Guido Merlotti

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.

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 (I2 = 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.

Type iv renal tubular acidosis: an emerging type of nephropathy

Type IV renal tubular acidosis (RTA IV) is a form of hyperchloremic RTA caused by an absolute or functional hypoaldosteronism that determines a distal acidification defect by inhibiting ammoniagenesis and H+ excretion by the collecting duct. Etiology is often multifactorial and includes disorders causing a reduction in aldosterone levels or in the sensitivity of the collecting duct to the hormone. Several drugs block the renin-angiotensin system (RAS) at different steps, thus increasing the risk of developing RTA IV especially when used in association in elderly patients with diabetes, congestive heart failure, chronic renal failure, or in renal transplant recipients. Diagnosis is based on the presence of hyperkaliemia disproportionate to the degree of renal function, associated with a metabolic acidosis with normal serum anion gap and a positive urinary anion gap; urinary pH can be lower than 5.5. Some laboratory features allow differential diagnosis with distal type I RTA. The clinical impact of RTA IV is becoming progressively heavier due to the widespread use of RAS-blocking drugs in elderly patients already at risk for this complication because of their comorbidities; this situation is inducing a critical revision of the indications to association therapies with these agents. The onset of hyperkalemia in this setting poses a therapeutic dilemma, as the patients at highest risk for RTA IV are also those who can derive the strongest cardiovascular benefits from RAS pharmacological blockade. It is therefore necessary to find a balance between risks and benefits in each patient, adopting preventive measures against RTA IV.

Endothelial Progenitor Cell-Derived Extracellular Vesicles Inhibit Kidney Ischemia-Reperfusion Injury through the transfer of Specific Micrornoa and Mrna Coding for the Transcription Factor NRF2: Relevance for Delayed Kidney Graft Function

Background and Aim Activation of the complement cascade and oxidative stress in tubular epithelial and peritubular endothelial cells are hallmarks of delayed kidney graft function (DGF) due to ischemia-reperfusion injury (IRI). Endothelial progenitor cells (EPCs) are bone marrow-derived precursors known to reverse IRI by paracrine mechanisms including the release of extracellular vesicles (EV), small particles playing a role in intercellular communication through the transfer of specific mRNA and microRNA. The aim of this study was to evaluate the regenerative role of EPC-derived EV in kidney IRI through the horizontal transfer of RNA involved in the inhibition of complement activation and of oxidative stress. Methods EPC were isolated from peripheral blood of healthy volouteers and EV obtained by supernatant ultracentrifugation were characterized for size, protein and RNA content. We evaluated the effects of EV in a rat model of kidney IRI and in vitro in human tubular epithelial and endothelial cells cultured in hypoxia in vitro. Results EPC-derived EV sized 60-130 nm and carried different subsets of mRNAs and microRNAs able to modulate cell proliferation, angiogenesis and apoptosis (eNOS, Akt, Bcl-XL, miR-126, miR-296). By RT-PCR, we also found within EV mRNAs coding for the complement inhibitors factor H, DAF (CD55), CD59 and for the anti-oxidant transription factgor nrf2. In experimental kidney IRI, EPC EV localized within peritubular capillaries and tubular cells exerting morphologic and functional protection from by reducing tubular cell apoptosis/senescence, endothelial-to-mesenchymal transition (EndoMT) and leukocyte infiltration. Moreover, EV administration reduced C5b9 deposition and enhanced the expression of factor H, DAF, CD59 and nrf2 in the ischemic kidney. In vitro, EV reduced hypoxia-induced apoptosis/senescence of tubular epithelial (caspase activation, Klotho expression) and endothelial cells (EndoMT) by up-regulating the expression of factor H, DAF, CD59 and nrf2, thus confirming the in vivo data. The role of specific mRNA transfer to hypoxic renal cells was confirmed by experiments using RNase-treated EV, EV released from EPC engineered to knock-down the complement inhibitors or nrf2 by specific siRNA, or EV produced by EPC transfected with siRNA Dicer, the intracellular enzyme essential for microRNA maturation. Conclusions EPC-derived EV protect the kidney from IRI by delivering pro-angiogenic, anti-apoptotic, complement inhibitors and anti-oxidant microRNAs and mRNAs coding for factor H, DAF, CD59 and nrf2 to injured tubular epithelial and endothelial cells. These results suggest the potential use of EPC-derived EVs as therapeutic tool to avoid or at least to limit IRI-associated DGF in kidney transplantation without the potential adverse effects of whole stem cell therapy including maldifferentiation and tumorigenesis.

