Na+/K+-ATPase stabilization by HSP70 in Outer Stripe of outer medulla (OSOM) during recovery from low protein (LP) diet.

CELESTE RUETE; LILIANA CARRIZO; VICTORIA BOCANEGRA; WALTER MANUCHA; PATRICIA G. VALLES

Budapest Hungría

Congreso; 14th Congress of International Pediatric Nephrology Association; 2007

International Pediatric Nephrology Association

Renal ischemia induces the rapid, duration-dependent relocation of apical and basolateral membrane proteins into the alternate domain (Fish, EM et al. Eng J Med 330:1580-1588, 1994). One of the most physiologically important transporters is the Na+/K+-ATPase, which is crucial for the absorptive, secretory, and concentrating capacity in the kidney (Doucet, A. Kidney Int 37: S118-124, 1992). For Na+/K+-ATPase to be translocated to the apical domain, it must first be detached from its cytoskeletal anchorage, which has been defined functionally by detergent extractability (Molitoris, BA et al. J Clin Inv 88: 462-469, 1992). Recent research suggests that renal ischemia rapidly induces the elaboration of 70-kDa Heat Shock Protein (HSP70) involved in the restoration of the cytoskeletal anchorage of Na+/K+-ATPase (Riordan M et al. Am J Physiol Renal Physiol 288: F1236-1242, 2005). HSP70 is known to bind to nascent and immature proteins to prevent premature and improper binding and folding. In low protein feeding (LP) renal ischemic injury events include hypoxia and ATP depletion on epithelial cells from duct segments (Vallés, P et al. Nephron Physiol 99: 90-100, 2005). Cells in the outer medulla suffer more extreme oxygen deprivation than cells in the cortex. Previously, we provided evidence for the apoptosis induction in epithelial cells from medullary collecting duct segments in LP and for the antiapoptotic, cytoprotective mechanism of HSP70 during protein recovery (Carrizo, L et al. Cell stress chaperones 11: 309-324, 2006). In the present study we tested whether HSP70 would stabilize renal Na+/K+-ATPase attachment to the cytoskeleton during recovery from LP feeding. To test whether HSP70 would stabilize renal Na+/K+-ATPase attachment to the cytoskeleton during recovery from low protein feeding. After weaning, Wistar rats (n=8), were fed for 14 days with a LP diet (8%), then the animals were recovered by means of a normal protein diet (24%,RP), each group had an age-matched control group (24%, NP). Rats were anesthetized and nefrectomized. The kidneys were perfused through the abdominal aorta with ice-cold phosphate buffered saline (PBS). Cortex  and outer strip of the outer medulla from all groups were isolated and homogenized in chilled extraction buffer containing 0.1% Triton X-100. The homogenate was centrifuged within the 10 minutes at 35,000g for 10 minutes at 4ºC to separate the Triton-soluble supernatant (non cytoskeletal) protein fraction from the Triton-insoluble pelleted fraction (cytoskeletal). The pellets were resuspended in extraction buffer of half the volume of the original homogenate, resulting in similar protein concentration as in supernatants. In the first set of experiments aliquots from NP and LP Triton X-100 insoluble (cytoskeletal pellets) were incubated in their own NP (NPs/NPi) or LP (LPs/LPi) Triton X-100 soluble (non cytoskeletal supernatants), respectively. At the same time, aliquots from LP pellets were incubated in NP supernatants (NPs/LPi). The second set of experiment consisted of parallel incubation of aliquots from the same LP pellet in both LP (LPs/LPi) and RP (RPs/LPi) supernatants, and aliquots from RP pellet were incubated in RP supernatant (RPs/RPi). To asses the translocation of HSP70 into the injured cytoskeletal fraction, incubation of LP pellet in RP supernatant (RPs/LPi) and of NP pellet in RP supernatant (RPs/NPi) were conducted. The therd set of experiment consisted in parallel incubation of 50 µl of LPi incubated in both 50 µl of RPs and 50 µl of RPs plus 25 µg of anti-HSP70 antibody (Sigma). This mixture was resuspended and incubated for 20 minutes at room temperature. Differential centrifugation was then repeated at 35000 x g for 15 minutes at 4ºC. Repelleted cytoskeletal fractions and dissociated supernatant fractions were saved for further analysis. In vitro coincubation of soluble non cytoskeletal and insoluble isolated cytoskeletal-associated fractions in the presence or absence of anti HSP 70 antibody was performed. Protein levels of Na+/K+-ATPase and HSP70 were measured by western blot. Incubated overnight in the primary antibodies against HSP70 (dilution 1:3,000) and the alpha-subunit of Na+/K+-ATPase (1:1,000), Sigma and Chemicon respectively. Detection was accomplished with secondary antibodies (DAKO) and detected with enhanced chemiluminescence system (ECL, Amersham) and exposure to x-ray film (Amersham). Interaction between de HSP70 y Na+/K+-ATPase was determined by coimmunoprecipitation. RESULTS Cytoskeletal dissociated Na+/K+-ATPase was demonstrated through the increased OSOM soluble fraction from LP compared to NP.         Meanwhile, decreased HSP70 levels in the same fraction was shown (Figure 2: NP: 100±4.6, LP: 79.84±4.2, RP: 98.63±5.3, p< 0.05). In renal cortex, absence of significant differences was shown in the Na+/K+-ATPase and HSP70 expression at among groups. Coimmunoprecipitation showed that the amount of HSP70 coprecipitating with Na+/K+-ATPase increased in membrane OSOM from LP, the level of HSP70 that coprecipitated with Na+/K+-ATPase in RP was similar to that seen in control. In LP and RP cortex tissues, interaction of both, Na+/K+-ATPase and HSP70, proteins by coimmunoprecipitation was similar to control. Translocation of HSP70 to the cytoeskeletal injured fraction associated with stabilization of Na+/K+-ATPase was shown in OSOM from LP, after in vitro coincubation of the cytoeskeletal fraction of LP and non cytoeskeletal fraction of RP. These effects were abolished by the addition of anti-HSP70 antibody in RPs/LPi. Aliquots from NPi and Lpi Triton X-100 insoluble (cytoskeletal pellets) were incubated in their own NPs (NPs/NPi) or LPs (Lps/LPi) Triton X-100 soluble (non cytoskeletal supernatants), respectively. At the same time, aliquots from LP pellets were incubated in NP supernatants (LPi/NPs). Repeat Triton X-100 extraction resulted in a significant increase in Triton extractability of Na,K-ATPase from the low protein cytoskeletal fraction after the incubation with LPs vs. NPs. To assess the in vitro repair, aliquots from the same LPi pellet were incubated in both LPs (LPi/LPs) and RPs (LPi/RPs) supernatants, and aliquots from RPi pellet were incubated in RPs supernatant (RPi/RPs). Incubation of LPs in RPs resulted in less Na-K ATPase levels in soluble fraction during the repeat Triton X-100 extraction. The HSP70 translocation into the injured cytoskeletal fraction was also evaluated through out the in vitro incubation of LPi in RPs (RPs/LPi) resulting in the appearance of a major signal of HSP70 triton-insoluble than in the incubation of NPi in RPs (NPi/ RPs). CONCLUSION: Our results allow us to suggest that HSP70 has a critical protective role in the integrity of the cytoskeletal anchorage of Na+/K+-ATPase during recovery from ischemic LP injury in outer stripe of the outer medulla.