Gene Expression and Organ Damage in Sickle Transgenic Mouse Kidneys Exposed to Progressive Hypoxia.

We have further examined the hypoxia-induced gene expression in the sickle transgenic mouse model with high expression of human βS and α globin that is homozygous for the mouse β-major deletion, NY1DD (Fabry et al, PNAS 89:12159, 1992) exposed to hypoxia (8% O2/92% N2) for 2 or 4 days vs normoxic conditions. Gene expression in the kidney was studied using cDNA expression microarrays, analyzed by GenePix software and the Significance Analysis of Microarrays (SAM) program, and validated by real-time PCR and immunocytochemistry. Data analyzed by hierarchical clustering (that segregates genes expressed in the hypoxic kidneys vs the normoxic kidney) and SOTA (self-organizing tree algorithm) revealed the effect of hypoxia on different clusters of genes. In one cluster, the effect of hypoxia on heme oxygenase-1 (HO-1), was particularly striking. HO-1 is slightly downregulated in normoxic kidneys and becomes up-regulated 2 and 4 fold on days 2 and 4 of hypoxia. HO-1 was also observed upon immunocytochemical staining with HO-1 antibodies (Calbiochem) in kidney sections from 4 kidneys harvested from 2 mice exposed to either 2 or 4 days hypoxia (4 mice total) and 2 kidneys each from a C57BL and a NY1DD mouse harvested under room air. Mice sacrificed under room air had negligible staining for HO-1. However, NY1DD mice maintained under hypoxia for 2 days had moderate HO-1 staining in some of the proximal tubules near the surface of the cortex and sickled cells in the vasculature. NY1DD mice exposed to 4 days of hypoxia had more intense staining for HO-1 in the proximal tubules that extend deeper towards the medulla. Histological examination of kidney sections confirmed red cell congestion and sickling predominantly in medullary vessels where hypoxia is maximal. HO-1 , the rate-limiting enzyme in the heme degradation pathway, is inducible in the kidney (Pimstone et al, J Clin Invest 50:2042, 1971) and increased in sickle mouse kidneys (Nath et al, Am J Path 158:893, 2001). HO-1 also protects cells from oxidant damage. Other very up-regulated genes include myosin Vb (involved in membrane remodeling) and lipocalin 2 (present in distal tubules/collecting ducts and modulates the cellular response to injury). Genes that were down-regulated include calbindin 28K (a cytosolic vitamin D dependent calcium binding protein that regulates calcium homeostasis potentially disrupted by hypoxia), metallothionein 2 (a zinc binding protein and oxidant scavenger that blocks the most reactive oxidant species including peroxynitrite and superoxide radical), fructose bisphosphatase (a gluconeogenic enzyme located in the proximal nephron and a marker for glomerular damage) and the sodium/calcium exchanger (downregulated in renal ischemia/reperfusion injury as a mechanism to protect the cell from unregulated calcium influx). Identification of genetic modifiers of renal pathophysiology in sickle cell anemia is the first step in understanding the clinical heterogeneity among SS patients. These studies may also reveal novel metabolic pathways in the SS kidney and may, therefore, hold potential for therapeutic intervention.