Francesco Trepiccione

Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease.

Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs‐induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD).

Quantitative proteomics reveals novel therapeutic and diagnostic markers in hypertension.

Hypertension is a prevalent disorder in the world representing one of the major risk factors for heart attack and stroke. These risks are increased in salt sensitive individuals. Hypertension and salt sensitivity are complex phenotypes whose pathophysiology remains poorly understood and, remarkably, salt sensitivity is still laborious to diagnose. Here we present a urinary proteomic study specifically designed to identify urinary proteins relevant for the pathogenesis of hypertension and salt sensitivity. Despite previous studies that underlined the association of UMOD gene variants with hypertension, this work provides novel evidence showing different uromodulin protein level in the urine of hypertensive patients compared to healthy individuals. Notably, we also show that patients with higher level of uromodulin are homozygous for UMOD risk variant and display a decreased level of salt excretion, highlighting the essential role of UMOD in the regulation of salt reabsorption in hypertension. Additionally, we found that urinary nephrin 1, a marker of glomerular slit diaphragm, may predict a salt sensitive phenotype and positively correlate with increased albuminuria associated with this type of hypertension.

Divergent behavior of hydrogen sulfide pools and of the sulfur metabolite lanthionine, a novel uremic toxin, in dialysis patients

Dialysis patients display a high cardiovascular mortality, the causes of which are still not completely explained, but are related to uremic toxicity. Among uremic toxins, homocysteine and cysteine are both substrates of cystathionine β-synthase and cystathionine γ-lyase in hydrogen sulfide biosynthesis, leading to the formation of two sulfur metabolites, lanthionine and homolanthionine, considered stable indirect biomarkers of its production. Hydrogen sulfide is involved in the modulation of multiple pathophysiological responses. In uremia, we have demonstrated low plasma total hydrogen sulfide levels, due to reduced cystathionine γ-lyase expression. Plasma hydrogen sulfide levels were measured in hemodialysis patients and healthy controls with three different techniques in comparison, allowing to discern the different pools of this gas. The protein-bound (the one thought to be the most active) and acid-labile forms are significantly decreased, while homolanthionine, but especially lanthionine, accumulate in the blood of uremic patients. The hemodialysis regimen plays a role in determining sulfur compounds levels, and lanthionine is partially removed by a single dialysis session. Lanthionine inhibits hydrogen sulfide production in cell cultures under conditions comparable to inxa0vivo ones. We therefore propose that lanthionine is a novel uremic toxin. The possible role of high lanthionine as a contributor to the genesis of hyperhomocysteinemia in uremia is discussed.

A randomized controlled pilot trial of lithium in spinocerebellar ataxia type 2

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder. Lithium is able to stimulate autophagy, and to reduce Ca2+ efflux from the inositol-1,4,5-triphosphate receptor. We designed a phase II, randomized, placebo-controlled, double-blind, 48-week trial with lithium carbonate in 20 patients with SCA2. The primary objective was to determine safety and tolerability of lithium. The secondary objectives were to determine disease progression, quality of life, mood, and brain volume change. Sixteen patients completed the trial, 8 randomized to lithium, 8 to placebo. Forty adverse events (AEs) were reported during the trial, twenty-eight in the lithium and 12 in the placebo group (pxa0=xa00.11). Mean AE duration was 57.4xa0±xa060.8 and 77.4xa0±xa068.5xa0days (pxa0=xa00.37). Non-significant differences were observed for the SARA and for brain volume change, whereas a significant reduction in the BDI-II was observed for lithium group (pxa0<xa00.05). Lithium was well tolerated and reported AEs were similar to those previously described for bipolar disorder patients. A correctly powered phase III trial is needed to assess if lithium may slow disease progression in SCA2.

A new recombinant MnSOD prevents the Cyclosporine A-induced renal impairment

BACKGROUNDnCyclosporine A (CsA) is one of the most frequently used anticalcineurinic drugs for preventing graft rejection and autoimmune disease. Its use is hampered by nephrotoxic effects, namely an impairment of the glomerular filtration rate (GFR) and hypertension. Evidence suggests that reactive oxygen species (ROS) play a causal role in the nephrotoxicity. The present study aims to investigate in vivo the effects of a new recombinant mitochondrial manganese-containing superoxide dismutase (rMnSOD), a strong antioxidant, on the CsA-induced nephotoxicity.nnnMETHODSnRats were treated with CsA (25 mg/kg/day) alone or in combination with rMnSOD (10 µg/kg/day) for 7 days. At the end of the treatment, GFR was estimated by inulin clearance (mL/min/100 g b.w.) and the mean arterial pressure (MAP) was recorded through a catheter inserted in the carotid artery. Superoxide concentration within the cells of the abdominal aorta was quantified from the oxidation of dihydroethidium (DHE). In kidney tissues, ROS levels were measured by the 27 dichloroflurescin diacetate assay. Renal morphology was examined at the histochemistry level.nnnRESULTSnCsA-treated rats showed a severe decrease in GFR (0.34 ± 0.17 versus 0.94 ± 0.10 in control, P < 0.001) which was prevented by rMnSOD co-administration (0.77 ± 0.10). CsA-injected animals presented with higher blood pressure which was unaffected by rMnSOD. ROS levels both in the aorta and in renal tissue were significantly increased by CsA treatment, and normalized by the co-administration with rMnSOD. This effect was, partly, paralleled by the recovery from CsA-induced morphological lesions.nnnCONCLUSIONSnAdministration of rMnSOD prevents CsA-mediated impairment of the GFR along with morphological alteration. This effect could be related to the inhibition of ROS.

