Increased Na+/H+ antiport activity in the renal brush border membrane of SHR

Increased Na+/H+ antiport activity in the renal brush border membrane of SHR. Defect in renal salt excretion may play an important role in the pathogenesis of hypertension. We examined sodium (Na+) uptake by brush border membrane (BBM) vesicles of young (6 week old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) of the same age. SHR had lower urinary Na+ excretion (223.1 ± 9.3 vs. 266.3 ± 3.7 µEq/day/lOO g, N = 8, P < 0.01) and higher systolic blood pressure (98.9 ± 1.2 vs. 82.9 ± 1.8mm Hg, N = 8, P < 0.01) than WKY. BBM vesicle Na+ uptake, measured by rapid filtration technique, was higher in SHR when compared to WKY (1.44 ± 0.03 vs. 1.01 ± 0.06 nmol/mg/5 sec, N = 4, N < 0.01). This increase in Na+ influx was apparent only in the present of an outward-directed proton (H+) gradient and was abolished by 1mM amiloride. BBM permeability to H+ as assessed by acridine orange quenching was not different between SHR and WKY. Kinetic analyses of the amiloride-sensitive BBM Na+ uptake revealed a higher Vmax (2.13 ± 0.27 vs. 0.70 ± 0.30 nmol/mg/5 sec, N = 4, P < 0.01) and a higher km for Na+ (3.55 ± 0.32 vs. 1.23 ± 0.14mM, N = 4, P < 0.05) in SHR. These findings thus demonstrate an intrinsic derangement in BBM Na+ transport in young SHR which is characterized by increased Na+/H+ antiport activity. This alteration in antiport activity is not attributable to changes in membrane permeability to H+, and is characterized by higher Vmax and km. Similar reports of increased Na+/H+ antiport activity in other tissues of SHR suggest that a generalized membrane transport disorder may exist in this model of genetic hypertension

Maintenance of Mouse Nephron Progenitor Cells in Aggregates with Gamma-Secretase Inhibitor.

Knowledge on how to maintain and expand nephron progenitor cells (NPC) in vitro is important to provide a potentially valuable source for kidney replacement therapies. In our present study, we examined the possibility of optimizing NPC maintenance in the "re-aggregate" system. We found that Six2-expressing (Six2(+))-NPC could be maintained in aggregates reconstituted with dispersed cells from E12.5 mouse embryonic kidneys for at least up to 21 days in culture. The maintenance of Six2(+)-NPC required the presence of ureteric bud cells. The number of Six2(+)-NPC increased by more than 20-fold at day 21, but plateaued after day 14. In an attempt to further sustain NPC proliferation by passage subculture, we found that the new (P1) aggregates reconstituted from the original (P0) aggregates failed to maintain NPC. However, based on the similarity between P1 aggregates and aggregates derived from E15.5 embryonic kidneys, we suspected that the differentiated NPC in P1 aggregates may interfere with NPC maintenance. In support of this notion, we found that preventing NPC differentiation by DAPT, a γ-secretase inhibitor that inhibits Notch signaling pathway, was effective to maintain and expand Six2(+)-NPC in P1 aggregates by up to 65-fold. The Six2(+)-NPC in P1 aggregates retained their potential to epithelialize upon exposure to Wnt signal. In conclusion, we demonstrated in our present study that the "re-aggregation" system can be useful for in vitro maintenance of NPC when combined with γ-secretase inhibitor

In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys

In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells.

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys

Maintenance of Mouse Nephron Progenitor Cells in Aggregates with Gamma-Secretase Inhibitor.

Knowledge on how to maintain and expand nephron progenitor cells (NPC) in vitro is important to provide a potentially valuable source for kidney replacement therapies. In our present study, we examined the possibility of optimizing NPC maintenance in the "re-aggregate" system. We found that Six2-expressing (Six2(+))-NPC could be maintained in aggregates reconstituted with dispersed cells from E12.5 mouse embryonic kidneys for at least up to 21 days in culture. The maintenance of Six2(+)-NPC required the presence of ureteric bud cells. The number of Six2(+)-NPC increased by more than 20-fold at day 21, but plateaued after day 14. In an attempt to further sustain NPC proliferation by passage subculture, we found that the new (P1) aggregates reconstituted from the original (P0) aggregates failed to maintain NPC. However, based on the similarity between P1 aggregates and aggregates derived from E15.5 embryonic kidneys, we suspected that the differentiated NPC in P1 aggregates may interfere with NPC maintenance. In support of this notion, we found that preventing NPC differentiation by DAPT, a γ-secretase inhibitor that inhibits Notch signaling pathway, was effective to maintain and expand Six2(+)-NPC in P1 aggregates by up to 65-fold. The Six2(+)-NPC in P1 aggregates retained their potential to epithelialize upon exposure to Wnt signal. In conclusion, we demonstrated in our present study that the "re-aggregation" system can be useful for in vitro maintenance of NPC when combined with γ-secretase inhibitor

Riboflavin transport by rabbit renal brush border membrane vesicles

AbstractThe present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent Kmof 25.7 ± 7.6 μM and Vmaxof 75.6 ± 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na+-dependent so that when Na+ was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na+-dependency was unlikely to be due to Na+-cotransport mechanism because RF uptake occurred without the characteristic ‘overshoot’ phenomenon as for other Na+-cotransport systems and the elimination of transmembrane Na+-gradient by preloading Na+ to the intravesicular space did not affect RF uptake. In contrast, removal of Na+ eliminated the binding component of RF uptake, suggesting the requirement of Na+ for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability (Cl− = NO3−SO4−gluconate−) or by using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K+ gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM

Controlled tubulogenesis from dispersed ureteric bud-derived cells using a micropatterned gel.