Autosomal dominant tubulointerstitial kidney disease (ADTKD)

Autosomal Dominant Tubulointerstitial Kidney Diseases (ADTKD) are a group of autosomal dominant hereditary nephropathies which share a rather aspecific pathological picture of interstitial fibrosis and tubular atrophy (IFTA) and a slowly progressing tubulointerstitial clinical profile: end-stage renal disease (ESRD) generally occurs between 30 and 50 years. Four types of ADTKD have been described so far, each showing some peculiarities: hepatocyte nuclear factor-1 β (HNF1β) ADTKD, in which ADTKD can be associated with renal cysts and a wide spectrum of congenital abnormalities of kidney and urinary tract (CAKUT) and extra-renal manifestations (diabetes, liver enzyme alterations, genital malformations), uromodulin (UMOD) ADTKD, characterised by early onset of hyperuricemia and gout, mucin-1 (MUC-1) ADTKD, a recently discovered form which appears to be an isolated tubulointerstitial nephropathy, and renin (REN) ADTKD, which appears in childhood and is characterised by anemia and mild signs of hyporeninism. A better awareness is needed in order to perform genetic analysis whenever clinical setting is compatible with this nephropathy. A timely diagnosis can have an important impact on both the patient and his family members in terms of therapy and prognosis, also at the prospect of renal transplant.

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

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).

Renal tubular acidosis

Renal tubular acidosis (RTA) refers to a group of disorders of multiple etiology characterised by a hyperchloremic, normal anion-gap metabolic acidosis caused by different defects affecting several mechanisms of urinary acidification: an impaired secretion of protons by collecting duct in distal hypokalemic RTA (type 1), impaired resorption of bicarbonate in proximal RTA (type 2), a mixed proximal and distal defect in RTA type 3 and an impaired ammoniagenesis due to an absolute or functional hypoaldosteronism in distal hyperkalemic RTA (type 4). Etiology is heterogeneous and ranges from primitive genetic forms to secondary RTA within a multi-system disorder such as autoimmune disease, paraproteinemia and calcium-phosphate disorders. Drug-induced RTA has been increasingly reported over the last decade due to widespread use of some molecules (e.g. antiviral and oncologic agents, inhibitors of renin-angiotensin system). The diagnosis of RTA is based on assessment of serum and urine anion-gap, whereas differential diagnosis between RTA forms requires the analysis of urine pH or osmolality and of serum and urine electrolytes. An acidification test should be performed to confirm the diagnosis. Clinical setting can also provide important clues in orienting towards a type of RTA: nephrolithiasis and nephrocalcinosis are associated with type 1 RTA, ostemalacia and osteoporosis are more frequent in type 2 RTA, osteopetrosis is typically linked to type 3 RTA and hyperkalemia is the predominant manifestation of type 4 RTA. An increased awareness of RTA is needed among clinical Nephrologists to recognise it and prevent potentially severe complications.

Comment on: Think to prevent before than to treat renal impairment in multiple myeloma: do not forget tubular damage mimicking Fanconi syndrome

In a recent paper, renal impairment is correctly described as a common complication of symptomatic myeloma (20 -40%) needing dialysis. Significant improvement has been attributed to novel therapeutic chemotherapy regimens coupled with extrarenal free light chain removal obtained by plasma exchange or dialysis [1]. As nephrologists, we would like to stress the importance of recognizing early symptoms of renal impairment other than by measuring renal function as glomerular filtration rate (GFR) [2]. An increase in serum creatinine and/or a decrease in GFR are not the only markers of renal impairment in patients with multiple myeloma. Although a serum creatinine > 2 mg/dl is one of the hypercalcemia, renal impairment, anemia, bone disease (CRAB) diagnostic criteria for symptomatic myeloma requiring therapy, it has been stated that “a variety of other types of end-organ dysfunctions can occur and lead to a need for therapy. Such a dysfunction is sufficient to support classifications of myeloma if proven to be myeloma-related” [3]. Tubular dysfunctions, and mainly proximal tubular dysfunctions with the picture of complete or incomplete Fanconi syndrome, constitute such “other end-organ dysfunctions” [1-3]. As far as the kidney is concerned, it is important to have early indicators of tubular dysfunction. This includes hypophosphatemia, hypouricemia, hypokalemia and metabolic acidosis coupled with urine loss of phosphate, urate, potassium, bicarbonate, low-molecular-weight proteins and glycosuria (in the presence of normal blood glucose levels). Therefore, even if free light chains detected in the urine (Bence Jones proteinuria) is not a sign of renal damage by itself, its transcellular “traffic” may cause tubular damage, eventually leading to tubular crystal-storing histiocytosis and Fanconi syndrome. Subsequently, tubular casts precipitating in the distal tubule may cause acute renal failure of the classical ‘myeloma kidney’ [4,5]. Therefore, a delay in diagnosis could allow irreversible kidney damage to occur and might shorten patient survival.