Genetic Analyses in Dent Disease and Characterization of CLCN5 Mutations in Kidney Biopsies.

Dent disease (DD), an X-linked renal tubulopathy, is mainly caused by loss-of-function mutations in CLCN5 (DD1) and OCRL genes. CLCN5 encodes the ClC-5 antiporter that in proximal tubules (PT) participates in the receptor-mediated endocytosis of low molecular weight proteins. Few studies have analyzed the PT expression of ClC-5 and of megalin and cubilin receptors in DD1 kidney biopsies. About 25% of DD cases lack mutations in either CLCN5 or OCRL genes (DD3), and no other disease genes have been discovered so far. Sanger sequencing was used for CLCN5 gene analysis in 158 unrelated males clinically suspected of having DD. The tubular expression of ClC-5, megalin, and cubilin was assessed by immunolabeling in 10 DD1 kidney biopsies. Whole exome sequencing (WES) was performed in eight DD3 patients. Twenty-three novel CLCN5 mutations were identified. ClC-5, megalin, and cubilin were significantly lower in DD1 than in control biopsies. The tubular expression of ClC-5 when detected was irrespective of the type of mutation. In four DD3 patients, WES revealed 12 potentially pathogenic variants in three novel genes (SLC17A1, SLC9A3, and PDZK1), and in three genes known to be associated with monogenic forms of renal proximal tubulopathies (SLC3A, LRP2, and CUBN). The supposed third Dent disease-causing gene was not discovered.

Pilot cohort study on the potential role of TCF7L2 rs7903146 on ischemic heart disease among non-diabetic kidney transplant recipients.

BACKGROUND: TCF7L2 rs7903146 C>T polymorphism is associated with diabetes in the general population but its independent impact on cardiovascular disease is debated. On this basis, we investigated its association with major adverse cardiac events (MACE) in a single-center cohort of non-diabetic kidney transplant recipients (KTRs). METHODS: Patients with pretransplant diabetes were excluded and patients who developed post-transplant diabetes were censored at time of diagnosis. RESULTS: rs7903146 C>T polymorphism appeared to modulate the risk of MACE: 5-year prevalence was 0.8% in CC patients, 7.2% in CT patients and 9.7% in TT patients (P<.001). TCF7L2 rs7903146 was an independent predictor of MACE in a multivariate Cox regression model (for each T allele, HR: 2.99, 95%CI: 1.62-5.52, P<.001), together with history of cardiac ischemic events (HR: 8.69, 95%CI: 3.57-21.16, P<.001), DGF (HR: 2.42, 95%CI: 0.98-5.95, P=.056) and HLA-mismatches (for each mismatch: HR: 1.55, 95%CI: 1.00-2.43, P=.053). Introduction of rs7903146 C>T polymorphism into a model based on these clinical variables significantly increased predictive power for MACE (P=.003). CONCLUSIONS: TCF7L2 rs7903146 T allele may be strongly and independently associated with MACE in non-diabetic KTRs. These findings suggest the possibility of employing this SNP to more accurately stratify cardiological risk in KTRs.

De novo noncutaneous malignancies after kidney transplantation are associated with an increased risk of graft failure: results from a time-dependent analysis on 672 patients.

The aim of this study was to evaluate the association between cancer occurrence and risk of graft failure in kidney transplant recipients. From November 1998 to November 2013, 672 adult patients received their first kidney transplant from a deceased donor and had a minimum follow-up of 6 months. During a median follow-up of 4.7 years (3523 patient-years), 47 patients developed a nonmelanoma skin cancer (NMSC) and 40 a noncutaneous malignancy (NCM). A total of 59 graft failures were observed. The failure rate was 6 per 100 patient-year (pt-yr) after NCM versus 1.5 per 100 pt-yr in patients without NCM. In a time-dependent multivariable model, the occurrence of NCM appeared to be associated with failure (HR = 3.27; 95% CI = 1.44-7.44). The effect of NCM on the cause-specific graft failure was different (P = 0.002) when considering events due to chronic rejection (HR = 0.55) versus other causes (HR = 15.59). The reduction of the immunosuppression after NCM was not associated with a greater risk of graft failure. In conclusion, our data suggest that post-transplant NCM may be a strong risk factor for graft failure, particularly for causes other than chronic rejection.

Calcium-sensing-related gene mutations in hypercalcaemic hypocalciuric patients as differential diagnosis from primary hyperparathyroidism: detection of two novel inactivating mutations in an Italian population.

