MECHANISMS OF RENAL ADAPTATION TO ANOXIA

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The broad objective of this project is an advanced understanding of molecular processes by which the immature nephron is protected from both oxygen deprivation and ischemic insults. Two classes of genes which are induced by ischemia or hypoxia have been shown to be over expressed constitutively in the immature kidney, the heat shock response (HSR) and the hypoxia responsive pathway (HRP). Each of these ubiquitous biological systems may play a fundamental role in the innate tolerance to injury manifested by the immature nephron. Specific Aim 1 will evaluate the HSR by determining if down-regulation of either constitutive activation of Heat Shock Transcription Factor (HSF) or diminution of HSP72 and/or HSP25 expression will impair tolerance to injury in immature nephrons. Specific Aim 2 will investigate HRP by delineating effects of inhibition of Hypoxia Inducible Factor (HIF-1) activity and synthesis of hemoxygenase and erythropoitin following hypoxic or ischemic insults in immature kidneys. Both specific aims will test a common hypothesis: if tolerance of the immature nephron is dependent on the activity of transcription factors and/or abundance of proteins which are constitutively over-expressed compared to the mature kidney, then dampening of transcription factor activity and/or inhibition of synthesis of specific proteins will increase the vulnerability of the immature nephron to a hypoxic or ischemic injury. The proposed studies utilize new techniques for gene silencing which allow a) global dampening of families of genes controlled by a common transcription factor (oligonucleotide decoys) and b) specific inhibition of synthesis of selected proteins (short interference RNA, siRNA). To ensure the feasibility of the experimental design, transcription factor decoys for HSF and HIF-1 have been developed and validated in LLCPK cells, as well as suspensions and cultures of immature proximal tubules. In addition, two target sequences for HSP72 siRNA have been identified and shown to specifically inhibit synthesis of HSP72 but not HSP25 in LLCPK cells after ATP depletion. The developing kidney, in which cytoprotective proteins are up-regulated without prior stress, represents an ideal and somewhat unique circumstance in which to delineate molecular mechanisms and pathobiologic processes which are fundamental to cellular injury in the mature kidney. Moreover, an in-depth understanding in the immature kidney will be important so that therapeutic interventions which might offset the protective effects can be avoided in care of infants with acute renal failure.