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H+ Exchanger NHE3 by Ubiquitination
AGA Abstracts
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Basal and Stimulated BB NHE3 Activity Requires Intracellular SNX27, a PDZDomain Containing Binding Partner of NHE3 That Regulates Endosome to Plasma Membrane Trafficking Varsha Singh, Jianbo Yang, Rafiquel Sarker, Tian-e Chen, Boyoung Cha, Nicholas C. Zachos, Mark Donowitz
Nedd4-2 Uniquely Regulates Human Na+/H+ Exchanger NHE3 by Ubiquitination Yi Ran No, Byong Kwon Yoo, Peijian He, Chris Yun Na+/H+ exchanger 3, NHE3, is an integral protein expressed in the brush border membrane of the epithelial cells in the small intestine, colon, and kidney where it conducts a bulk of Na+ and HCO3- absorption. NHE3 at the brush border membrane is in flux with NHE3 in intracellular vesicles as such the primary means of NHE3 regulation involves the movement of NHE3 molecules between the two compartments. Ubiquitination is a regulated posttranslational modification that conjugates ubiquitin (Ub) to Lys residues of target proteins and controls their intracellular fate. Nedd4-1 (neural precursor cell expressed, developmentally down-regulated 4-1) and Nedd4-2 belong to the Nedd4 family of E3 Ub ligases that typically interact with protein substrates through a PY (PPxY) motif. Endocytic process is a vital process for NHE3 regulation, but the status of NHE3 ubiquitination and the role of ubiquitination in NHE3 endoycytosis and degradation have not been reported. The goal of this study was to determine the ubiquitination status of NHE3 and whether NHE3 degradation and/or recycling are regulated by ubiquitination. A comparison of NHE3 sequences from 25 species revealed the presence of PY motifs in NHE3s from human and several non-human primates, but not in non-primate NHE3s. By co-immunoprecipitation, we found that Nedd4-2, but not Nedd4-1, interacted with human NHE3 (hNHE3) but not with NHE3 from rabbit or opossum. Surprisingly, rat NHE3 despite the lack of a PY motif co-immunoprecipitated with Nedd4-2. However, a subsequent analysis showed that silencing of Nedd4-2 by RNAi in PS120 and SKCO-15 cells markedly decreased hNHE3 ubiquitination while increasing Na+/H+ exchange by hNHE3. However, no effect on non-primate NHE3s, including rat NHE3, was observed. The interaction between hNHE3 and Nedd4-2 took place at the plasma membrane and knockdown of Nedd4-2 resulted in a change in ubiquitination level of surface hNHE3. Ubiquitination by Nedd4-2 did not alter NHE3 protein stability, but rendered hNHE3 to undergo internalization at a significantly greater rate than nonprimate NHE3s. Using a series of C-terminal deletion and point mutations, we identified the PY motif of hNHE3 as the primary Nedd4-2 binding motif. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. In conclusion, our study revealed a unique molecular mechanism regulating human NHE3 and showed that NHE3 endocytosis is regulated by ubiquitination.
Background: Regulation of the transport activity of the intestinal and renal proximal tubule BB Na+-H+ exchanger, NHE3 depends on dynamic association with members of the multiPDZ domain-NHERF gene family. Three members of the NHERF family are present in or just below the BB and dynamically fix NHE3 in the BB. BLAST searches showed that this gene family evolved from common ancestors and their PDZ domains are highly related. One additional protein is highly related to this gene family, sorting nexin 27 (SNX27) via its single PDZ domain. The SNX gene family is a diverse group of cytoplasmic and membraneassociated proteins, all of which are involved in endosomal protein trafficking. They contain a phosphoinositide-binding PX domain, which targets proteins to PI-3P-rich endosomal membranes. SNX27 is the only family member that contains a PDZ domain. In this study we tested the hypothesis that SNX27 regulates NHE3 activity and does so via its NHERF family related PDZ domain. Methods: Human embryonic kidney 293 cells and human intestinal polarized epithelial cell line, SKCO-15 which expresses NHE3 endogenously were used for these studies. Na+/H+ exchange activity was determined with BCECF/fluorometry. Percent surface expression of NHE3 and rates of endocytosis and exocytosis were determined using initial cell surface biotinylation. SNX27 was KD using lentivirus-shRNAs-puromycin 80% KD). Recombinant GST-fusion proteins (SNX27 full length (FL), SNX27-PDZ and SNX27-FERM) were purified for pulldown assays to study direct protein-protein interactions in vitro. Intracellular localization of SNX27 was done by IF/confocal microscopy.. Site directed mutagenesis was used to mutate the PDZ domain (GAGA) of SNX27. Results: Basal and serum stimulated NHE3 activity were reduced in the SNX27-KD cells by ~50% and ~60%, respectively. Reduced NHE3 activity was associated with a ~50% decrease in basal surface expression. This reduction in surface NHE3 was associated with a ~50% reduction in basal exocytosis (endocytic recycling); however, basal endocytosis was normal. GST pull down assay showed that full length as well as the PDZ-domain of SNX27 binds to the intracellular C-terminus of NHE3. Reduced surface NHE3 expression in SNX27 KD could be rescued by overexpression of shRNA-resistant rat SNX27 but not the rat SNX27- double PDZ domain-GAGA mutant. Wild type SNX27 colocalized with intracellular NHE3, and is present in EEA1 and Rab5 positive compartments. Conclusions: 1) Endosomal SNX27 is required for basal and stimulated NHE3 activity by affecting endocytic recycling of NHE3. 2) PDZ domain of SNX27 binds NHE3 to regulate NHE3 activity. 3) Endogenous intracellular NHE3 co-localizes with SNX27 which is present in an EEA1/ Rab5 positive early endosomal compartment. 4) SNX27 regulates endosome to plasma membrane trafficking of NHE3 to help set basal NHE3 activity.
192 Increased Functional AE2 Activity Does Not Compensate for Increased pHi in CFTR Knockout Enteroids Nancy M. Walker, Sydney R. Stein, Ashlee M. Williams, Jinghua Liu, Lane L. Clarke Primary murine small intestinal enteroids enable physiological studies of enterocytes within a well-differentiated epithelium which is not influenced by neuroendocrine, microbial, or immune-mediated factors. Previously, using enteroids from mice with targeted disruption of the anion channel Cftr (CF), we found that CF crypt enterocytes maintained an alkaline intracellular pH (pHi) (Am J Physiol. 302: C1492-503, 2012). An alkaline pHi facilitates cell proliferation and thereby may be a contributing factor to the 6-fold increased risk for gastrointestinal cancer in CF patients (NEJM 332: 494-99, 1995). Enterocytes normally express a wide-range of acid-base transporters that can assist in pHi regulation so we asked why pHi is not regulated to normal levels in CF enterocytes. Using wild-type (WT) and CF murine enteroids, real-time quantitative RT-PCR was used to interrogate the mRNA expression of 10 genes involved in acid-base transport by the intestinal epithelium. Four baseloading genes were down regulated in CF enteroids as compared to WT: carbonic anhydrase IX, the anion exchanger Slc26a6, the electrogenic NaHCO3 cotransporter NBCe1, and the Na+/H+ exchanger, NHE2 (p<0.05; enteroids from 8 sex-matched WT/CF littermates). In contrast, the basolateral Cl-/HCO3- exchanger AE2, a base unloader, was up-regulated in CF enteroids. Since AE2 is known to play an important role in pHi regulation, functional studies using BCECF microfluorimetry evaluated AE2 Cl-/HCO3- exchange activity in enterocytes from WT and CF enteroids. The rate of Cl- -dependent HCO3- efflux across the basolateral membrane in CF crypt enterocytes maintained in Matrigel® culture was significantly greater than in WT (WT =-1.64±0.81, CF =-5.30±1.33 mM/min; p<0.05, enteroids from 6 sex-matched WT/CF littermates). Studies using enteroids from AE2 knockout mice confirmed that basolateral Cl-/HCO3- exchange resulted from AE2 activity (AE2 KO= 0.36±1.15 mM/min, enteroids from 4 AE2 KO mice). To investigate the mechanism of altered acid-base transporter expression, protein expression of the acid-sensitive transcription factor Egr-1 was measured by immunoblot densitometry and showed a trend of reduced expression in the CF as compared to WT enteroids (CF = 75.3 ± 13.7 % of WT, p=0.065, n=6 sex-matched WT/CF littermates). A Genomatix® search of the promoter regions from the differentially expressed genes indicated that all contained Egr-1 response elements. Ongoing studies are investigating the hypothesis that increased intracellular Cl- concentration in CF enterocytes impedes the functional activity of AE2. We conclude that increased AE2 activity and down-regulation of base-loading transporters in CF crypt enterocytes do not fully compensate for the effect of CFTR loss on the pHi regulation. Supported by NIDDK-44816.
