(A1 and A2) Transmission electron microscopy depicting ultrastructure of PTECs cultured in MPS device. Polarization of PTECs was shown by localization of the tight junction protein ZO-1 to the luminal (apical) aspect of the PTEC tubule, and localization of Na+/K+-ATPase to the lateral interface between neighboring cells and the basal border between PTECs and collagen substrate (Fig. 1C). Cilia formation in response to fluid shear stress was evidenced by positive staining of acetylated tubulin in rod-like structures that originate close to the cell nucleus; the ciliary process averaged 10 3.5 m in length (Fig. 2B1/B2). Open in a separate windows Fig. 2 Ultrastructure of human PTECs in human kidney 3D MPS. (A1 and A2) Transmission electron microscopy depicting ultrastructure of PTECs cultured in MPS device. Cellular structure labels: MV-microvilli, M-mitochondria, TJ-tight junction, ER-endoplasmic reticulum, and GA-Golgi Apparatus. (A1 10,000x, A2 30,000x magnification). (B1 and B2) PTECs in MPS form cilia as seen from 2 representative images of single cells stained for acetylated tubulin in reddish. ((8). The reclamation process is usually mediated by -glutamyl transpeptidase (GGT), which normally mediates the transfer of glutamyl moiety from glutathione to an acceptor amino acid as part of the -glutamyl cycle, a pathway for the synthesis and degradation of glutathione. Selective IHC staining for GGT in the MPS revealed enriched localization of the enzyme at the luminal aspect of the PTEC structure (Fig. 3A). GGT mediates a comparable reaction for the oxidized form of glutathione, glutathione disulfide (GSSG) (Fig. 3B) (9), which is a more chemically stable substrate for assessing the activity of -glutamyl transpeptidase in proximal tubule MPS. PTECs within the MPS were perfused with media made up of 4 M GSSG in the presence or absence of the irreversible GGT inhibitor acivicin (1 mM). The recovery of GSSG in the effluent was low ( 1.5%), demonstrating extensive catalytic activity of GGT; in the presence of acivicin, an approximate 2-fold increase in GSSG recovery was observed over 2-4 hours (Fig. Tyrphostin AG 879 3C). Open in a separate window Fig. 3 GGT activity in human kidney 3D MPS. (A) Immunocytochemistry reveals proper apical localization of GGT (green) in juxtaposition to nuclei (blue) within the PTEC tubule. (B) -glutamyl transpeptidase (GGT) is functionally essential to cleaving the -glutamyl moiety from oxidized glutathione and can be inhibited by acivicin. (C) GGT activity as determined by oxidized glutathione abundance in the presence and absence of inhibitor, acivicin (n = 4 MPS devices) (*, Tyrphostin AG 879 0.001, 2-tailed predictions of 90% inhibition (11). Open in a separate window Fig. 4 Glucose Reabsorption in human kidney 3D MPS. (A) Glucose is actively reabsorbed from the urine via SGLT2 located on the apical membrane in the PTEC tubule. Immunocytochemistry reveals proper apical localization of SGLT2 (green) in juxtaposition to nuclei (blue). DIC images showing the structure of PTECs in the MPS in the presence and absence of SGLT2 inhibitor, apigenin (B and D). Fluorescent images showing the distribution of the fluorescent glucose analog, NBDG (C and E). NBDG was actively reabsorbed in the absence of inhibitor (C) and was not absorbed in the presence of inhibitor (E). (F) Quantification of cell-associated fluorescent signal following subtraction of auto fluorescence, demonstrating significant reduction of glucose uptake in the presence of inhibitors apigenin and dapaglifozin (n = 3 MPS devices/ group) (*, 0.001, unpaired to a drop in either blood or luminal filtrate pH with an increased generation and secretion of ammonia (Fig. 6A). To demonstrate this physiological response, PTECs were exposed to a decrease in MPS luminal perfusate pH from 7.4 to 6 6.9. PTEC cells in the MPS responded with an approximate 3-fold increase in effluent ammonia concentration (pH 7.40.55.AC was a recipient of the Warren G. screening of new chemical compounds prior to initiating human clinical trials. (Figure 2A1/A2 and Supplementary figure 2) (7). Polarization of PTECs was shown by localization of the tight junction protein ZO-1 to the luminal (apical) aspect of the PTEC tubule, and localization of Na+/K+-ATPase to the lateral interface between neighboring cells and the basal border between PTECs and collagen substrate (Fig. 1C). Cilia formation in response to NF2 fluid shear stress was evidenced by positive staining of acetylated tubulin in rod-like structures that originate close to the cell nucleus; the ciliary process averaged 10 3.5 m in length (Fig. 2B1/B2). Open in a separate window Fig. 2 Ultrastructure of human PTECs in human kidney 3D MPS. (A1 and A2) Transmission electron microscopy depicting ultrastructure of PTECs cultured in MPS device. Cellular structure labels: MV-microvilli, M-mitochondria, TJ-tight junction, ER-endoplasmic reticulum, and GA-Golgi Apparatus. (A1 10,000x, A2 30,000x magnification). (B1 and B2) PTECs in MPS form Tyrphostin AG 879 cilia as seen from 2 representative images of single cells stained for acetylated tubulin in red. ((8). The reclamation process is mediated by -glutamyl transpeptidase (GGT), which normally mediates the transfer of glutamyl moiety from glutathione to an acceptor amino acid as part of the -glutamyl cycle, a pathway for the synthesis and degradation of glutathione. Selective IHC staining for GGT in the MPS revealed enriched localization of the enzyme at the luminal aspect of the PTEC structure (Fig. 3A). GGT mediates a comparable reaction for the oxidized form of glutathione, glutathione disulfide (GSSG) (Fig. 3B) (9), which is a more chemically stable substrate for assessing the activity of -glutamyl transpeptidase in proximal tubule MPS. PTECs within the MPS were perfused with media containing 4 M GSSG in the presence or absence of the irreversible GGT inhibitor acivicin (1 mM). The recovery of GSSG in the effluent was low ( 1.5%), demonstrating extensive catalytic activity of GGT; in the presence of acivicin, an approximate 2-fold increase in GSSG recovery was observed over 2-4 hours (Fig. 3C). Open in a separate window Fig. 3 GGT activity in human kidney 3D MPS. (A) Immunocytochemistry reveals proper apical localization of GGT Tyrphostin AG 879 (green) in juxtaposition to nuclei (blue) within the PTEC tubule. (B) -glutamyl transpeptidase (GGT) is functionally essential to cleaving the -glutamyl moiety from oxidized glutathione and can be inhibited by acivicin. (C) GGT activity as determined by oxidized glutathione abundance in the presence and absence of inhibitor, acivicin (n = 4 MPS devices) (*, 0.001, 2-tailed predictions of 90% inhibition (11). Open in a separate window Fig. 4 Glucose Reabsorption in human kidney 3D MPS. (A) Glucose is actively reabsorbed from the urine via SGLT2 located on the apical membrane in the PTEC tubule. Immunocytochemistry reveals proper apical localization of SGLT2 (green) in juxtaposition to nuclei (blue). DIC images showing the structure of PTECs in the MPS in the presence and absence of SGLT2 inhibitor, apigenin (B and D). Fluorescent images showing the distribution of the fluorescent glucose analog, NBDG (C and E). NBDG was actively reabsorbed in the absence of inhibitor (C) and was not absorbed in the presence of inhibitor (E). (F) Quantification of cell-associated fluorescent signal following subtraction of auto fluorescence, demonstrating significant reduction of glucose uptake in the presence of inhibitors apigenin and dapaglifozin (n = 3 MPS devices/ group) (*, 0.001, unpaired to a drop in either blood or luminal filtrate pH with an increased generation and secretion of ammonia (Fig. 6A). To demonstrate this physiological response, PTECs were exposed to a decrease in MPS luminal perfusate pH from 7.4 to 6 6.9. PTEC cells in the MPS responded with an approximate 3-fold increase in effluent ammonia concentration (pH 7.40.55 mM NH3, pH 6.91.56 mM NH3) (Fig. 6B). Open in a Tyrphostin AG 879 separate window Fig. 6 Ammoniagenesis in human kidney 3D MPS. (A) The physiological response to a drop in either blood or luminal pH resulting in the generation and secretion of ammonia in the tubular outflow. (B) With the MPS, luminal media was initially at pH 7. 4 and then switched to pH 6.9. Secreted ammonia in the outflow was quantified spectrophotometrically from 4 separate devices after 4 h and was significantly different when exposed to acidic conditions. (*, = 0.05, 2-tailed out to 28+ days (7, 17). We also demonstrated proximal tubular origin for the majority of epithelial cells in culture by IHC staining of differential markers of kidney epithelial cells (viz., aquaporin 1, aquaporin 2, and SGLT2). Structurally, PTECs in the MPS exhibited polarized.