Acid-base disturbances occur in a wide range of diseases, and lead to disturbances in arterial (a) and intracellular pH that can have devastating consequences for the patient. In response to such disturbances, the renal proximal tubule (PT)?which normally handles ~80% of the H+ secreted by the kidney?appropriately adjusts its rate of H+ secretion (JH). Previous work showed that the PT does not sense the pH on the basolateral (BL) side of the PT. Rather, PT JH markedly rises with increases in [CO2]BL or decreases in [HCO3?]BL. Nevertheless, mechanisms for sensing ?[CO2]BL and ?[HCO3?]BL and transducing these to ?JH are poorly understood. Important clues are that the basolateral CO2-evoked increase in JH requires that endogenously secreted ANG II bind to apical AT1A receptors, and is blocked by inhibitors that target a subset of receptor tyrosine kinases that include ErbB1 and ErbB2. New data show that the knockout (KO) of receptor protein tyrosine phosphatase ? (RPTP?), normally present in the PT basal membrane, eliminates the ?JH produced by ?[CO2]BL and ?[HCO?]BL, and markedly reduces the ability of the whole mouse to regulate pHa during metabolic acidosis (MAc). Curiously, the extracel- lular side of RPTP? has a region?the carbonic-anhydrase?like domain (CALD)?that is ~40% identical to clas- sical carbonic anhydrases (CAs). The three aims of this proposal are a multidisciplinary approach to address, at three levels of integration, how PTs senses ?[CO2]BL and ?[HCO3?]BL and transduce them into ?JH, and the role played by these processes in pHa regulation of pHa during whole-body MAc and respiratory acidosis (RAc): At the molecular level, we ask (1) what is the mechanism of RPTP?? How do ?[CO2] and ?[ HCO3?] control RPTP??s dimerization, which controls its phosphatase activity? Does RPTP? interact with ErbB1 and ErbB2? And does the CALD lack of CA activity, and if so, why? At the cellular level we ask (2) how do RPTP? and putative down- stream elements function in isolated PTs? We will use viral constructs, injected into kidneys of RPTP? ?/? mice, to determine the roles of RPTP??s CALD and phosphatase domains. Are ErbB1 and ErbB2 required to transduce ?[CO2]BL and ?[ HCO3?]BL signals to ?JH? Do [CO2]BL and [HCO3?]BL control JH by modulating ability of luminal ANG II to signal at or downstream to apical AT1A? At the whole-animal level, we ask (3) are RPTP? and downstream effectors (ErbB1, ErbB2, ACE, and AT1A) essential in the whole-body responses to MAc and RAc? The approach, using KO mice, is to impose MAc or RAc and assess arterial blood gases, urine chemistry, and targeted transcription and protein profiles. Our work will produce major insights into how RPTP? senses and transduces changes in acid-base status, how downstream elements function in PTs, and the impact for the whole animal. Because RPTP? is expressed in a broad range of cell types, is closely related to RPTP? (mainly in CNS astro- cytes), and because RPTP??s CALD is similar to three orphaned CAs, the impact of our work will extend across a diverse range of organ systems and cell types. It will provide important insights clinical problems that include acid-base disturbances and to diseases linked to RPTP?, which include schizophrenia and cancer.

Public Health Relevance

The body continuously generates H+ (acid) through metabolism. The proximal tubule (PT) cells of the kidney secrete most of this H+ into the fluid that is eventually excreted as urine. This process, which helps maintain the acid-base status of the body, increases under conditions of acidosis. However, PT cells do not sense H+ per se, but the ratio of CO2/HCO3? (the components of soda water) in the blood. Indeed, this ratio rises during acidosis. Preliminary work suggests that a protein known as receptor protein tyrosine phosphatase ? (RPTP?), which weaves through the cell membrane of PT cells that faces the blood, is the CO2/HCO3? sensor. Part of RPTP? is on the outside of the PT cell, facing the blood. Interestingly, this part of RPTP? has a region that resembles the well-studied enzyme carbonic anhydrase (CA). This is the CA-like domain (CALD). However, it appears that mutations that have occurred during evolution have destroyed RPTP??s enzymatic activity. Another part of RPTP?, on the inside of the PT cell, can remove phosphate groups from target proteins. This activity is important for sending signals among molecules within the PT cell. The goals of this project are: (1) To identify how the binding of CO2 and HCO3? to the CALD of RPTP? control RPTP??s ability to remove phosphate groups on target proteins. (2) To understand how RPTP? and its target proteins control the secretion of H+ by the living proximal tubules. And (3) to understand how RPTP?, and the proteins that it sends signals to, control the pH of the blood in living animals during acidosis. The proposed work could have important implications for clinical approaches to acidosis, which is common in a wide range of diseases; hypertension, schizophrenia, and cancer.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Kidney Molecular Biology and Genitourinary Organ Development (KMBD)
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Ketchum, Christian J
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Case Western Reserve University
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