190 Human Small Intestinal Enteroids Enable Mechanistic Studies of cAMPInduced Changes in Intracellular pH Jennifer Foulke-Abel, Julie In, Nicholas C. Zachos, Olga Kovbasnjuk, Khalil Ettayebi, Hugo de Jonge, Mary K. Estes, Mark Donowitz Background: Drug development has been hindered due to lack of normal human models and reliance on animal or human cancer cell models. An ex vivo model of normal human intestine is now available in the form of three-dimensional human enteroids. We have previously demonstrated that human enteroids exhibit the main epithelial transport functions of Na+ absorption via NHE3 and Cl- secretion via CFTR, and now show that the enteroid model is suitable for more detailed mechanistic assessments of cAMP-regulated transport processes such as those encountered under pathophysiological conditions. Methods: We assembled a bank of human enteroids with normal histology cultured from endoscopic biopsies and surgical specimens of duodenum (n=11), jejunum (n=13), ileum (n=14), and proximal (n=7) and distal colon (n=5). Segment-specific characteristics were interrogated by immunostaining to compare enteroids with biopsies of intact tissue. Intracellular pH (pHi) was measured using multiphoton microscopy and the pH-sensitive dye SNARF-4F to follow changes in basal and pharmalogically regulated ion transport in three-dimensional enteroids differentiated by Wnt3A withdrawal. NHE3 function was isolated by inclusion of HOE-694 (50 μM) to eliminate contributions of NHE1, NHE2, and NHE8. Results: 1) Immunostaining demonstrates that human enteroids retain transport protein expression and localization profiles (NHE3, NKCC1, DRA, Na+/K+-ATPase) consistent with their respective native tissues. 2) Na+/H+ exchange in HCO3--free Na+-containing buffer was modulated primarily by NHE3 and was inhibited by S3226 (20 μM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (100 μM), 8-Br-cAMP (100 μM), forskolin (25 μM), and cholera toxin (0.1 μg/mL, 5 h). 3) In HCO3--free Na+-containing buffer, forskolin induced intracellular acidification that was blocked by preincubation with CFTRinh-172 (25 μM), H-89 (10 μM),or low concentration DIDS (500 μM). Acidification continued to occur when NHE3 was inhibited by preincubation with EIPA. In contrast, addition of HCO3- prevented the forskolin-mediated pHi decrease. Conclusions: 1) Human enteroids recapitulate the transport mechanisms present in normal human small intestine, exhibiting Na+ absorption mediated by NHE3 and anion secretion via CFTR. 2) NHE3 is inhibited by cAMP (cholera toxin, forskolin, 8Br-cAMP), and forskolin and cholera toxin induce Cl- secretion. 3) The observed cAMPinduced pHi acidification is due to increased HCO3- secretion by CFTR and functionally linked apical Cl-/HCO3- exchangers. The proposed mechanism also implicates a low concentration DIDS-sensitive component such as NBCe1 as the primary source of basolateral HCO3uptake. The human enteroid response to cAMP and cholera toxin provides an effective ex vivo culture system to define the mechanism of pHi regulation in enterocytes and study pathophysiology of diarrhea.
