Cyclosporin-induced hypertension is associated with the up-regulation of Na+-K+-2Cl- cotransporter (NKCC2).

BACKGROUND: The use of cyclosporin A (CsA) is hampered by the development of nephrotoxicity including hypertension, which is partially dependent on renal sodium retention. To address this issue, we have investigated in vivo sodium reabsorption in different nephron segments of CsA-treated rats through micropuncture study coupled to expression analyses of sodium transporters. To translate the findings in rats to human, kidney-transplanted patients having CsA treatment were enrolled in the study. METHODS: Adult male Sprague-Dawley rats were treated with CsA (15 mg/kg/day) for 21 days, followed by micropuncture study and expression analyses of sodium transporters. CsA-treated kidney-transplanted patients with resistant hypertension were challenged with 50 mg furosemide. RESULTS: CsA-treated rats developed hypertension associated with reduced glomerular filtration rate. In vivo microperfusion study demonstrated a significant decrease in rate of absolute fluid reabsorption in the proximal tubule but enhanced sodium reabsorption in the thick ascending limb of Henle's loop (TAL). Expression analyses of sodium transporters at the same nephron segments further revealed a reduction in Na+-H+ exchanger isoform 3 (NHE3) in the renal cortex, while TAL-specific, furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and NHE3 were significantly upregulated in the inner stripe of outer medulla. CsA-treated patients had a larger excretion of urinary NKCC2 protein at basal condition, and higher diuretic response to furosemide, showing increased FeNa+, FeCl- and FeCa2+ compared with both healthy controls and FK506-treated transplanted patients. CONCLUSION: Altogether, these findings suggest that up-regulation of NKCC2 along the TAL facilitates sodium retention and contributes to the development of CsA-induced hypertension.

Bardet-Biedl syndrome: The pleiotropic role of the chaperonin-like BBS6, 10, and 12 proteins.

Bardet-Biedl syndrome (BBS) is a rare pleiotropic disorder known as a ciliopathy. Despite significant genetic heterogeneity, BBS1 and BBS10 are responsible for major diagnosis in western countries. It is well established that eight BBS proteins, namely BBS1, 2, 4, 5, 7, 8, 9, and 18, form the BBSome, a multiprotein complex serving as a regulator of ciliary membrane protein composition. Less information is available for BBS6, BBS10, and BBS12, three proteins showing sequence homology with the CCT/TRiC family of group II chaperonins. Even though their chaperonin function is debated, scientific evidence demonstrated that they are required for initial BBSome assembly in vitro. Recent studies suggest that genotype may partially predict clinical outcomes. Indeed, patients carrying truncating mutations in any gene show the most severe phenotype; moreover, mutations in chaperonin-like BBS proteins correlated with severe kidney impairment. This study is a critical review of the literature on genetics, expression level, cellular localization and function of BBS proteins, focusing primarily on the chaperonin-like BBS proteins, and aiming to provide some clues to understand the pathomechanisms of disease in this setting.

A New Synthetic Spiroketal: Studies on Antitumor Activity on Murine Melanoma Model In Vivo and Mechanism of Action In Vitro.

BACKGROUND: In a previous study, we synthesised a new spiroketal derivative, inspired to natural products, that has shown high antiproliferative activity, potent telomerase inhibition and proapoptotic activity on several human cell lines. OBJECTIVE: This work focused on the study of in vivo antitumor effect of this synthetic spiroketal on a murine melanoma model. In order to shed additional light on the origin of the antitumor effect, in vitro studies were performed. METHODS: Spiroketal was administered to B16F10 melanoma mice at a dose of 5 mg/Kg body weight via intraperitoneum at alternate days for 15 days. Tumor volume measures were made every 2 days starting after 12 days from cells injection. The effects of the spiroketal on tumor growth inhibition, apoptosis induction, and cell cycle modification were investigated in vitro on B16 cells. HIF1α gene expression, the inhibition of cells migration and the changes induced in cytoskeleton conformation were evaluated. RESULTS: Spiroketal displayed proapoptotic activity and high antitumor activity in B16 cells with nanomolar IC50. Moreover it has shown to inhibit cell migration, to strongly reduce the HIF1α expression and to induce strongly deterioration of cytoskeleton structure. A potent dose-dependent antitumor efficacy in syngenic B16/C57BL/6J murine model of melanoma was observed with the suppression of tumor growth by an average of 90% at a dose of 5 mg/kg. CONCLUSION: The synthesized spiroketal shows high antitumor activity in the B16 cells in vitro at nM concentration and a dose-dependent antitumor efficacy in syngenic B16/C57BL/6J mice. The results suggest that this natural product inspired spiroketal may have a potential application in melanoma therapy.

Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways.

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2-/-) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2-/- mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

Acute and chronic effects of metabolic acidosis on renal function and structure.

