Stephane Lefrancois

Effects of Chronic Renal Failure on Liver Drug Transporters

Chronic renal failure (CRF) is associated with a decrease in liver drug metabolism, particularly mediated by the cytochrome P450. CRF also impedes intestinal drug transporters [mainly P-glycoprotein (P-gp) and multidrug resistance protein (MRP)]. However, very few studies have evaluated the effects of CRF on liver drug transport. The present study aimed to investigate the repercussions of CRF on liver drug transporters involved in hepatic uptake [organic anion transporting polypeptide (Oatp) 2] and in drug extrusion (P-gp and MRP2). Two groups of rats were studied: control and CRF. Oatp2, P-gp, and MRP2 protein expressions and mRNA levels, as well as some of their metabolic activity, were assessed. The effects of CRF serum on drug transporters were also evaluated in cultured hepatocytes. Compared with control, creatinine clearance was reduced by 70% (p < 0.01) in rats with CRF. Protein expression and mRNA levels of P-gp were increased by 25 and 40% (p < 0.01), respectively, in liver from rats with CRF. MRP2 protein expression was identical in both groups, whereas its mRNA levels were increased by 35% (p < 0.01) in CRF rats. Finally, Oatp2 protein expression was reduced by 35%, whereas its mRNA levels remained unchanged. Similar results were obtained when hepatocytes were incubated with uremic serum. In conclusion, CRF is associated with a decrease in liver transporters involved in drug absorption and an increase in those involved in drug extrusion. Uremic mediators appear to be responsible for these modifications.

Effects of chronic renal failure on kidney drug transporters and cytochrome P450 in rats.

Chronic renal failure (CRF) leads to decreased drug renal clearance due to a reduction in the glomerular filtration rate. However, little is known about how renal failure affects renal metabolism and elimination of drugs. Because both depend on the activity of uptake and efflux by renal transporters as well as enzymes in tubular cells, the purpose of this study was to investigate the effects of CRF on the expression and activity of select renal drug transporters and cytochrome P450. Two groups of rats were studied: control and CRF (induced by 5/6 nephrectomy). Compared with control rats, we observed reductions in the expression of both protein and mRNA of Cyp1a, sodium-dependent phosphate transport protein 1, organic anion transporter (Oat)1, 2, and 3, OatK1/K2, organic anion-transporting polypeptide (Oatp)1 and 4c1, P-glycoprotein, and urate transporter 1, whereas an induction in the protein and mRNA expression of Mrp2, 3, and 4 and Oatp2 and 3 was observed. Cyp3a expression remained unchanged. Similar results were obtained by incubating a human proximal tubule cell line (human kidney-2) with sera from CRF rats, suggesting the presence of uremic modulators. Finally, the renal elimination of [3H]digoxin and [14C]benzylpenicillin was decreased in CRF rats, compared with controls, as shown by a 4- and 9-fold accumulation, respectively, of these drugs in kidneys of rats in CRF. Our results demonstrate that CRF affects the expression and activity of several kidney drug transporters leading to the intrarenal accumulation of drugs and reduced renal clearance that could, at least partially, explain the tubular toxicity of many drugs.

The phosphatidylinositol 4-kinase PI4KIIIα is required for the recruitment of GBF1 to Golgi membranes

Sorting from the Golgi apparatus requires the recruitment of cytosolic coat proteins to package cargo into trafficking vesicles. An important early step in the formation of trafficking vesicles is the activation of Arf1 by the guanine nucleotide exchange factor GBF1. To activate Arf1, GBF1 must be recruited to and bound to Golgi membranes, a process that requires Rab1b. However, the mechanistic details of how Rab1 is implicated in GBF1 recruitment are not known. In this study, we demonstrate that the recruitment of GBF1 also requires phosphatidylinositol 4-phosphate [PtdIns(4)P]. Inhibitors of PtdIns(4)P synthesis or depletion of PI4KIIIα, a phosphatidylinositol 4-kinase localized to the endoplasmic reticulum and Golgi, prevents the recruitment of GBF1 to Golgi membranes. Interestingly, transfection of dominant-active Rab1 increased the amount of PtdIns(4)P at the Golgi, as detected by GFP-PH, a PtdIns(4)P-sensing probe. We propose that Rab1 contributes to the specificity and timing of GBF1 recruitment by activating PI4KIIIα. The PtdIns(4)P produced then allows GBF1 to bind to Golgi membranes and activate Arf1.

