Patients with uremia often have signs of protein malnutrition including decreased muscle mass. The long-term goal is to identify cellular mechanisms causing protein malnutrition so new therapies can be designed. In rats with chronic renal failure (CRF), the investigators found that acidosis stimulates muscle protein degradation and increases nitrogen excretion. In CRF patients, acidosis also accelerates proteolysis, pointing to clinical relevance of the experimental results. In rats, the mechanism involves ATP-dependent proteolysis in muscle; the investigators believe the proteolytic pathway is the ubiquitin-proteasome system because mRNAs encoding components of the pathway are high in muscle. The higher mRNAs were found to be related to increased transcription and the mechanism requires glucocorticoids. The proposed studies will document that the ubiquitin-proteasome system is involved in muscle using Western blot and enzyme activity measurements in rats with acidosis, uremia and the nephrotic syndrome (which causes a net decrease in protein intake). Surprisingly, muscle cell pH (measured by NMR) in rats with CRF or acidosis is not subnormal. This prompted a search for stimuli increasing transcription of genes encoding these mRNAs. Information about the promoter of the human C3 proteasome subunit reveals candidate transcription factors; the investigators have cloned part of the rat promoter to determine if these sequences are there. Second, catabolic illnesses with high levels of the same mRNAs in muscle (e.g., sepsis, cancer, etc) are associated with activation of TNF and NF-KB. Preliminary results reveal activation of NF-KB in muscle of CRF rats and evidence for activation of protein kinase pathways that could stimulate transcription factors. It is planned to clone the rat C3 promoter and test it functionally in cultured myocytes using a reporter gene to determine critical promoter regions. The importance of signal pathways that could stimulate transcription factors will be tested in myocytes and in rat muscle. Results from these experiment could provide critical information about stimulation of protein catabolism in uremia and other catabolic conditions associated with loss of muscle.
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