BACKGROUND: Emerging evidence suggests that chronic metabolic acidosis (CMA) may have significant implications in terms of worsening renal disease in CKD patients, but the effect of CMA on renal function and structure has not been fully elucidated. METHOD: We studied the acute and chronic consequences of an acid load (AL) on glomerular filtration rate (GFR) and renal histology in C57BL/6 mice. FITC-inulin clearance was performed at several time points; markers of renal fibrosis were studied at mRNA and protein levels; finally, kidney expression of candidate molecules triggering changes in renal function was studied. RESULTS: Glomerular hyperfiltration occurred within 1-3 days from AL; after 1 week, the GFR returned to baseline and then declined progressively within 15-21 days. The GFR decline was accompanied by the onset of renal fibrosis, as shown by Masson trichrome staining. Markers of renal fibrosis, namely α-smooth muscle actin and collagen-1, increased after 1 day of acid loading in both mRNA and protein levels and remained higher than baseline for up to 21 days. Well-known mediators of renal fibrosis, including transforming growth factor (TGF)-ß and the intrarenal renin-angiotensin system (RAS) axis, were increased even before the decline of the GFR. CONCLUSION: Acid load caused hyperfiltration acutely and a progressive decline of the GFR chronically; the evidence of renal fibrosis indicates that structural and not only functional renal changes occurred. The concomitant upregulation of TGF-ß and intrarenal RAS axis indicates that those factors may be potentially involved in the progression of kidney disease in this setting.

Effect of rMnSOD on Sodium Reabsorption in Renal Proximal Tubule in Ochratoxin A-Treated Rats.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium that represent toxic real threat for human beings and animal health. In this study we evaluated the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) on oxidative stress and on the alterations of fluid reabsorption in renal proximal tubule (PT) as possible causes of OTA nephrotoxicity. Finally, we have measured the concentration of O2- in the kidney through dihydroethidium assay (DHE) and nitric oxide (NO) concentration through nitrites and nitrates assay. Male Sprague Dawley rats weighing 120-150 g were treated for 14 days by gavage, as follows: Control group, 12 rats received a corresponding amount of saline solution (including 10% DMSO); rMnSOD group, 12 rats treated with rMnSOD (10 µg/kg bw); OTA group, 12 rats treated with OTA (0.5 mg/kg bw) dissolved in 10% DMSO and then scaled to required volume with corn oil; rMnSOD + OTA, 12 rats treated with rMnSOD (10 µg/kg bw) plus OTA (0.5 mg/kg bw). Our results have shown that rMnSOD restores the alteration of reabsorption in PT in rats treated with OTA plus rMnSOD, probably through the response to pressure natriuresis, where nitric oxide plays a key role. Moreover, rMnSOD prevents the nephrotoxicity induced by OTA probably restoring the balance between superoxide and NO that is most probably the cause of hypertension and renal functional alterations through the inhibition of NO synthase. In conclusion these data provide important information for understanding of mechanism of toxic action of OTA. J. Cell. Biochem. 119: 424-430, 2018. © 2017 Wiley Periodicals, Inc.

[Patho-physiology of renal dysfunction in Bardet-Biedl Syndrome]. / DIl coinvolgimento renale nella sindrome di Bardet-Biedl.

Bardet-Biedl Syndrome (BBS) is a rare autosomal recessive disorder with renal and extra-renal involvement. The wide spectrum of clinical manifestations is associated to the high genetic heterogeneity. To date 21 genes have been identified in humans and the majority of them encode proteins located on the basal body of the primary cilium. For this reason the disease is has been included among the 'ciliopathies'. The renal involvement is extremely heterogeneous in BBS and is considered the main cause of morbidity and mortality. Recent evidences have suggested that mutations in BBS6, 10 and 12 are associated with a more severe renal dysfunction. The most common renal dysfunction is the urine concentrating defect, even though the underlying mechanism is not completely known. Recently we have demonstrated that hyposthenuria in BBS patients has a renal origin, and depends on desmopessin resistance. The majority of hyposthenuric BBS patients have a combined defect to both concentrate and dilute the urine. The combined defect is associated with a blunted increased urine Aquaproine-2 (u-AQP2) excretion in antidiuresis. A ccordingly, in vitro BBS10 silencing prevented AQP2 trafficking to the apical plasma membrane. However, after long term water restriction hyposthenuric BBS patients showed the same u-AQP2 excretion compared with controls, suggesting that other mechanisms are implicated into the pathogenesis of hyposhtenuria. The complete molecular mechanism underlying hyposhtenuria remains largely unknown in BBS. Whether this defect may represent a predictor factor for poor renal outcome remains to be elucidated.

Simvastatin inhibits the expression of stemness­related genes and the metastatic invasion of human cancer cells via destruction of the cytoskeleton.

Statins are a class of drugs that inhibit the rate-limiting steps in the cholesterol biosynthesis pathway. They act by inhibiting 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate. Blocking of mevalonate synthesis leads to inhibition of the farnesylation and geranylgeranylation of several functional proteins, such as RhoA and other small guanosine triphosphate-binding proteins, that are important in maintaining the undifferentiated status of the cells. In the present study, we hypothesized that simvastatin, likely through the inhibition of farnesylation and geranylgeranylation of Rac1, Cd42 and RhoA, induces a destruction/restructuration of the cytoskeleton that decreases mechanical strain transfer to the nuclei, inducing the loss of transmission of regulatory signals from the cytoskeleton to the nucleoskeleton. Although this remains at present a hypothesis and is not easy to define if the de-structuration of the cytoskeleton is a secondary effect of simvastatin treatment or the inhibition of post-translational protein modification have a precise role in the structuration of actin cytoskeleton, we speculate that these signal variations could inhibit the expression of certain stemness genes, which could therefore be considered nucleoskeleton-associated and mechanically regulated genes. On the other hand, the restructuration of the cytoskeleton inhibits the formation of lamellipodia and filopodia, which likely decreases the capability of cancer cells to invade the extracellular matrix, thereby modulating the equilibrium between proliferation, differentiation and metastatic invasion in human cancer cells. On the basis of our results we think that simvastatin, alone or in combination with conventional drugs, may have a possible role in cancer therapy.

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.