BACKGROUND: Inactivating mutations of the calcium-sensing receptor (CaSR), of the G-protein subunit α11 (GNA11) and of the adaptor-related protein complex 2, sigma 1 subunit (AP2S1) genes are responsible for familial hypocalciuric hypercalcaemia (FHH). The aim of this study was to analyse prevalence and pathogenicity of CaSR, GNA11 and AP2S1 mutations in patients with an FHH phenotype and to compare them with a sample of patients with primary hyperparathyroidism (PHPT) in order to identify the most useful laboratory parameter for a differential diagnosis. METHODS: Patients with an FHH phenotype were studied with polymerase chain reaction amplification and direct sequencing of the entire CaSR, GNA11 and AP2S1 coding sequences. Novel mutations were introduced in a Myc-tagged human wild-type (WT) CaSR cDNA-expressing vector, and functional assay was performed on human embryonic kidney cells evaluating expression and function of mutated proteins. RESULTS: Among 16 FHH patients, none had an inactivating GNA11 or AP2S1 mutation while 3 (18.8%) carried a CaSR mutation and 10 (62.5%) at least one CaSR polymorphism. Within the latter group, 7 of 10 patients had more than one polymorphism (4.1 ± 2.1 per patient). Two novel CaSR mutations [c.2120A>T (E707V) and c.2320G>A (G774S)] were identified: the E707V mutation prevented CaSR expression (western blot), whereas the G774S mutation determined a reduced receptor sensitivity to calcium (IP3 assay). PHPT patients showed significantly (P < 0.001) higher serum calcium, parathyroid hormone, urinary calcium and calcium-creatinine clearance ratio (CCCR) and significantly lower serum phosphate than FHH ones. CONCLUSIONS: FHH should be clearly differentiated by PHPT to avoid unnecessary surgery: CCCR could be a useful screening tool while genetic analysis should include the two novel CaSR mutations herein described. The role of multiple polymorphisms deserves further investigation in patients with an FHH phenotype.

Unexpectedly high prevalence of rare genetic disorders in kidney transplant recipients with an unknown causal nephropathy.

BACKGROUND: Patients with a rare genetic disease may receive renal transplantation (KTx) without a correct diagnosis of causal nephropathy and therefore develop unexpected and even severe complications. The aim of the study was to describe the cases of rare genetic disorders diagnosed after KTx, in order to draw clinical lessons for the transplant physician. METHODS: We retrospectively assessed all patients who had received a diagnosis of a rare genetic disorder after KTx. RESULTS: In our center, more than 30% (278/911) of kidney transplant (KTx) recipients were diagnosed with a causal nephropathy: Prevalence of rare genetic disorders in this group was 4.32% (12/278), including 2,8-dihydroxyadeninuria (2,8-DHA) disease (n = 2), HNF-1B-associated nephropathy (n = 2), UMOD-related nephropathy (n = 5), Fabry disease (n = 1), INF2 focal segmental glomerulosclerosis (n = 1), and Senior-Løken syndrome (n = 1). 2,8-DHA nephropathy relapsed in both patients causing an acute renal failure and jeopardizing the graft. CONCLUSIONS: Kidney transplant recipients without a diagnosis of causal nephropathy appear to be a selected population in which rare genetic diseases might be more common than expected. As even a belated diagnosis after KTx can have a significant impact on graft and patient survival and on other family members, this possibility should be evaluated in KTx recipients without a known causal nephropathy.

Chronic renal failure of unknown origin is caused by HNF1B mutations in 9% of adult patients: a single centre cohort analysis.

BACKGROUND: HNF1B gene mutations might be an underdiagnosed cause of nephropathy in adult patients mainly because of their pleomorphic clinical presentations. As most studies are based on paediatric populations, it is difficult to assess the likelihood of finding HNF1B mutations in adult patients and consequently define clinical settings in which genetic analysis is indicated. The aim of this study was the search for mutations in the HNF1B gene in a cohort of unrelated adult patients with nephropathy of unknown aetiology. METHODS: Patients were tested for the HNF1B gene if they had chronic kidney disease of unknown origin and renal structure abnormalities (RSA) or a positive family history of nephropathy. The HNF1B coding sequence and intron-exon boundaries were analysed by direct sequencing. The search for gene deletions was performed by Multiple Ligation Probe Analysis (MLPA). RESULTS: Heterozygous mutations were identified in 6 out of 67 screened patients (9.0%) and included two whole gene deletions, one nonsense (p.Gln136Stop), two missense (p.Gly76Cys and p.Ala314Thr) mutations and a frameshift microdeletion (c.384_390 delCATGCAG), the latter two (c.384_390 del and p.Ala314Thr) not ever being reported to date. Mean age of the mutated patients at screening was 48.5 years with a M/F ratio of 2/4. The clinical manifestations of affected patients were extremely pleomorphic, including several urological and extra-renal manifestations. CONCLUSIONS: Mutations of HNF1B could explain chronic kidney disease in up to 9% of adult patients with a nephropathy of unknown aetiology and RSA: therefore an HNF1B mutation analysis should be considered in this group of patients.

