Identification of a renal-specific oxido-reductase in newborn diabetic mice

Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is wi dely distributed in mammalian tissues and has been implicated in complicati ons of diabetes, including diabetic nephropathy. To identify a renal-specif ic reductase belonging to the AKR family, representational difference analy ses of cDNA from diabetic mouse kidney were performed. A full-length cDNA w ith an ORF of 855 nt and yielding a approximate to 1.5-kb mRNA transcript w as isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had approximate to 91% and approximate to 97% amino aci d identity with mouse protein. In vitro translation of the cDNA yielded a p rotein product of approximate to 33 kDa, Northern and Western blot analyses , using the cDNA and antirecombinant protein antibody, revealed its express ion exclusively confined to the kidney. Like ALR2, the expression was up-re gulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm-Horsfa ll protein nor aquaporin-2, The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members wher e it is confined to the C terminus. Fluorescence quenching and reactive blu e agarose chromatography studies revealed that it binds to NADPH with high affinity (K-dNADPH = 66.9 +/- 2.3 nM). This binding domain is a tetrapeptid e (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the o ther AKR members. The identified protein is designated as RSOR because it i s renal-specific with properties of an oxido-reductase, and like ALR2 it ma y be relevant in the renal complications of diabetes mellitus.