Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

Abstract

Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.

Cysteamine-bicalutamide combination treatment restores alpha-ketoglutarate and corrects proximal tubule phenotype in cystinosis

“…Although several cellular defects have been associated with cystinosis, the exact mechanism linking cystinosin loss, lysosomal defects and epithelial dysfunction remains elusive. Defective cystinosin alters lysosomal autophagy dynamics 31,32 , impairs activity of mTORC1 and increases the numbers of autophagosomes in CTNS -/cells. Although our data suggest an abnormal induction of autophagy in cystinotic cells, the accumulation of the autophagy substrate SQSTM1 and the decreased lysosomal cargo processing, confirms the lack of autophagy completion in CTNS -/cells, in agreement with the recent studies postulating an impairment of autophagy flux in many LSDs, including cystinosis 33,34 .…”

The renal Fanconi syndrome in cystinosis: pathogenic insights and therapeutic perspectives

“…3b). First, in addition to severely decreased expression of the endocytic receptors megalin and cubilin in PTCs 91,122 , impaired megalin recycling has been reported in cystinotic cells 123 , as in two other hereditary diseases associated with renal Fanconi syndrome: Dent disease 124 and Lowe oculocerebrorenal (OCRL) syndrome 125 . However, the finding that administration of cysteamine fails to control established Fanconi syndrome despite correction of megalin recycling in vitro 123 argues against a major role for recycling defects in cystinosis.…”

“…Second, cystinotic lysosomes become enlarged and cluster to the perinuclear region of cells, reflecting impaired lysosome motility and indicating reduced accessibility to endocytic and autophagic loads 122,123 . Three possible explanations exist for the pericentriolar trapping of cystinotic lysosomes: first, through physical retention, since enlarged lysosomes have impaired free diffusion 126 and are predicted to engage with increased numbers of microtubular contacts via their cytoskeletal motor protein, dynein, and since the microtubular network is most dense around centrioles 127 ; second, through altered regulation of lysosomal calcium release that controls lysosomal dynamics 127 , although no change in the total level of lysosomal calcium store has been detected in cultured cystinotic cells 128 ; and third, through downregulation of Rab–GTPase(s) that normally catalyse lysosomal trafficking, such as Rab27a, which is specifically expressed in PTCs and is downregulated in PTCs from mice and humans with cystinosis 129 .…”

“…Direct i n situ evidence of an impaired rate of proteolysis, independent of less frequent encounters between internalized protein cargo carried by late endosomes and the set of proteolytic enzymes brought by lysosomes, has been demonstrated in primary PTC cultures from Ctns -knockout mice 122 , and in human cystinotic cells 123 . Assuming that lysosomal influx of cysteine does not increase in cystinotic cells to compensate for higher intralysosomal levels of free cystine due to impaired exodus, thus leading to luminal oxidation of lysosomal thiols, the defect in intrinsic lysosomal proteolysis could result from three possible mechanisms (FIG.…”

First Successful Conception Induced by a Male Cystinosis Patient

“…However, in this case, the patient had clear signs of primary hypogonadism, sufficient to explain his infertility. As several studies demonstrated recently that cystinosin is involved in numerous cellular processes, one can argue whether merely the lysosomal cystine accumulation should be considered as the only pathophysiological mechanism involved in infertility (Raggi et al 2014;Giade Chevronnay et al 2015;Ivanova et al 2015Ivanova et al , 2016Rega et al 2016). In this regard, it is of note that the cystinosin-LKG isoform, of which its localization is not limited to the lysosomal membrane, has the highest expression in the testes compared to other organs (Taranta et al 2008).…”