Experience of 70-cm-long femoral tunnelled twin Tesio catheters for chronic haemodialysis.

BACKGROUND: Tunnelled femoral catheters with their tip in the lower inferior vena cava (IVC) are proposed only in few cases, but they often provide less than optimal blood flows and frequently have complications. The aim of this prospective observational study is to evaluate the use of 70-cm-long tunnelled cuffed femoral twin Tesio catheters with their tip in the upper IVC for haemodialysis. METHODS: Between May 2007 and May 2009, 25 tunnelled femoral catheters (fCVC) have been placed in 25 patients (77.7 +/- 10.8 years) with exhausted thoracic venous accesses or old patients with several comorbidities. Two 10 Fr carbothane 70-cm-long Tesio catheters with a Dacron cuff at 45 cm from the tip were placed in the femoral vein of each patient and then tunnelled; tips were in the upper third of the IVC. fCVCs were removed for either malfunction (Qb < 200 ml/min) or infection that did not resolve with antibiotics. RESULTS: Technical success of placement was 100%. The 6- and 12-month assisted primary patency rate were respectively 67 +/- 13% and 54 +/- 17%. The mean session Kt/V was 1.45 +/- 0.19, and the blood flow was 270 +/- 17 ml/min. Six fCVCs have been removed: three for infection, one for accidental damaging and two for the making of a different vascular access. The main complications were 2 catheter tip thrombi, 3 tunnel infections and 11 fCVC-related bacteraemia (1.77 episodes per 1000 CVC-days). CONCLUSION: The placement of twin fCVCs with their tip in the high IVC can provide an adequate dialysis and can be considered for patients with no remaining thoracic accesses.

[Peritoneal dialysis: a time-limited therapy because of encapsulating peritoneal sclerosis?]. / La dialisi peritoneale: una metodica ''a termine'' a causa della peritonite sclerosante incapsulante?

Encapsulating peritoneal sclerosis (EPS) is a rare but severe disease with a mortality rate of 24%-54%, whose main risk factor is peritoneal dialysis (PD) (cumulative incidence 0.5%-7.3%). Although the role of the time spent on peritoneal dialysis is not completely clear, the available evidence suggests that peritoneal dialysis should not be discontinued early for the sake of reducing the EPS risk. We proposed a ''pro-con'' debate which confirmed that PD is not a time-limited technique. Nevertheless, in patients on long-term PD, surveillance of the peritoneal membrane is crucial. The development of EPS requires two ''hits'': first a chronic, inflammatory stimulus, which is typical of peritoneal dialysis, then a second event like PD interruption or kidney transplant. The main pharmacological and dialysis strategies that have been used as primary prevention did not show any significant results, and benefits are more likely to be achieved by reducing the peritoneal inflammation and better preservation of the membrane integrity, for example by means of more biocompatible PD solutions. Among the main surveillance and early diagnosis procedures other than the peritoneal equilibration test, the Ca-125 appearance rate and new tests that evaluate the peritoneal water transport seem to be promising.

[Incremental peritoneal dialysis: a common solution for different types of patients]. / La dialisi peritoneale incrementale: una soluzione comune per pazienti diversi.

It is still a matter of debate whether peritoneal dialysis should be initiated with a full dose regimen or with incremental doses. The use of low-dose dialysis is not always feasible, but it may have the advantages of improved quality of life and lower infective and metabolic complication rates. However, incremental peritoneal dialysis has to meet the minimal suggested adequacy targets in terms of depuration (Kt/V, creatinine clearance), ultrafiltration, and electrolyte balance. Incremental peritoneal dialysis has been proved feasible and safe in asymptomatic patients with a glomerular filtration rate (GFR)<6 mL/min, but the residual renal function has to be monitored strictly. A second population is composed of asymptomatic, mostly older patients with GFR between 6 and 10 mL/min, in whom a low-dose start may preserve the residual renal function and favor a more gradual training. Lastly, patients with severe, terminal, chronic cardiomyopathy who are not candidates for a heart transplant may experience beneficial effects on cardiac function and hospitalization with low-dose peritoneal dialysis treatment even when they have GFR>10 mL/min. In conclusion, incremental peritoneal dialysis is a feasible therapeutic option that the nephrologist should know and be able to perform in those patients who may benefit from it.

