Nephrolithiasis, kidney failure and bone disorders in Dent disease patients with and without CLCN5 mutations

open9noDent disease (DD) is a rare X-linked recessive renal tubulopathy characterised by low-molecular-weight proteinuria (LMWP), hypercalciuria, nephrocalcinosis and/or nephrolithiasis. DD is caused by mutations in both the CLCN5 and OCRL genes. CLCN5 encodes the electrogenic chloride/proton exchanger ClC-5 which is involved in the tubular reabsorption of albumin and LMW proteins, OCRL encodes the inositol polyphosphate 5-phosphatase, and was initially associated with Lowe syndrome. In approximately 25 % of patients, no CLCN5 and OCRL mutations were detected. The aim of our study was to evaluate whether calcium phosphate metabolism disorders and their clinical complications are differently distributed among DD patients with and without CLCN5 mutations. Sixty-four male subjects were studied and classified into three groups: Group I (with CLCN5 mutations), Group II (without CLCN5 mutations) and Group III (family members with the same CLCN5 mutation). LMWP, hypercalciuria and phosphaturic tubulopathy and the consequent clinical complications nephrocalcinosis, nephrolithiasis, bone disorders, and chronic kidney disease (CKD) were considered present or absent in each patient. We found that the distribution of nephrolithiasis, bone disorders and CKD differs among patients with and without CLCN5 mutations. Only in patients harbouring CLCN5 mutations was age-independent nephrolithiasis associated with hypercalciuria, suggesting that nephrolithiasis is linked to altered proximal tubular function caused by a loss of ClC-5 function, in agreement with ClC-5 KO animal models. Similarly, only in patients harbouring CLCN5 mutations was age-independent kidney failure associated with nephrocalcinosis, suggesting that kidney failure is the consequence of a ClC-5 dysfunction, as in ClC-5 KO animal models. Bone disorders are a relevant feature of DD phenotype, as patients were mainly young males and this complication occurred independently of age. The triad of symptoms, LMWP, hypercalciuria, and nephrocalcinosis, was present in almost all patients with CLCN5 mutations but not in those without CLCN5 mutations. This lack of homogeneity of clinical manifestations suggests that the difference in phenotypes between the two groups might reflect different pathophysiological mechanisms, probably depending on the diverse genes involved. Overall, our results might suggest that in patients without CLCN5 mutations several genes instead of the prospected third DD underpin patients' phenotypes.openAnglani, Franca; D’Angelo, Angela; Bertizzolo, Luisa Maria; Tosetto, Enrica; Ceol, Monica; Cremasco, Daniela; Bonfante, Luciana; Addis, Maria Antonietta; Del Prete, DorellaAnglani, Franca; D'Angelo, Angela; Bertizzolo, Luisa Maria; Tosetto, Enrica; Ceol, Monica; Cremasco, Daniela; Bonfante, Luciana; Addis, Maria Antonietta; DEL PRETE, Dorell

The measurement of global shortening as a new parameter to evaluate bone specimen response to uniaxial loading

Mechanical load is nowadays considered one of the factor mainly affecting bone tissue properties, both as architecture and functionality. Mechanotransduction is the capability of cells to translate mechanical stresses into biochemical signals, and several studies performed on mouse models demonstrated that also bone cells show a high responsiveness to mechanical stimuli. To date, bone cells mechanotransduction is mainly investigated in animal models, by the use of organ cultures or directly in vivo, and the actual strains induced by the external loads are measured through the use of micro strain gauges placed on the tibia mid-diaphysis. With the aim of proposing a new parameter to come along with the measurement of the actual strains, we exploited the capability of tibial global shortening to return useful information. We employed an experimental system based on a dual mode actuator/transducer with an adequate force range and a high length resolution to retrieve the small shortening of the bone specimens subjected to uniaxially load. Preliminary results showed that the tibia global shortening has a linear relationship with the increasing load, in the range of force usually used in these studies. In addition, the tibia global shortening showed the capability of gathering the changes occurring in the bone tissue mechanical properties when subjecting the specimens to loading signals of different frequencies. When tested with load signals of a frequency equal or higher than 1 Hz, in fact, the bone specimens showed a more rigid behavior. At 9 N of load, for example, the average value of tibia global shortening measured at 0.1 Hz is, on average, 18 % higher than when measured at all the other tested frequencies

Development and mechanical validation of an in vitro system for bone cell vibration loading

Vibration loading, both low magnitude and high magnitude at high frequency, has been demonstrated to have an anabolic effect on bone cells. The study of the mechanotransduction, the process by which mechanical loadings are detected by cells and converted in a chemical signal, is made accessible through the use of in vitro loading system. The aim of an in vitro loading system is to recreate the forces acting in the cell microenvironment. The goal of this study was to develop and mechanically validate a vibration loading system able to engender sinusoidal vertical vibration at different combinations of magnitude (0.3 g, 1 g, and 3 g) and frequency (30 Hz, 60 Hz and 90 Hz). A system like this can be therefore employed to study cell response to high and low magnitudes at high frequencies, thus providing a comprehensive evaluation of bone cell mechanotransduction. The mechanical validation, that is the characterization of the right loading input to the system to obtain the desired stimulation on cell culture, was performed in two different methods: open-loop and closed-loop mode. The results obtained in the open-loop mode showed a good intra-day repeatability of the measurements with values of index of dispersion always lower than 0.6%. While in the closed-loop mode a systematic search was implemented to reach the optimal amplitude stimulation. The vibration signals acquired on long-term test following the systematic search showed a good stability with index of dispersion always lower than 1%. Following the mechanical validation, the system was used to stimulate osteoblast like cells (Saos-2) with vibration loading of nine combinations of magnitude and frequency and the cell proliferation was studied 24h after the treatment by cell counting. Our preliminary results showed that no alterations in the proliferation were induced by 90 Hz vibration loading. On the other hand, small modulations in the proliferation were reported for lower stimulation frequency, being statistically significant when using 0.3 g of amplitude at 30 Hz