AGA Abstracts
193 Autophagic Down-Regulation and Transient Traffic Perturbation of NHE3 by Rapamycin: Implication in Non-Infectious Diarrhea in Organ Transplantation Archana Patel, Jun Yang, Rachana Potru, Michael Dolinger, David Conti, David M. Jones, Yunfei Huang, Xinjun Zhu The immunosuppressant rapamycin (sirolimus), an mTOR inhibitor, frequently causes noninfectious diarrhea which tends to be severe and intractable, resulting in repeated hospitalizations and is a risk factor for graft loss in transplant patients. The mechanism of rapamycininduced diarrhea remains unclear. Here we report that non-infectious diarrheic episodes were closely associated with a sharp rise in serum rapamycin levels without a change in dosing in a large cohort of renal transplant recipients. Moreover, surface expression of the Na+/H+ exchanger 3 (NHE3), which is a major transporter that mediates intestinal absorption
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Basal and Stimulated BB NHE3 Activity Requires Intracellular SNX27, a PDZDomain Containing Binding Partner of NHE3 That Regulates Endosome to Plasma Membrane Trafficking Varsha Singh, Jianbo Yang, Rafiquel Sarker, Tian-e Chen, Boyoung Cha, Nicholas C. Zachos, Mark Donowitz
Nedd4-2 Uniquely Regulates Human Na+/H+ Exchanger NHE3 by Ubiquitination Yi Ran No, Byong Kwon Yoo, Peijian He, Chris Yun Na+/H+ exchanger 3, NHE3, is an integral protein expressed in the brush border membrane of the epithelial cells in the small intestine, colon, and kidney where it conducts a bulk of Na+ and HCO3- absorption. NHE3 at the brush border membrane is in flux with NHE3 in intracellular vesicles as such the primary means of NHE3 regulation involves the movement of NHE3 molecules between the two compartments. Ubiquitination is a regulated posttranslational modification that conjugates ubiquitin (Ub) to Lys residues of target proteins and controls their intracellular fate. Nedd4-1 (neural precursor cell expressed, developmentally down-regulated 4-1) and Nedd4-2 belong to the Nedd4 family of E3 Ub ligases that typically interact with protein substrates through a PY (PPxY) motif. Endocytic process is a vital process for NHE3 regulation, but the status of NHE3 ubiquitination and the role of ubiquitination in NHE3 endoycytosis and degradation have not been reported. The goal of this study was to determine the ubiquitination status of NHE3 and whether NHE3 degradation and/or recycling are regulated by ubiquitination. A comparison of NHE3 sequences from 25 species revealed the presence of PY motifs in NHE3s from human and several non-human primates, but not in non-primate NHE3s. By co-immunoprecipitation, we found that Nedd4-2, but not Nedd4-1, interacted with human NHE3 (hNHE3) but not with NHE3 from rabbit or opossum. Surprisingly, rat NHE3 despite the lack of a PY motif co-immunoprecipitated with Nedd4-2. However, a subsequent analysis showed that silencing of Nedd4-2 by RNAi in PS120 and SKCO-15 cells markedly decreased hNHE3 ubiquitination while increasing Na+/H+ exchange by hNHE3. However, no effect on non-primate NHE3s, including rat NHE3, was observed. The interaction between hNHE3 and Nedd4-2 took place at the plasma membrane and knockdown of Nedd4-2 resulted in a change in ubiquitination level of surface hNHE3. Ubiquitination by Nedd4-2 did not alter NHE3 protein stability, but rendered hNHE3 to undergo internalization at a significantly greater rate than nonprimate NHE3s. Using a series of C-terminal deletion and point mutations, we identified the PY motif of hNHE3 as the primary Nedd4-2 binding motif. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. In conclusion, our study revealed a unique molecular mechanism regulating human NHE3 and showed that NHE3 endocytosis is regulated by ubiquitination.
Background: Regulation of the transport activity of the intestinal and renal proximal tubule BB Na+-H+ exchanger, NHE3 depends on dynamic association with members of the multiPDZ domain-NHERF gene family. Three members of the NHERF family are present in or just below the BB and dynamically fix NHE3 in the BB. BLAST searches showed that this gene family evolved from common ancestors and their PDZ domains are highly related. One additional protein is highly related to this gene family, sorting nexin 27 (SNX27) via its single PDZ domain. The SNX gene family is a diverse group of cytoplasmic and membraneassociated proteins, all of which are involved in endosomal protein trafficking. They contain a phosphoinositide-binding PX domain, which targets proteins to PI-3P-rich endosomal membranes. SNX27 is the only family member that contains a PDZ domain. In this study we tested the hypothesis that SNX27 regulates NHE3 activity and does so via its NHERF family related PDZ domain. Methods: Human embryonic kidney 293 cells and human intestinal