Multiparametric magnetic resonance imaging allows non-invasive functional and structural evaluation of diabetic kidney disease.

Background: We sought to develop a novel non-contrast multiparametric MRI (mpMRI) protocol employing several complementary techniques in a single scan session for a comprehensive functional and structural evaluation of diabetic kidney disease (DKD). Methods: In the cross-sectional part of this prospective observational study, 38 subjects ages 18‒79 years with type 2 diabetes and DKD [estimated glomerular filtration rate (eGFR) 15‒60 mL/min/1.73 m2] and 20 age- and gender-matched healthy volunteers (HVs) underwent mpMRI. Repeat mpMRI was performed on 23 DKD subjects and 10 HVs. By measured GFR (mGFR), 2 DKD subjects had GFR stage G2, 16 stage G3 and 20 stage G4/G5. A wide range of MRI biomarkers associated with kidney haemodynamics, oxygenation and macro/microstructure were evaluated. Their optimal sensitivity, specificity and repeatability to differentiate diabetic versus healthy kidneys and categorize various stages of disease as well as their correlation with mGFR/albuminuria was assessed. Results: Several MRI biomarkers differentiated diabetic from healthy kidneys and distinct GFR stages (G3 versus G4/G5); mean arterial flow (MAF) was the strongest predictor (sensitivity 0.94 and 1.0, specificity 1.00 and 0.69; P = .04 and .004, respectively). Parameters significantly correlating with mGFR were specific measures of kidney haemodynamics, oxygenation, microstructure and macrostructure, with MAF being the strongest univariate predictor (r = 0.92; P < .0001). Conclusions: A comprehensive and repeatable non-contrast mpMRI protocol was developed that, as a single, non-invasive tool, allows functional and structural assessment of DKD, which has the potential to provide valuable insights into underlying pathophysiology, disease progression and analysis of efficacy/mode of action of therapeutic interventions in DKD.

Fab1 phosphatidylinositol 3-phosphate 5-kinase controls trafficking but not silencing of endocytosed receptors.

The trafficking of endocytosed receptors through phosphatidylinositol 3-phosphate [PtdIns(3)P]-containing endosomes is thought to attenuate their signaling. Here, we show that the PtdIns(3)P 5-kinase Fab1/PIKfyve controls trafficking but not silencing of endocytosed receptors. Drosophila fab1 mutants contain undetectable phosphatidylinositol 3,5-bisphosphate levels, show profound increases in cell and organ size, and die at the pupal stage. Mutant larvae contain highly enlarged multivesicular bodies and late endosomes that are inefficiently acidified. Clones of fab1 mutant cells accumulate Wingless and Notch, similarly to cells lacking Hrs, Vps25, and Tsg101, components of the endosomal sorting machinery for ubiquitinated membrane proteins. However, whereas hrs, vps25, and tsg101 mutant cell clones accumulate ubiquitinated cargo, this is not the case with fab1 mutants. Even though endocytic receptor trafficking is impaired in fab1 mutants, Notch, Wingless, and Dpp signaling is unaffected. We conclude that Fab1, despite its importance for endosomal functions, is not required for receptor silencing. This is consistent with the possibility that Fab1 functions at a late stage in endocytic receptor trafficking, at a point when signal termination has occurred.

Miple1 and miple2 encode a family of MK/PTN homologues in Drosophila melanogaster.

Midkine (MK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. To date, this family of proteins has been implicated in multiple processes, such as growth, survival, and migration of various cells, and has roles in neurogenesis and epithelial-mesenchymal interaction during organogenesis. In this report, we have characterized two members of the MK/PTN family of proteins in Drosophila, named Miple1 and Miple2, from Midkine and Pleiotrophin. Drosophila miple1 and miple2 encode secreted proteins which are expressed in spatially restricted, nonoverlapping patterns during embryogenesis. Expression of miple1 can be found at high levels in the central nervous system, while miple2 is strongly expressed in the developing midgut endoderm. The identification of homologues of the MK/PTN family in this genetically tractable model organism should allow an analysis of their function during complex developmental processes.

Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons.