The Role of Ceroid Lipofuscinosis Neuronal Protein 5 (CLN5) in Endosomal Sorting

ABSTRACT Mutations in the gene encoding CLN5 are the cause of Finnish variant late infantile Neuronal Ceroid Lipofuscinosis (NCL), and the gene encoding CLN5 is 1 of 10 genes (encoding CLN1 to CLN9 and cathepsin D) whose germ line mutations result in a group of recessive disorders of childhood. Although CLN5 localizes to the lysosomal compartment, its function remains unknown. We have uncovered an interaction between CLN5 and sortilin, the lysosomal sorting receptor. However, CLN5, unlike prosaposin, does not require sortilin to localize to the lysosomal compartment. We demonstrate that in CLN5-depleted HeLa cells, the lysosomal sorting receptors sortilin and cation-independent mannose 6-phosphate receptor (CI-MPR) are degraded in lysosomes due to a defect in recruitment of the retromer (an endosome-to-Golgi compartment trafficking component). In addition, we show that the retromer recruitment machinery is also affected by CLN5 depletion, as we found less loaded Rab7, which is required to recruit retromer. Taken together, our results support a role for CLN5 in controlling the itinerary of the lysosomal sorting receptors by regulating retromer recruitment at the endosome.

Expression and regulation of LRP-2/megalin in epithelial cells lining the efferent ducts and epididymis during postnatal development.

Low density lipoprotein receptor‐related protein‐2/megalin (LRP‐2) is a receptor belonging to the low density lipoprotein receptor family that mediates endocytosis and lysosomal degradation of a variety of ligands including apolipoprotein J (Apo J)/clusterin/SGP‐2. LRP‐2 has been shown to be expressed regionally in the adult rat epididymis. In this study, we describe the pattern of expression of LRP‐2 in the efferent ducts and epididymis during postnatal development of the rat and examine the role of testicular luminally derived substances on its expression. The expression of LRP‐2 was analyzed immunocytochemically in tissues of normal animals ranging in age from postnatal day 7–90 and in 15‐day‐old efferent‐duct‐ligated animals sacrificed at later ages. In the efferent ducts, LRP‐2 expression, appearing as a dense band on the apical surface of the nonciliated epithelial cells, was noted as early as day 7, well before the entry of sperm, Sertoli‐cell‐derived secretory products, and high levels of androgens. Efferent duct ligation studies further revealed that expression under this condition was comparable to controls at all later ages examined, suggesting that the factor regulating its expression was not a luminally derived testicular substance. In normal untreated animals, LRP‐2 expression was not apparent at any of the ages examined in the proximal initial segment of the epididymis. By comparison, the distal initial segment, although having no LRP‐2 expression from 7–15 days, showed expression in principal cells by day 21 which intensified at days 29 and 39. However, by day 49 and at later ages (56 and 90), LRP‐2 immunoreactivity over principal cells became spotty or with weak or moderate reactivity in some cells and none in others. LRP‐2 expression in the intermediate zone, proximal caput, corpus, and cauda regions also appeared in principal cells by day 21, intensified at days 29 and 39 and persisted as such at all later ages examined, correlating with high levels of androgens shown to occur by day 39. Although LRP‐2 expression in the distal caput region was evident in principal cells at days 21 and 29, it became spotty with weak, moderate, or absent reactivity over principal cells at all later ages. These data suggest that LRP‐2 expression is under the influence of both stimulatory and region‐specific inhibitory factors. Analysis of 15‐day‐old efferent‐duct‐ligated animals at all later ages examined revealed that there was no change in LRP‐2 expression along the entire epididymis, suggesting that both the stimulatory and inhibitory factors are not luminally derived testicular substances. The observed pattern of LRP‐2 expression in all regions of the epididymis, except the distal caput region, was similar to that described for Apo J internalization by principal cells during postnatal development, showing a correlation between LRP‐2 expression and its ligand, Apo J. In summary, LRP‐2 expression in the epididymis undergoes region‐specific changes during postnatal development and appears to be influenced by both stimulatory and inhibitory factors. Mol. Reprod. Dev. 53:282–293, 1999.