Developmental engineering is a potential option for neo-organogenesis of complex organs such as the kidney. The application of this principle requires the ability to construct a tubular structure from dispersed renal progenitor cells with defined size and geometry. In this present study we report the generation of tubular structures from dispersed ureteric bud cells in vitro by using a micropatterned gel. Dispersed CMUB-1 cells, a mouse ureteric bud-derived cell line, or mIMCD cells, a mouse collecting duct-derived cell line, were suspended in collagen I and seeded into an agarose-based micropatterned gel. We found that within 24-36 h of incubation, the cells developed a tubular structure that conformed to the geometry of the micropattern of the gel. The lumen formation of the tubular structure was confirmed by immunohistochemical staining and observed by confocal microscopy. We found that higher concentrations of collagen I negatively influenced the efficiency of tubular formation. Tubule formation in CMUB-1, but not mIMCD, cells was positively influenced by the addition of aldosterone (10, 50 and 200 µg/ml), FGF (50 and 100 µg/ml) and fibronectin (10 and 50 µg/ml) to the growth medium. We further demonstrated the functionality of the generated tubes by in vitro budding, which was induced by growth factors, such as glial cell-derived neurotrophic factor (GDNF) or fibroblast growth factor 7 (FGF7), in the presence of beads soaked with the activin A inhibitor follistatin. Our current study thus demonstrates the possibility of constructing a functional tubular structure from dispersed ureteric bud cells in vitro in a controlled manner. Copyright © 2014 John Wiley &amp; Sons, Ltd

Controlled tubulogenesis from dispersed ureteric bud-derived cells using a micropatterned gel.

Developmental engineering is a potential option for neo-organogenesis of complex organs such as the kidney. The application of this principle requires the ability to construct a tubular structure from dispersed renal progenitor cells with defined size and geometry. In this present study we report the generation of tubular structures from dispersed ureteric bud cells in vitro by using a micropatterned gel. Dispersed CMUB-1 cells, a mouse ureteric bud-derived cell line, or mIMCD cells, a mouse collecting duct-derived cell line, were suspended in collagen I and seeded into an agarose-based micropatterned gel. We found that within 24-36 h of incubation, the cells developed a tubular structure that conformed to the geometry of the micropattern of the gel. The lumen formation of the tubular structure was confirmed by immunohistochemical staining and observed by confocal microscopy. We found that higher concentrations of collagen I negatively influenced the efficiency of tubular formation. Tubule formation in CMUB-1, but not mIMCD, cells was positively influenced by the addition of aldosterone (10, 50 and 200 µg/ml), FGF (50 and 100 µg/ml) and fibronectin (10 and 50 µg/ml) to the growth medium. We further demonstrated the functionality of the generated tubes by in vitro budding, which was induced by growth factors, such as glial cell-derived neurotrophic factor (GDNF) or fibroblast growth factor 7 (FGF7), in the presence of beads soaked with the activin A inhibitor follistatin. Our current study thus demonstrates the possibility of constructing a functional tubular structure from dispersed ureteric bud cells in vitro in a controlled manner. Copyright © 2014 John Wiley &amp; Sons, Ltd

Developmental stage affects NPC maintenance and UB branching morphogenesis in aggregates.

<p><b>(A</b>) Aggregates were reconstituted from embryonic kidneys at different developmental stages, starting from E11.5 up to E15.5, and immuno-stained for Six2 (green) and DBA (red) after 7 days in culture. Aggregates from E11.5 (a,f,k) and 12.5 (b,g,l), showed similar characteristics in Six2⁺-NPC maintenance and UB branching morphogenesis. In contrast, aggregates from later developmental stages, i.e., at E13.5 (c,h,m), 14.5 (d,i,n) and 15.5 (e,j,o), showed less well maintenance of Six2⁺-NPC and failure of UB cells to reorganize into orderly branching structures. (Scale bar = 500 μm). <b>(B)</b> (a) A representative E12.5 aggregate after 7 days in culture was immuno-stained for NPC marker, Six2 (green), UB marker, DBA (red) and DNA (blue). Abundant Six2⁺ NPC were seen surrounding UB structures, with Six2^-/DBA^- cells in adjacent areas. (b) EdU (5-ethynyl-2’-deoxyuridine) staining of a representative E12.5 aggregate showed Six2⁺/EdU⁺ cells in MM areas that were both close to and distant from the UB structure (white arrow). In contrast, DBA⁺/EdU⁺ cells were detected mostly in UB tips surrounded by Six2⁺-NPC cells (white arrowhead). (c-e) A representative aggregate was immuno-stained for: (c) renal vesicle marker, Lef1 (green)/UB marker, DBA (red); (d) proximal tubule marker, LTL (red)/epithelial marker, pancytokeratin (blue)/NPC marker, Six2 (green); (e) podocyte marker, podocalyxin (green)/UB marker, DBA (red), and showed the development of Lef⁺-renal vesicle like structures (c), LTL⁺- (d) and podocalyxin⁺-(e) epithelial structures. (Scale bar = 200 μm)</p