Regulation of glomerulotubular balance. II. Impact of angiotensin II on flow-dependent transport

Abstract

Underlying glomerulotubular balance (GTB) is the impact of axial flow to regulate Na ϩ and HCO 3 Ϫ transport by modulating Na ϩ -H ϩ exchanger 3 (NHE3) and H-ATPase activity. It is not known whether the cascade of events following a change in flow relies on local angiotensin (ANG II) generation or receptor availability. Mouse tubules were microperfused in vitro at flows of 5 and 20 nl/min, and net fluid (Jv) and HCO 3 Ϫ (JHCO3) absorption and cell height were measured. Na ϩ (JNa) and Cl Ϫ (JCl) absorption and changes in microvillous torque were estimated. Raising flow increased Na ϩ and HCO 3 Ϫ reabsorption but did not change either Cl Ϫ transport or cell volume. Losartan reduced absolute Na ϩ and HCO 3 Ϫ absorption at both low and high flows but did not affect fractional flowstimulated transport. Compared with controls, in AT1a knockout (KO) mouse tubules, 53% of flow-stimulated Na ϩ absorption was abolished, but flow-stimulated HCO 3 Ϫ absorption was retained at similar levels. The remaining flow-stimulated JHCO3 was eliminated by the H-ATPase inhibitor bafilomycin. Inhibition of the AT 2 receptor by PD123319 increased both J Na and JHCO3 but did not affect flowmediated fractional changes. NHE3 expression at the protein level was reduced in AT 1a KO mice kidneys. We conclude that 1) although the AT1a receptor is necessary for flow to impact NHE3, the effect on H ϩ -ATPase is independent of AT1a; 2) the small flow-mediated changes in cell volume suggest a coordinate flow effect on both luminal and basolateral transporters; and 3) there is no evidence of flow-dependent Cl Ϫ transport, and thus no evidence for convective paracellular Cl Ϫ transport in mouse tubules.angiotensin II; kidney proximal tubule; glomerulotubular balance; AT 1a,; AT2 receptor; NHE3; H-ATPase GLOMERULOTUBULAR BALANCE (GTB) refers to the proportional variation of filtered load and fluid and electrolyte reabsorption by the proximal tubule; it derives in large part from flowdependent modulation of epithelial cell transport. We have previously studied this phenomenon by examining mouse proximal tubules in vitro, measuring volume and HCO 3 Ϫ absorption under different perfusion rates and have demonstrated that both Na/H exchanger 3 (NHE3) and H-ATPase activities are modulated by axial flow (10,12). This modulation requires an intact actin cytoskeleton (10), and, as initially proposed (18), the afferent signal is flow-dependent torque (bending moment at the apical membrane due to fluid flow) on brush border microvilli (10). These previous studies are summarized elsewhere (49). ANG II directly regulates electrolyte transport along the nephron and is a key antinatriuretic signal in the proximal tubule (24, 48). ANG II can be produced within the proximal tubule from the action of a local renin and angiotensin-converting enzyme on angiotensinogen, synthesized in the liver and filtered at the glomerulus (33). Knockout of the AT 1a receptor in the proximal tubule reduced sodium absorption and blood pressure, thus demonstrating an important role of ANG...

Regulation of glomerulotubular balance. III. Implication of cytosolic calcium in flow-dependent proximal tubule transport

“…We found that blocking the AT 1 receptor by losartan reduced absolute Na ϩ and HCO 3 Ϫ absorption at both low and high flows, but did not affect fractional flowstimulated transport. Compared with WT control, in AT 1a KO mouse tubules, 53% of flow-stimulated Na ϩ absorption was abolished, but flow-stimulated HCO 3 Ϫ absorption was retained at similar levels. The remaining flow-stimulated HCO 3 Ϫ absorption was eliminated by the H-ATPase inhibitor bafilomycin.…”

“…We also found that NHE3 expression at the protein level was reduced in AT 1a KO mice kidneys. These results indicate that the AT 1 receptor is important in maximizing the NHE activity activated by flow; however, it is not critical for the flow-stimulated HCO 3 Ϫ transport, which still exists when the inhibitors are present or when the AT 1a receptor is knocked out (3).…”

“…Regulatory mechanisms of the GTB, including the role of dopamine and ANG II receptors and the role of cAMP-PKA have been examined. From these studies we have demonstrated that dopamine completely blocks flowstimulated Na ϩ transport and partially abolishes flow-stimulated HCO 3 Ϫ absorption, and the remaining portion of the increment of HCO 3 Ϫ reabsorption (J HCO3 ) by flow is blocked by the H-ATPase inhibitor bafilomycin. The effects of dopamine were completely abolished by the dopamine DA1 receptor antagonist Sch 23390 (5).…”

“…With respect to Na ϩ reabsorption, high luminal Ca 2ϩ decreased transport at low flow, but preserved the flow-induced increase; low luminal Ca 2ϩ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect; thapsigargin decreased baseline flow, leaving the flow effect intact. With respect to HCO 3 Ϫ reabsorption, high luminal Ca 2ϩ decreased transport at low flow and mildly diminished the flow-induced increase; low luminal Ca 2ϩ had little impact; both BAPTA and 2-APB had no effect on baseline flux, but abrogated the flow effect. These data implicate IP3 receptor-mediated intracellular Ca 2ϩ signaling as a critical step in transduction of microvillous drag to modulate Na ϩ and HCO 3 Ϫ transport.…”

Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion

“…The cycling conditions for all genes were: preincubation at 95°C for 10 min, followed by 40 cycles at 94°C for 15 s and 60°C for 60 s. The final mRNA abundance of each gene was normalized to the abundance of an endogeneous gene, Cyclophilin A, using the DDC T method (2 ϪDDCT ). Fold-change in the gene expression was calculated (15). The primers used in this study were listed in Table 1.…”