[Cardioprotective effect of vitamin D in nondialyzed patients with stage 3-5 CKD]. / Effetti cardioprotettivi della vitamina D nei pazienti con malattia renale cronica allo stadio 3-5 non in dialisi.

Epidemiological and observational data indicate that there is a close relationship between progressive renal dysfunction in chronic kidney disease (CKD), cardiovascular disease, and mortality. In addition, deficits in vitamin D (25-hydroxyvitamin D) and vitamin D receptor (VDR) activation play a crucial role in adversely affecting cardiovascular health in CKD patients. Even in patients with mild CKD, renal dysfunction is associated with cardiovascular events. Modulation of vitamin D levels results in correlative regulatory effects on mineral homeostasis, hypertension, and vascular calcification. The use of VDR activators such as paricalcitol to treat these and other parameters outside of cardiovascular and renal disease not only results in enhanced patient health but significantly reduces the mortality risk in CKD patients.

Oxidative stress in elderly chronic renal failure patients: effects of renal replacement therapies on cell membrane fluidity.

BACKGROUND: Renal replacement therapies (RRTs) produce a partial loss of antioxidants and formation of reactive oxygen species (ROS), which are a major factor involved in alterations of plasma membrane fluidity and endothelial activation, but their role on plasma membrane fluidity in vivo is still unclear. We compared erythrocyte plasma membrane fluidity, ROS and total plasma antioxidant defenses (Lagtime) in aged patients with chronic renal failure (CRF) on conservative treatment, peritoneal dialysis (PD) and hemodialysis (HD) before (HD-pre) and after (HD-post) a treatment, to evaluate the role of different RRTs on oxidative stress and plasma membrane fluidity in aged patients. METHODS: We assessed erythrocyte plasma membrane fluidity, plasma lipid hydroperoxide levels and Lag-Time in 11 CRF patients on conservative treatment, 15 on PD, 12 on HD and 30 healthy controls. RESULTS: Hydroperoxides were higher in CRF, PD and HD-post, whereas Lag-time was significantly lower in PD, CRF and in HD-post. CRF, PD and HD-pre also had higher membrane fluidity (rsDPH), compared with HD-post and controls. CONCLUSION: These findings are in keeping with the hypothesis that the Lag-time decrease is due not only to the effect of the RRT but also to the uremic state, and that PD patients undergo a chronic, greater oxidative stress. Contrary to expectations, all patients showed greater erythrocyte membrane fluidity, which can be attributed to uremic toxicity. These observations reinforce the hypothesis that oxidative stress is an intrinsic component of this disease state and indeed is already present also in CRF not yet requiring RRT.

Malignancy after kidney transplantation: results of 400 patients from a single center.

BACKGROUND: Post-transplant malignancies (PTM) occur in a percentage as high as 50% in patients followed 20 yr and have become a main cause of mortality and are expected to be the first cause of death within the next 20 yr in kidney transplant recipients. PATIENTS AND METHODS: We analyzed the PTM incidence in our kidney transplant recipients, and its main risk factors. The records of 400 patients (min follow up = one yr) have been retrospectively reviewed and categorized into three groups: patients without any tumor, with a non-melanoma skin cancer and with a solid or hematologic cancer. A cancer-free multivariate survival study was performed stratified by age, sex, immunosuppressive therapy, time on dialysis, body mass index (BMI), smoke, diabetes and nephropathy. RESULTS: Thirty patients developed PTM: 12 non-melanoma skin cancer, three lymphomas and 15 solid malignancies (seven genitourinary, three lung, two breast, two gastrointestinal and one sarcoma). The mean age at diagnosis was 55 yr, with a mean time from transplant of 27 months. We observed six deaths and two graft losses. Non-melanoma skin cancer-free survival and the solid/hematologic cancer-free survival was 99.5% and 98.5% at one yr, and 95.2% and 94.6% at five yr, respectively. At univariate analysis, age and induction therapy were significant risk factors for both types of PTM, while only recipient age significantly increased the risk of all PTM, and anti CD25 significantly reduced the risk of non-melanoma skin cancer at the multivariate study. CONCLUSIONS: These data confirm the role of age and induction strategies in modulating the risk of neoplasia. To look for which strategies might reduce the PTM risk, including a personalized therapy to minimize the effects of chronic immunosuppressant, will be a crucial goal.

Erdheim-Chester Disease as a Mimic of IgG4-Related Disease: A Case Report and a Review of a Single-Center Cohort.