polarized epithelial cell line, SKCO-15 which expresses NHE3 endogenously were used for these studies. Na+/H+ exchange activity was determined with BCECF/fluorometry. Percent surface expression of NHE3 and rates of endocytosis and exocytosis were determined using initial cell surface biotinylation. SNX27 was KD using lentivirus-shRNAs-puromycin 80% KD). Recombinant GST-fusion proteins (SNX27 full length (FL), SNX27-PDZ and SNX27-FERM) were purified for pulldown assays to study direct protein-protein interactions in vitro. Intracellular localization of SNX27 was done by IF/confocal microscopy.. Site directed mutagenesis was used to mutate the PDZ domain (GAGA) of SNX27. Results: Basal and serum stimulated NHE3 activity were reduced in the SNX27-KD cells by ~50% and ~60%, respectively. Reduced NHE3 activity was associated with a ~50% decrease in basal surface expression. This reduction in surface NHE3 was associated with a ~50% reduction in basal exocytosis (endocytic recycling); however, basal endocytosis was normal. GST pull down assay showed that full length as well as the PDZ-domain of SNX27 binds to the intracellular C-terminus of NHE3. Reduced surface NHE3 expression in SNX27 KD could be rescued by overexpression of shRNA-resistant rat SNX27 but not the rat SNX27- double PDZ domain-GAGA mutant. Wild type SNX27 colocalized with intracellular NHE3, and is present in EEA1 and Rab5 positive compartments. Conclusions: 1) Endosomal SNX27 is required for basal and stimulated NHE3 activity by affecting endocytic recycling of NHE3. 2) PDZ domain of SNX27 binds NHE3 to regulate NHE3 activity. 3) Endogenous intracellular NHE3 co-localizes with SNX27 which is present in an EEA1/ Rab5 positive early endosomal compartment. 4) SNX27 regulates endosome to plasma membrane trafficking of NHE3 to help set basal NHE3 activity.
192 Increased Functional AE2 Activity Does Not Compensate for Increased pHi in CFTR Knockout Enteroids Nancy M. Walker, Sydney R. Stein, Ashlee M. Williams, Jinghua Liu, Lane L. Clarke Primary murine small intestinal enteroids enable physiological studies of enterocytes within a well-differentiated epithelium which is not influenced by neuroendocrine, microbial, or immune-mediated factors. Previously, using enteroids from mice with targeted disruption of the anion channel Cftr (CF), we found that CF crypt enterocytes maintained an alkaline intracellular pH (pHi) (Am J Physiol. 302: C1492-503, 2012). An alkaline pHi facilitates cell proliferation and thereby may be a contributing factor to the 6-fold increased risk for gastrointestinal cancer in CF patients (NEJM 332: 494-99, 1995). Enterocytes normally express a wide-range of acid-base transporters that can assist in pHi regulation so we asked why pHi is not regulated to normal levels in CF enterocytes. Using wild-type (WT) and CF murine enteroids, real-time quantitative RT-PCR was used to interrogate the mRNA expression of 10 genes involved in acid-base transport by the intestinal epithelium. Four baseloading genes were down regulated in CF enteroids as compared to WT: carbonic anhydrase IX, the anion exchanger Slc26a6, the electrogenic NaHCO3 cotransporter NBCe1, and the Na+/H+ exchanger, NHE2 (p<0.05; enteroids from 8 sex-matched WT/CF littermates). In contrast, the basolateral Cl-/HCO3- exchanger AE2, a base unloader, was up-regulated in CF enteroids. Since AE2 is known to play an important role in pHi regulation, functional studies using BCECF microfluorimetry evaluated AE2 Cl-/HCO3- exchange activity in enterocytes from WT and CF enteroids. The rate of Cl- -dependent HCO3- efflux across the basolateral membrane in CF crypt enterocytes maintained in Matrigel® culture was significantly greater than in WT (WT =-1.64±0.81, CF =-5.30±1.33 mM/min; p<0.05, enteroids from 6 sex-matched WT/CF littermates). Studies using enteroids from AE2 knockout mice confirmed that basolateral Cl-/HCO3- exchange resulted from AE2 activity (AE2 KO= 0.36±1.15 mM/min, enteroids from 4 AE2 KO mice). To investigate the mechanism of altered acid-base transporter expression, protein expression of the acid-sensitive transcription factor Egr-1 was measured by immunoblot densitometry and showed a trend of reduced expression in the CF as compared to WT enteroids (CF = 75.3 ± 13.7 % of WT, p=0.065, n=6 sex-matched WT/CF littermates). A Genomatix® search of the promoter regions from the differentially expressed genes indicated that all contained Egr-1 response elements. Ongoing studies are investigating the hypothesis that increased intracellular Cl- concentration in CF enterocytes impedes the functional activity of AE2. We conclude that increased AE2 activity and down-regulation of base-loading transporters in CF crypt enterocytes do not fully compensate for the effect of CFTR loss on the pHi regulation. Supported by NIDDK-44816.