The role of the kidney in salt-sensitive hypertension

Primary hypertension is one of the leading risk factors for cardiovascular disease. Although the pathogenesis is not completely understood, an imbalance of sodium and chloride homeostasis seems to be relevant both in the induction and in the maintenance of salt-sensitive hypertension. Besides individual renal phenotypes, salt intake is one of the most important environmental determinants of this condition. The Milan hypertensive strain (MHS) of rats is an interesting model to investigate the molecular mechanisms underling the development of salt-sensitive hypertension. In young MHS rats, hypertension is anticipated by a phase of increased salt reabsorption localized along the medullary thick ascending limb associated with the up-regulation of the apical sodium−potassium−chloride cotransporter (NKCC2). Later, the frank hypertensive status of adult MHS rats is accompanied by the activation of the luminal and basal lateral transporters of sodium chloride (NaCl) in the distal convoluted tubule (DCT). Several lines of evidence have proven the key role of DCT in the maintenance of hypertension in MHS rats; more importantly, hypertensive patients carrying a mutation of α-adducin (resembling the MHS model) have a high sensitivity to thiazides, suggesting that the Na+–Cl− cotransporter also plays a pivotal role in humans.

Selective dicer suppression in the kidney alters gsk3β/β-Catenin Pathways Promoting a Glomerulocystic Disease

Dicer is a crucial enzyme for the maturation of miRNAs. Mutations in the Dicer gene are highly associated with Pleuro Pulmonary Blastoma-Family Dysplasia Syndrome (PPB-FDS, OMIM 601200), recently proposed to be renamed Dicer syndrome. Aside from the pulmonary phenotype (blastoma), renal nephroma and thyroid goiter are frequently part of Dicer syndrome. To investigate the renal phenotype, conditional knockout (cKO) mice for Dicer in Pax8 expressing cells were generated. Dicer cKO mice progressively develop a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria and severe renal failure. Higher cellular turnover of the parietal cells of Bowman’s capsule precedes the development of the cysts and the primary cilium progressively disappears with cyst-enlargement. Upregulation of GSK3β precedes the development of the glomerulocystic phenotype. Downregulation of β-catenin in the renal cortex and its cytosolic removal in the cells lining the cysts may be associated with observed accumulation of GSK3β. Alterations of β-catenin regulating pathways could promote cystic degeneration as in other models. Thus, miRNAs are fundamental in preserving renal morphology and function. Alteration of the GSK3β/β-catenin pathway could be a crucial mechanism linking miRNA dysregulation and the development of a glomerulocystic disease.

A fate-mapping approach reveals the composite origin of the connecting tubule and alerts on “single-cell”-specific KO model of the distal nephron

The distal nephron is a heterogeneous part of the nephron composed by six different cell types, forming the epithelium of the distal convoluted (DCT), connecting, and collecting duct. To dissect the function of these cells, knockout models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell markers has been recently questioned. Here, by mapping the fate of the aquaporin 2 (AQP2) and Na+-Cl- cotransporter (NCC)-positive cells using transgenic mouse lines expressing the yellow fluorescent protein fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2-expressing precursor results give rise not only to the principal cells, but also to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other hand, some principal cells and B-type intercalated cells can develop from NCC-expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.

Renal phenotype in Bardet-Biedl syndrome: a combined defect of urinary concentration and dilution is associated with defective urinary AQP2 and UMOD excretion

The renal phenotype in Bardet-Biedl syndrome (BBS) is highly variable. The present study describes renal findings in 41 BBS patients and analyzes the pathogenesis of hyposthenuria, the most common renal dysfunction. Five of 41 patients (12%) showed an estimated glomerular filtration rate < 60 ml·min-1·1.73 m-2 Urine protein and urine albumin-to-creatinine ratio were over 200 and 30 mg/g in 9/24 and 7/23 patients, respectively. Four of 41 patients showed no renal anomalies on ultrasound. Twenty of 34 patients had hyposthenuria in the absence of renal insufficiency. In all 8 of the hyposthenuric patients studied, dDAVP failed to elevate urine osmolality (Uosm), suggesting a nephrogenic origin. Interestingly, water loading (WL) did not result in a significant reduction of Uosm, indicating combined concentrating and diluting defects. dDAVP infusion induced a significant increase of plasma Factor VIII and von Willebrand Factor levels, supporting normal function of the type 2 vasopressin receptor at least in endothelial cells. While urinary aquaporin 2 (u-AQP2) abundance was not different between patients and controls at baseline, the dDAVP-induced increased u-AQP2 and the WL-induced reduction of u-AQP2 were blunted in patients with a combined concentrating and diluting defect, suggesting a potential role of AQP2 in the defective regulation of water absorption. Urine Uromodulin excretion was reduced in all hyposthenuric patients, suggesting a thick ascending limb defect. Interestingly, renal Na, Cl, Ca, but not K handling was impaired after acute WL but not at basal. In summary, BBS patients show combined urinary concentration and dilution defects; a thick ascending limb and collecting duct tubulopathy may underlie impaired water handling.

SGK3: a novel regulator of renal phosphate transport?

Phosphate is a key constituent of several important molecules, and hyperphospatemia has been associated with increased cardiovascular mortality. The kidney plays a crucial role in phosphate metabolism, as it is able to modulate phosphate excretion. Serum- and glucocorticoid-inducible kinase 3 (SGK3) has been shown to regulate a wide variety of transport systems. Bhandaru et al. suggest that SGK3 may have a significant role in the regulation of renal tubular phosphate transport.