Slit proteins steer the migration of many cell types through their binding to Robo receptors, but how Robo controls cell motility is not clear. We describe the functional analysis of vilse, a Drosophila gene required for Robo repulsion in epithelial cells and axons. Vilse defines a conserved family of RhoGAPs (Rho GTPase-activating proteins), with representatives in flies and vertebrates. The phenotypes of vilse mutants resemble the tracheal and axonal phenotypes of Slit and Robo mutants at the CNS midline. Dosage-sensitive genetic interactions between vilse, slit, and robo mutants suggest that vilse is a component of robo signaling. Moreover, overexpression of Vilse in the trachea of robo mutants ameliorates the phenotypes of robo, indicating that Vilse acts downstream of Robo to mediate midline repulsion. Vilse and its human homolog bind directly to the intracellular domains of the corresponding Robo receptors and promote the hydrolysis of RacGTP and, less efficiently, of Cdc42GTP. These results together with genetic interaction experiments with robo, vilse, and rac mutants suggest a mechanism whereby Robo repulsion is mediated by the localized inactivation of Rac through Vilse.

Rhomboid 3 orchestrates Slit-independent repulsion of tracheal branches at the CNS midline.

EGF-receptor ligands act as chemoattractants for migrating epithelial cells during organogenesis and wound healing. We present evidence that Rhomboid 3/EGF signalling, which originates from the midline of the Drosophila ventral nerve cord, repels tracheal ganglionic branches and prevents them from crossing it. rho3 acts independently from the main midline repellent Slit, and originates from a different sub-population of midline cells: the VUM neurons. Expression of dominant-negative Egfr or Ras induces midline crosses, whereas activation of the Egfr or Ras in the leading cell of the ganglionic branch can induce premature turns away from the midline. This suggests that the level of Egfr intracellular signalling, rather than the asymmetric activation of the receptor on the cell surface, is an important determinant in ganglionic branch repulsion. We propose that Egfr activation provides a necessary switch for the interpretation of a yet unknown repellent function of the midline.

Jeb signals through the Alk receptor tyrosine kinase to drive visceral muscle fusion.

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). We have previously shown that the Drosophila Alk RTK is crucial for visceral mesoderm development during early embryogenesis. Notably, observed Alk visceral mesoderm defects are highly reminiscent of the phenotype reported for the secreted molecule Jelly belly (Jeb). Here we show that Drosophila Alk is the receptor for Jeb in the developing visceral mesoderm, and that Jeb binding stimulates an Alk-driven, extracellular signal-regulated kinase-mediated signalling pathway, which results in the expression of the downstream gene duf (also known as kirre)--needed for muscle fusion. This new signal transduction pathway drives specification of the muscle founder cells, and the regulation of Duf expression by the Drosophila Alk RTK explains the visceral-mesoderm-specific muscle fusion defects observed in both Alk and jeb mutant animals.

A crucial role for the Anaplastic lymphoma kinase receptor tyrosine kinase in gut development in Drosophila melanogaster.

The Drosophila melanogaster gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian Alk, which encodes a member of the Alk/Ltk family of receptor tyrosine kinases (RTKs). In humans, the t(2;5) translocation, which involves the ALK locus, produces an active form of ALK, which is the causative agent in non-Hodgkin's lymphoma. The physiological function of the Alk RTK, however, is unknown. In this paper, we describe loss-of-function mutants in the Drosophila Alk gene that cause a complete failure of the development of the gut. We propose that the main function of Drosophila Alk during early embryogenesis is in visceral mesoderm development.

Attractive and repulsive functions of Slit are mediated by different receptors in the Drosophila trachea.

Oxygen delivery in many animals is enabled by the formation of unicellular capillary tubes that penetrate target tissues to facilitate gas exchange. We show that the tortuous outgrowth of tracheal unicellular branches towards their target tissues is controlled by complex local interactions with target cells. Slit, a phylogenetically conserved axonal guidance signal, is expressed in several tracheal targets and is required both for attraction and repulsion of tracheal branches. Robo and Robo2 are expressed in different branches, and are both necessary for the correct orientation of branch outgrowth. At the CNS midline, Slit functions as a repellent for tracheal branches and this function is mediated primarily by Robo. Robo2 is necessary for the tracheal response to the attractive Slit signal and its function is antagonized by Robo. We propose that the attractive and repulsive tracheal responses to Slit are mediated by different combinations of Robo and Robo2 receptors on the cell surface.