190 Human Small Intestinal Enteroids Enable Mechanistic Studies of cAMPInduced Changes in Intracellular pH Jennifer Foulke-Abel, Julie In, Nicholas C. Zachos, Olga Kovbasnjuk, Khalil Ettayebi, Hugo de Jonge, Mary K. Estes, Mark Donowitz Background: Drug development has been hindered due to lack of normal human models and reliance on animal or human cancer cell models. An ex vivo model of normal human intestine is now available in the form of three-dimensional human enteroids. We have previously demonstrated that human enteroids exhibit the main epithelial transport functions of Na+ absorption via NHE3 and Cl- secretion via CFTR, and now show that the enteroid model is suitable for more detailed mechanistic assessments of cAMP-regulated transport processes such as those encountered under pathophysiological conditions. Methods: We assembled a bank of human enteroids with normal histology cultured from endoscopic biopsies and surgical specimens of duodenum (n=11), jejunum (n=13), ileum (n=14), and proximal (n=7) and distal colon (n=5). Segment-specific characteristics were interrogated by immunostaining to compare enteroids with biopsies of intact tissue. Intracellular pH (pHi) was measured using multiphoton microscopy and the pH-sensitive dye SNARF-4F to follow changes in basal and pharmalogically regulated ion transport in three-dimensional enteroids differentiated by Wnt3A withdrawal. NHE3 function was isolated by inclusion of HOE-694 (50 μM) to eliminate contributions of NHE1, NHE2, and NHE8. Results: 1) Immunostaining demonstrates that human enteroids retain transport protein expression and localization profiles (NHE3, NKCC1, DRA, Na+/K+-ATPase) consistent with their respective native tissues. 2) Na+/H+ exchange in HCO3--free Na+-containing buffer was modulated primarily by NHE3 and was inhibited by S3226 (20 μM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (100 μM), 8-Br-cAMP (100 μM), forskolin (25 μM), and cholera toxin (0.1 μg/mL, 5 h). 3) In HCO3--free Na+-containing buffer, forskolin induced intracellular acidification that was blocked by preincubation with CFTRinh-172 (25 μM), H-89 (10 μM),or low concentration DIDS (500 μM). Acidification continued to occur when NHE3 was inhibited by preincubation with EIPA. In contrast, addition of HCO3- prevented the forskolin-mediated pHi decrease. Conclusions: 1) Human enteroids recapitulate the transport mechanisms present in normal human small intestine, exhibiting Na+ absorption mediated by NHE3 and anion secretion via CFTR. 2) NHE3 is inhibited by cAMP (cholera toxin, forskolin, 8Br-cAMP), and forskolin and cholera toxin induce Cl- secretion. 3) The observed cAMPinduced pHi acidification is due to increased HCO3- secretion by CFTR and functionally linked apical Cl-/HCO3- exchangers. The proposed mechanism also implicates a low concentration DIDS-sensitive component such as NBCe1 as the primary source of basolateral HCO3uptake. The human enteroid response to cAMP and cholera toxin provides an effective ex vivo culture system to define the mechanism of pHi regulation in enterocytes and study pathophysiology of diarrhea.
AGA Abstracts
193 Autophagic Down-Regulation and Transient Traffic Perturbation of NHE3 by Rapamycin: Implication in Non-Infectious Diarrhea in Organ Transplantation Archana Patel, Jun Yang, Rachana Potru, Michael Dolinger, David Conti, David M. Jones, Yunfei Huang, Xinjun Zhu The immunosuppressant rapamycin (sirolimus), an mTOR inhibitor, frequently causes noninfectious diarrhea which tends to be severe and intractable, resulting in repeated hospitalizations and is a risk factor for graft loss in transplant patients. The mechanism of rapamycininduced diarrhea remains unclear. Here we report that non-infectious diarrheic episodes were closely associated with a sharp rise in serum rapamycin levels without a change in dosing in a large cohort of renal transplant recipients. Moreover, surface expression of the Na+/H+ exchanger 3 (NHE3), which is a major transporter that mediates intestinal absorption
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