ROLE OF PROSAPOSIN IN THE MALE REPRODUCTIVE SYSTEM: EFFECT OF PROSAPOSIN INACTIVATION ON THE TESTIS, EPIDIDYMIS, PROSTATE, AND SEMINAL VESICLES

SGP-l/prosaposin can be secreted or targeted to the lysosomes where it is processed into smaller saposins (A, B, C, and D) required for the hydrolysis of glycosphingolipids. The deficiency of saposins B and C results in variant forms of metachromatic leukodystrophy and Gaucher?s disease, respectively, which are characterized by lysosomal storage of undegraded glycosphingolipids. In the nervous system, prosaposin presents trophic activity. A mouse model was recently developed by creating a null allele in embryonic stem cells through gene targeting to investigate the phenotypic diversity of prosaposin mutations and the involvement of this protein in lysosomal storage diseases, and for the development of therapeutic approaches. Mice homozygous mutants die at the age of 35?40 days and neurological disorders contribute to the early demise of the mutant mice. The male reproductive organs in homozygous mutants show several abnormalities, such as a decrease in testis size with reduced spermiogenesis and an involution of the prostate, seminal vesicles, and epididymis. In these animals, the blood levels of testosterone remain normal. In the prostate of homozygous mutants, only the basal epithelial cells appear to be present, while the secretory cells are absent. These findings suggest that prosaposin may be involved in the development and maintenance of the male reproductive organs, as well as, in cellular differentiation.SGP-1/prosaposin can be secreted or targeted to the lysosomes where it is processed into smaller saposins (A, B, C, and D) required for the hydrolysis of glycosphingolipids. The deficiency of saposins B and C results in variant forms of metachromatic leukodystrophy and Gauchers disease, respectively, which are characterized by lysosomal storage of undegraded glycosphingolipids. In the nervous system, prosaposin presents trophic activity. A mouse model was recently developed by creating a null allele in embryonic stem cells through gene targeting to investigate the phenotypic diversity of prosaposin mutations and the involvement of this protein in lysosomal storage diseases, and for the development of therapeutic approaches. Mice homozygous mutants die at the age of 35-40 days and neurological disorders contribute to the early demise of the mutant mice. The male reproductive organs in homozygous mutants show several abnormalities, such as a decrease in testis size with reduced spermiogenesis and an involution of the prostate, seminal vesicles, and epididymis. In these animals, the blood levels of testosterone remain normal. In the prostate of homozygous mutants, only the basal epithelial cells appear to be present, while the secretory cells are absent. These findings suggest that prosaposin may be involved in the development and maintenance of the male reproductive organs, as well as, in cellular differentiation.

Elevated levels of the polyadenylation factor CstF 64 enhance formation of the 1kB Testis brain RNA-binding protein (TB-RBP) mRNA in male germ cells

