephrology of the University of Foggia from January of 2005 to March of 2007. Results Two proteins, with 21,598 and 23,458 m/z, were significantly decreased in IgA nephropathy and identified as Perlecan laminin G-like 3 peptide and Ig κ light chains, respectively. Western blot analysis confirmed the lower urinary excretion of laminin G-like 3 in IgA nephropathy patients compared with CKD patients and healthy individuals. Immunonephelometry analysis confirmed the lower urinary excretion of free κ light chains in IgA nephropathy patients compared with CKD patients and healthy individuals. Immunohistochemistry analysis justified the urinary excretion profile of such proteins in IgA nephropathy. Finally, urinary free κ light chains and laminin G-like 3 concentration inversely correlated with severity of clinical and histologic features of our IgA nephropathy cohort. Conclusions Laminin G-like 3 and free κ light chains can contribute to the noninvasive assessment of IgA nephropathy disease activity.

2 Essalud Introduction and Aims: Progression and long-term renal outcome of proliferative lupus nephritis (PLN) in people from peruvian amazonic region is an uninvestigated subject in the literature. The aim of this study was to evaluate the influence of amazonic race of peruvian people on the renal outcome of PLN in comparison with the rest of the patients. Methods: All patients who fulfilled American College of Rheumatology lupus criteria and who were referred for a kidney biopsy from 2000 to 2010 were enrolled in the study. Subjects with end- stage renal disease (ESRD) at baseline, or follow-up time below 6 months, were excluded. Cases were randomly matched to Amazonic race (A) and Others (O) patients according to the class of LN, SLEDAI score, baseline estimated glomerular filtration rate (eGFR, Modification of Diet in Renal Disease simplified formula), ANA, Anti DNA, C3 levels, serum albumin, clinical onset and follow-up time. Treatment was decided by the clinical staff based on usual literature protocols. The primary endpoint was doubling of serum creatinine and/or ESRD. The secondary endpoint was defined as a variation of (GFR) per year (δGFR/y index), calculated as the difference between final and initial eGFR adjusted by follow-up time for each patient. Results: We included 92 patients (28 A: 64 O). At baseline, A and O patients were not statistically different regarding WHO LN class (III 18.5% IV 71.7%, V 9.8%), eGFR (A 62.4 + 36.4 versus F 60.1 + 27.2 ml/min/1.73m2), follow-up time (A 64.2 + 27.8 versus O 59.9 + 26.9 months), and 24-hour proteinuria (A 3.3 + 1.6 versus O 2.9 + 1.4 g/day), as well as age, albumin, C3, antinuclear antibody, anti-DNA antibody, haematuria and SLEDAI score. There was significative difference in the primary outcome (A 53.6% versus F 12.5%, log-rank p = 0.62). Same way, amazonic race was significantly associated with a worse renal function progression, as measured by δGFR/y index (β coefficient for amazonic race -11.9, 95% confidence interval -23.1.8 to -2.3, p = 0.03). The multivariate linear regression model showed that peruvian amazonic race remained statistically associated with a worse renal outcome even after adjustment for eGFR, proteinuria, albumin and C3 complement at baseline Conclusions: In our study, amazonic race presented a worse evolution of LN when compared with the rest of patients with similar baseline features and treatment. Factors that influence the progression of LN in amazonic people should be further studied.

OBJECTIVEThe intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immunoglobulin A nephropathy (IgAN) pathogenesis, which is still largely undefined. Recently, vasopressin (AVP) has been advocated to contribute to the genesis and progression of chronic kidney diseases (CKD) directly, and indirectly, via RAS activation. Our aim is to explore the intrarenal activity of AVP, its relationship with RAS activity, as well as its modulation by therapies in IgAN.DESIGNIn this observational study, we measured plasma copeptin, a surrogate marker of AVP, the urine excretion of aquaporin 2 (AQP2), a protein reflecting renal AVP action, and angiotensinogen (AGT), a parameter of renal RAS activation, and their relationship with renal function in 44 IgAN patients at the time of renal biopsy, without any drug therapy, and after 6-month treatment with ACEi or steroid+ACEi. Twenty-one patients with other CKD and 40 healthy subjects were recruited as controls.METHODSELISAs were used to measure all variables of interest.RESULTSAt baseline, IgAN patients showed higher urinary levels of AQP2, compared with controls and patients with other CKD. Urinary AQP2 and AGT levels strongly correlated with the presence of arterial hypertension. Steroids+ACEi caused the decrease of all the variables examined. The fall of urinary AQP2 and AGT following drug treatments was associated with the decrease of daily proteinuria.CONCLUSIONOur findings would support the involvement of AVP-AQP2 axis, interacting with the RAS, in the progression of IgAN and candidate AQP2 as a possible novel marker of the disease.