Immunoglobulin-G4 (IgG4)-related disease (IgG4RD) is a fibro-inflammatory disorder characterized by tissue-infiltrating IgG4 plasma cells, and, often, high serum IgG4. Several autoimmune, infectious, or proliferative conditions mimic IgG4RD. Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis, characterized by foamy histiocytic infiltration, fibrosis, and chronic inflammation. ECD and IgG4RD manifestations may overlap.A patient presented with huge fibrous retroperitoneal masses causing compression on neighboring structures; the case posed the challenge of the differential diagnosis between IgG4RD and ECD mainly because of a prominent serum and tissue IgG4 response.Retroperitoneal biopsy led to the diagnosis of ECD; the V600E BRAF mutation was found. Treatment with the BRAF inhibitor vemurafenib was started.Treatment failed to induce mass regression and the patient died after 3 months of therapy. Prompted by this case, we examined serum and tissue IgG4 in a series of 15 ECD patients evaluated at our center, and found that approximately one-fourth of the cases have increased IgG4 in the serum and often in the tissue.The differential diagnosis between IgG4RD and ECD can be challenging, as some ECD patients have prominent IgG4 responses. This suggests the possibility of common pathogenic mechanisms between ECD and IgG4RD.

The Role of TCF7L2 rs7903146 in Diabetes After Kidney Transplant: Results From a Single-Center Cohort and Meta-Analysis of the Literature.

BACKGROUND: Several genetic polymorphisms modulate the risk of posttransplant diabetes mellitus (PTDM), a complication associated with an increased morbidity and mortality after kidney transplantation; however, their clinical utility is still undefined. METHODS: Genetic analysis was performed in 464 kidney transplantation recipients to evaluate whether transcription factor 7-like 2 (TCF7L2) rs7903146 gene polymorphism is associated with the risk of PTDM and a meta-analysis of similar studies including our results was performed (total kidney transplantation recipients, n = 3105). A predictive model of PTDM was built on the basis of this polymorphism and clinical parameters. RESULTS: In our cohort, 163 patients possessed the CC genotype of rs7903146 (35.1%), 237 were CT (51.1%), and 64 were TT (13.8%): their 2 years PTDM incidence was, respectively, 7.8%, 11.9%, and 22.7%. At multivariate analysis, age (per year; hazard ratio [HR], 1.029; 95% confidence interval [95% CI], 1.005-1.054; P = 0.017), body mass index (25.0-29.9 vs <25.0; HR, 2.43; 95% CI, 1.40-4.23; P = 0.0018; ≥30 vs <25.0; HR, 5.70; 95% CI, 2.77-11.74; P < 0.0001), TCF7L2 rs7903146 (per each T allele; HR, 1.81; 95% CI, 1.26-2.59; P = 0.001) and previous transplants (HR, 2.80; 95% CI, 1.39-5.64; P = 0.004) emerged as independent predictive factors for PTDM.Meta-analysis of present and 5 previous studies showed higher risk of PTDM in carriers of rs7903146 TT genotype (odds ratio, 1.95; 95% CI, 1.39-2.74; P < 0.0001) and absence of heterogeneity among studies (I = 0%).Inclusion of this polymorphism in a predictive model appeared to improve its ability to stratify patients according to the risk of PTDM. CONCLUSIONS: In renal transplant patients, TCF7L2 rs7903146 is strongly and independently associated with PTDM and might hold the potential to identify patients at risk for this complication.

Testing for the cytosine insertion in the VNTR of the MUC1 gene in a cohort of Italian patients with autosomal dominant tubulointerstitial kidney disease.

INTRODUCTION: Medullary cystic kidney disease type 1 (MCKD1; OMIM #174000) is a familial progressive tubule-interstitial nephropathy belonging to the recently defined group of autosomal dominant tubulointerstitial kidney diseases (ADTKD). CASE REPORT: A specific type of cytosine insertion in the extracellular variable number tandem repeat (VNTR) domain of the MUC1 gene causing the disease was tested in a group of 21 families with ADTKD. We identified this type of MUC1 mutation in two families, whose affected members are described in detail in this case report. Affected (ADTKD-MUC1) members developed end-stage renal disease (ESRD) with a higher incidence (p = 0.033) and at a younger age (p = 0.013) than probands with ADTKD but without this type of mutation. All patients with MUC1-associated kidney disease shared a rather unspecific tubule-interstitial laboratory pattern without medullary cysts, leading to ESRD between the age of 33 and 47 years. We were not able to identify any single common extra-renal feature among affected patients, even if they had various comorbidities, which are described in detail. CONCLUSIONS: We identified this type of MUC1 mutation in 9.5 % of families from an ADTKD Italian cohort; larger studies are needed to better define the criteria for genetic testing for this type of mutation.