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Nephrology

Research

Important research focuses on the regulation of NHE3, a Na⁺/H⁺ exchanger found on the apical membrane of proximal tubule cells. NHE3 plays a fundamental role in volume and acid-base homeostasis by mediating the majority of sodium and bicarbonate reabsorption by the proximal tubule of the kidney. Tight regulation of NHE3-mediated sodium bicarbonate reabsorption is critical for the maintenance of normal volume and acid-base status under widely varying conditions. Many physiologic factors are known to affect NHE3 activity including dopamine, angiotensin II, chronic acidosis and parathyroid hormone. On a molecular and cellular level, regulation of NHE3 can occur via multiple mechanisms including intracellular protein trafficking, interaction with accessory regulatory proteins, and direct protein phosphorylation. To enable the study of endogenous NHE3 phosphorylation and its relationship to the other cellular forms of regulation, we have developed phosphospecific antibodies to two different PKA phosphorylation sites within the C-terminus of NHE3, serines 552 and 605. Using these antibodies, we are able to use techniques such as Western blotting, immunofluorescence, and immunoprecipitation to evaluate changes in NHE3 phosphorylation in response to various stimuli such as dopamine and acidosis, alterations in NHE3 trafficking in response to phosphorylation, and to elucidate specific interactions between phosphorylated NHE3 and regulatory proteins.

Another laboratory has documented that over expression of heat shock proteins may be of critical importance in renal epithelia which are depleted of ATP. Recent studies have demonstrated that over expression of HSP-27 has a specific cellular effect in porcine kidney cells which have been energy deprived. This small heat shock protein interacts with actin and stabilizes the cytoskeleton in injured cells. The result of this interaction which was demonstrated by fluorescent energy transfer techniques is stabilization of the domain specific localization of Na/K-ATPase. These studies represent the unique direct observation of interactions between a small heat shock protein, actin and mechanisms of cellular disruption.

A similar relationship between the constitutive overexpression of HSP-70 and protection of the lateralization of Na/K-ATPase has been demonstrated in immature nephrons. The kidneys from postnatal day 10 have been shown to have a marked increase in HSP-70 and to have diminished attachment of Na/K-ATPase from the cytoskeleton after injury when compared to adult kidneys. Taken together, these observations suggest that HSP-70 and HSP-27 may have important synergistic interactions with the cytoskeletal related proteins and play an important role in sublethally injured cells and the processes of recovery from ischemia or ATP depletion.

Newly developed techniques for gene silencing (decoy and SiRNA) have been used to investigate the effects of dampening the HSP response to ATP-depletion. Long-term studies continue on the risk of cardiovascular disease in adults who have had childhood nephrotic syndrome.

The Norman Siegel Pediatric Renal Career Enhancement Fund