The single copy mouse Testis Brain RNA‐Binding Protein (TB‐RBP) gene encodes three mRNAs of 3.0, 1.7, and 1.0 kb which only differ in their 3′ UTRs. The 1 kb TB‐RBP mRNA predominates in testis, while somatic cells preferentially express the 3.0 kb TB‐RBP mRNA. Here we show that the 1 kb mRNA is translated several‐fold more efficiently than the 3 kb TB‐RBP in rabbit reticulocyte lysates and cells with elevated levels of the 1 kB TB‐RBP mRNA express high levels of TB‐RBP. To determine if the cleavage stimulatory factor CstF 64 can modulate the alternative splicing of the TB‐RBP pre‐mRNA and therefore TB‐RBP expression, CstF 64 levels and binding to alternative polyadenylation sites were examined. CstF 64 is abundant in the testis and preferentially binds to a distal site in the TB‐RBP pre‐mRNA that produces the 3 kb TB‐RBP. Moreover, upregulation or overexpression of CstF 64 increases the poly(A) site selection for the 1 kb TB‐RBP mRNA. We propose that the level of the polyadenylation factor CstF 64 modulates the level of TB‐RBP synthesis in male germ cells by an alternative processing of the TB‐RBP pre‐mRNA. Mol. Reprod. Dev. 58:460–469, 2001.

The Lysosomal Transport of Prosaposin Requires the Conditional Interaction of Its Highly Conserved D Domain with Sphingomyelin

Lysosomal prosaposin (65 kDa) is a nonenzymic protein that is transported to the lysosomes in a mannose 6-phosphate-independent manner. Selective deletion of the functional domains of prosaposin indicates that the D domain and the carboxyl-terminal region are necessary for its transport to the lysosomes. Inhibitors of sphingolipid biosynthesis, such as fumonisin B1 (FB1) and tricyclodecan-9-yl xanthate potassium salt (D609), also interfere with the trafficking of prosaposin to lysosomes. In this study, we examine sphingomyelin as a direct candidate for the trafficking of prosaposin. Chinese hamster ovary and COS-7 cells overexpressing prosaposin or an albumin/prosaposin construct were incubated with these inhibitors, treated with sphingolipids, and then immunostained. Sphingomyelin restored the immunostaining in lysosomes in both FB1- and D609-treated cells and ceramide reestablished the immunostaining in FB1-treated cells only.d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits glycosphingolipids, had no effect on the immunostaining pattern. To determine whether sphingomyelin has the same effect on the transport of endogenous prosaposin, testicular explants were treated with FB1 and D609. Sphingomyelin restored prosaposin immunogold labeling in the lysosomes of FB1- and D609-treated Sertoli cells, whereas ceramide restored the label in FB1 treatment only. Albumin linked to the D and COOH-terminal domains of prosaposin was used as a dominant negative competitor. The construct blocked the targeting of prosaposin and induced accumulation of membrane in the lysosomes, demonstrating that the construct uses the same transport pathway as endogenous prosaposin. In conclusion, our results showed that sphingomyelin, the D domain, and its adjacent COOH-terminal region play a crucial role in the transport of prosaposin to lysosomes. Although the precise nature of this lipid-protein interaction is not well established, it is proposed that sphingomyelin microdomains (lipid rafts) are part of a mechanism ensuring correct intercellular trafficking of prosaposin.

Sortilin turnover is mediated by ubiquitination

Sortilin is a transmembrane domain protein that has been implicated in the sorting of prosaposin and other soluble cargo from the Golgi to the lysosomal compartment. While the majority of the receptor is recycled back to the Golgi from endosomes, it is known that upon successive rounds of transport, a proportion of sortilin is degraded in lysosomes. Recently, it was shown that sortilin is palmitoylated and that this post-translational modification prevents its degradation and enables sortilin to efficiently traffic back to the Golgi. Thus palmitoylation can be used to modulate the amount of receptor and hence cargo reaching the lysosome. In this work, we demonstrate that non-palmitoylated sortilin is ubiquitinated and internalized into the lysosomal compartment via the ESCRT pathway for degradation. Furthermore, we identified Nedd4 as an E3 ubiquitin ligase that mediates this post-translational modification. We propose a model where palmitoylation and ubiquitination play opposite roles in the stability and turnover of sortilin and serve as a control mechanism that balances the amount of lysosomal sorting and trafficking in cells.

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.