The difficulty in accessing mammalian nephrons in vivo hinders the study of podocyte biology. The Drosophila nephrocyte shares remarkable similarities to the glomerular podocyte, but the lack of a functional readout for nephrocytes makes it challenging to study this model of the podocyte, which could potentially harness the power of Drosophila genetics. Here, we present a functional analysis of nephrocytes and establish an in vivo system to screen for renal genes. We found that nephrocytes efficiently take up secreted fluorescent protein, and therefore, we generated a transgenic line carrying secreted fluorescent protein and combined it with a nephrocyte-specific driver for targeted gene knockdown, allowing the identification of genes required for nephrocyte function. To validate this system, we examined the effects of knocking down sns and duf, the Drosophila homologs of nephrin and Neph1, respectively, in pericardial nephrocytes. Knockdown of sns or duf completely abolished the accumulation of the fluorescent protein in pericardial nephrocytes. Examining the ultrastructure revealed that the formation of the nephrocyte diaphragm and lacunar structure, which is essential for protein uptake, requires sns. Our preliminary genetic screen also identified Mec2, which encodes the homolog of mammalian Podocin. Taken together, these data suggest that the Drosophila pericardial nephrocyte is a useful in vivo model to help identify genes involved in podocyte biology and facilitate the discovery of renal disease genes.

The insect nephrocyte and the mammalian glomerular podocyte are similar with regard to filtration, but it remains unclear whether there is an organ or cell type in flies that reabsorbs proteins. Here, we show that the Drosophila nephrocyte has molecular, structural, and functional similarities to the renal proximal tubule cell. We screened for genes required for nephrocyte function and identified two Drosophila genes encoding orthologs of mammalian cubilin and amnionless (AMN), two major receptors for protein reabsorption in the proximal tubule. In Drosophila, expression of dCubilin and dAMN is specific to nephrocytes, where they function as co-receptors for protein uptake. Targeted expression of human AMN in Drosophila nephrocytes was sufficient to rescue defective protein uptake induced by dAMN knockdown, suggesting evolutionary conservation of Cubilin/AMN co-receptors function from flies to humans. Furthermore, we found that Cubilin/ AMN-mediated protein reabsorption is required for the maintenance of nephrocyte ultrastructure and fly survival under conditions of toxic stress. In conclusion, the insect nephrocyte combines filtration with protein reabsorption, using evolutionarily conserved genes and subcellular structures, suggesting that it can serve as a simplified model for both podocytes and the renal proximal tubule.

By characterization of the uptake of glutathione-S-conjugates, principally dinitrophenyl-S-glutathione (DNP-CS), by vacuolar membrane vesicles, we demonstrate that a subset of energy-dependent transport processes in plants are not H+-coupled but instead are directly energized by MgATP. The most salient features of this transport pathway are: (a) its specific, obligate requirement for MgATP as energy source; (b) the necessity for hydrolysis of the y-phosphate of MgATP for uptake; (c) the insensitivity of uptake to uncouplers of the transtonoplast H+ gradient (carbonylcyanide 4-trifluoromethoxyphenylhydrazone, gramicidin-D, and NH,CI); (d) its pronounced sensitivity to vanadate and partia1 inhibition by vinblastine and verapamil; (e) the lack of chemical modification of DNP-CS either during or after transport; (0 the capacity of S conjugates of chloroacetanilide herbicides, such as metolachlor-CS, but not free herbicide, to inhibit uptake; and (g) the ability of vacuolar membrane vesicles purified from a broad range of plant species, including Arabidopsis, Befa, Vigna, and Zea, to mediate MgATP-dependent, H+-electrochemical potential difference-independent DNP-CS uptake. O n the basis of these findings it is proposed that the transport of DNP-CS across the vacuolar membrane of plant cells is catalyzed by a glutathione-conjugate transporter that directly employs MgATP rather than the energy contained i n the transtonoplast H+-electrochemical potential difference to drive uptake. The broad distribution of the vacuolar DNP-CS transporter and its inhibition by metolachlor-CS are consistent with the notion that it plays a general role in the vacuolar sequestration of glutathione-conjugable cytotoxic agents.The prevailing working model for energy-dependent solute transport by plant cells is chemiosmosis (Mitchell, 1976;Spanswick, 1981). According to this scheme, the primary energizers for solute transport are pumps that mediate electrogenic H+-translocation across the membranes concerned to generate a gradient of electrical potential (AI)) and chemical potential (ApH). Depending on the net charge on the solute-porter complex, the motive force for transport is believed to be derived from the ApH and/or AI) components of the H+-electrochemical potential difference (A,%"+). For with or in exchange for H+, transport is driven by ApH; for solute-porter complexes that carry a net charge, transport is driven by AI).The notion of phosphoanhydride-energized H+-translocation across the principal membranes of plant cells is irrefutable. Primary ATP-energized Hf pumps and a PPienergized H+ pump in the case of the vacuolar membrane (Rea and Poole, 1993) have been demonstrated in the membranes bounding most of the compartments of plant cells, and in a11 cases the pumps concerned have been shown to be critica1 for establishment of the resting membrane potential and transmembrane pH gradients (Spanswick, 1981;Sze, 1985;Rea and Poole, 1993). What is not so clear, however, is whether essentially a11 energy-dependent secondary transport...

Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells. [BMB reports 2012; 45(4): 247-252]

Purpose: The aim of this study was to investigate the value of radiomics analysis of iodine-based material decomposition (MD) images with dual-energy computed tomography (DECT) imaging for preoperatively predicting microsatellite instability (MSI) status in colorectal cancer (CRC).Methods: This study included 102 CRC patients proved by postoperative pathology, and their MSI status was confirmed by immunohistochemistry staining. All patients underwent preoperative DECT imaging scanned on either a Revolution CT or Discovery CT 750HD scanner, and the iodine-based MD images in the venous phase were reconstructed. The clinical, CT-reported, and radiomics features were obtained and analyzed. Data from the Revolution CT scanner were used to establish a radiomics model to predict MSI status (70% samples were randomly selected as the training set, and the remaining samples were used to validate); and data from the Discovery CT 750HD scanner were used to test the radiomics model. The stable radiomics features with both inter-user and intra-user stability were selected for the next analysis. The feature dimension reduction was performed by using Student's t-test or Mann–Whitney U-test, Spearman's rank correlation test, min–max standardization, one-hot encoding, and least absolute shrinkage and selection operator selection method. The multiparameter logistic regression model was established to predict MSI status. The model performances were evaluated: The discrimination performance was accessed by receiver operating characteristic (ROC) curve analysis; the calibration performance was tested by calibration curve accompanied by Hosmer–Lemeshow test; the clinical utility was assessed by decision curve analysis.Results: Nine top-ranked features were finally selected to construct the radiomics model. In the training set, the area under the ROC curve (AUC) was 0.961 (accuracy: 0.875; sensitivity: 1.000; specificity: 0.812); in the validation set, the AUC was 0.918 (accuracy: 0.875; sensitivity: 0.875; specificity: 0.857). In the testing set, the diagnostic performance was slightly lower with AUC of 0.875 (accuracy: 0.788; sensitivity: 0.909; specificity: 0.727). A nomogram based on clinical factors and radiomics score was generated via the proposed logistic regression model. Good calibration and clinical utility were observed using the calibration and decision curve analyses, respectively.Conclusion: Radiomics analysis of iodine-based MD images with DECT imaging holds great potential to predict MSI status in CRC patients.