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Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. It is caused by a defect in the lysosomal cystine transporter, cystinosin, which results in an accumulation of cystine in all organs. Despite the ubiquitous expression of cystinosin, a renal Fanconi syndrome is often the first manifestation of cystinosis, usually presenting within the first year of life and characterized by the early and severe dysfunction of proximal tubule cells, highlighting the unique vulnerability of this cell type. The current therapy for cystinosis, cysteamine, facilitates lysosomal cystine clearance and greatly delays progression to kidney failure but is unable to correct the Fanconi syndrome. This Review summarizes decades of studies that have fostered a better understanding of the pathogenesis of the renal Fanconi syndrome associated with cystinosis. These studies have unraveled some of the early molecular changes that occur before the onset of tubular atrophy and identified a role for cystinosin beyond cystine transport, in endolysosomal trafficking and proteolysis, lysosomal clearance, autophagy and the regulation of energy balance. These studies have also led to the identification of new potential therapeutic targets and here, we outline the potential role of stem cell therapy for cystinosis and provide insights into the mechanism of haematopoietic stem cell-mediated kidney protection.

Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins.

Cystinosis, a main cause of Fanconi syndrome, is reproduced in congenic C57BL/6 cystinosin knockout (KO) mice. To identify the sequence of pathogenic and adaptation mechanisms of nephropathic cystinosis, we defined the onset of Fanconi syndrome in KO mice between 3 and 6 months of age and analyzed the correlation with structural and functional changes in proximal tubular cells (PTCs), with focus on endocytosis of ultrafiltrated disulfide-rich proteins as a key source of cystine. Despite considerable variation between mice at the same age, typical event sequences were delineated. At the cellular level, amorphous lysosomal inclusions preceded cystine crystals and eventual atrophy without crystals. At the nephron level, lesions started at the glomerulotubular junction and then extended distally. In situ hybridization and immunofluorescence revealed progressive loss of expression of megalin, cubilin, sodium-glucose cotransporter 2, and type IIa sodium-dependent phosphate cotransporter, suggesting apical dedifferentiation accounting for Fanconi syndrome before atrophy. Injection of labeled proteins revealed that defective endocytosis in S1 PTCs led to partial compensatory uptake by S3 PTCs, suggesting displacement of endocytic load and injury by disulfide-rich cargo. Increased PTC apoptosis allowed luminal shedding of cystine crystals and was partially compensated for by tubular proliferation. We conclude that lysosomal storage triggered by soluble cystine accumulation induces apical PTC dedifferentiation, which causes transfer of the harmful load of disulfide-rich proteins to more distal cells, possibly explaining longitudinal progression of swan-neck lesions. Furthermore, our results suggest that subsequent adaptation mechanisms include lysosomal clearance of free and crystalline cystine into urine and ongoing tissue repair.

Noninvasive analysis of renal function in conscious mice is necessary to optimize the use of mouse models. In this study, we evaluated whether single photon emission-computed tomography (SPECT) using specific radionuclear tracers can be used to analyze changes in renal proximal tubule functions. The tracers included 99mTC-dimercaptosuccinic acid (99mTc-DMSA), which is used for cortex imaging; 99mTc_mercaptoacetyltriglycine ([sup.99m]Tc-MAG3), used for dynamic renography; and [sup.123]I-[[beta].sub.2]-microglobulin, which monitors receptor-mediated endocytosis. 99mTc-DMSA SPECT imaging was shown to delineate the functional renal cortex with a ~1-mm spatial resolution and accumulated in the cortex reaching a plateau 5 h after injection. The cortical uptake of 99mTc-DMSA was abolished in Clcn5 knockout mice, a model of proximal tubule dysfunction. Dynamic renography with [sup.99m]Tc-MAG3 in conscious mice demonstrated rapid extraction from blood, renal accumulation, and subsequent tubular secretion. Anesthesia induced a significant delay in the 99mTc-MAG3 clearance. The tubular reabsorption of [sup.123]I-[[beta].sub.2]-microglobulin was strongly impaired in the Clcn5 knockout mice, with defective tubular processing and loss of the native tracer in urine, reflecting proximal tubule dysfunction. Longitudinal studies in a model of cisplatin-induced acute tubular injury revealed a correlation between tubular recovery and [sup.123]I-[[beta].sub.2]-microglobulin uptake. These data show that SPECT imaging with well-validated radiotracers allows in vivo investigations of specific proximal tubule functions in conscious mice. receptor-mediated endocytosis; tubular secretion; CIC-5; Dent's disease; radionuclear tracers doi: 10.1152/ajprenal.00413.2009

Renal tabular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-contalning vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. [Clcn5.sup.Y/-] mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney. chloride/proton antiporter; kinesin family; proximal tubular reabsorption; hypercalciuric nephrolithiasis; receptor-mediated endocytosis doi: 10.1152/ajprenal.00038.2009

A study is conducted to show that a gradient of activin/TGF(beta) signaling controls hepatoblast differentiation. It is found that in the absence of Onecut transcription factors, the shape of the activin/TGFB signaling is perturbed and the hepatoblasts differentiate into hybrid cells that display characteristics of both hepatocytes and biliary cells and therefore, a gradient of activin/TGF(beta) signaling modulated by Onecut factors is required to segregate the hepatocytic and the biliary lineages.

The presence of a local renin-angiotensin system has been established in organs that serve as angiotensin targets. In this study, the expression of angiotensinogen mRNA and subcellular localization of renin, angiotensin-converting enzyme, and angiotensin II were investigated in bovine adrenal medullary cells in primary culture. By light microscopy, expression of angiotensinogen mRNA, immunoreactive renin, angiotensin-converting enzyme, and angiotensin II were readily detectable only in the chromaffin cells. The density distribution of renin and angiotensin II in sucrose gradients suggested a concentration in chromaffin granules, a localization directly confirmed by immunoelectron microscopy. Reverse transcriptase-polymerase chain reaction and sequencing confirmed the expression of angiotensinogen in bovine chromaffin cells and the adrenal medulla. In addition, in vitro autoradiography indicated that both angiotensin-converting enzyme and angiotensin type I receptors were present in the adrenal medulla. These results provide the first direct evidence that chromaffin cells in the adrenal medulla are not only the target for angiotensin but should also be considered as potential local angiotensin-generating and -storing cells. in situ hybridization; immunocytochemistry; radioligand autoradiography; angiotensinogen