The protective role of Sirt1 in renal damage was investigated. by Sirt1-mediated epigenetic regulation in podocytes contributed to albuminuria. These phenomena were not observed in 5/6 nephrectomized mice. We also exhibited retrograde Pneumocandin B0 interplay from PT to glomeruli using nicotinamide mononucleotide (NMN) from conditioned medium measurement of the auto-fluorescence of photoactivatable NMN and injection of fluorescence-labeled NMN. In human subjects with diabetes Sirt1 and Claudin-1 levels were correlated with proteinuria level. Sirt1 in PT protects against albuminuria in diabetes through maintaining NMN concentrations around glomeruli and controlling podocyte function. INTRODUCTION Diabetic nephropathy is the most common cause of end-stage renal disease affecting about one-third of subjects with diabetes mellitus1. Early diabetic nephropathy is usually characterized by mesangial hypertrophy and glomerular hyperfiltration with microalbuminuria. Sir2 is usually a nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase and a member of the sirtuin class of proteins. Sirt1 a mammalian ortholog of Sir2 deacetylates histones and various transcription factors protecting against acute and chronic stress2 3 Sirt1 mitigates diabetes by attenuating hepatic insulin resistance4 5 and enhancing pancreatic insulin secretion6. Recently the renal protective effects of Sirt17 8 and a pathogenic role for Sirt1 in diabetic nephropathy9-12 have been reported although the relationship between renal Sirt1 and the pathogenesis of kidney damage in diabetes have not been investigated. Our group recently produced transgenic (TG) mice overexpressing Sirt1 specifically in the proximal tubules (PT) and reported that Sirt1 alleviated acute kidney injury13 14 In diabetic nephropathy PT changes are evident even in the early stages15. PT changes are reportedly closely linked to the loss of renal function and more accurately predict the progression of diabetic nephropathy than glomerular changes15. Therefore our TG mice are good models for Pneumocandin B0 exploring the PT-specific role of Sirt1 in diabetic nephropathy. We used these mice as well as PT-specific Sirt1 knock-out mice to investigate the protective role of Sirt1 in diabetes-induced albuminuria. RESULTS Sirt1 in diabetes and effects of Sirt1 overexpression Downregulation of Sirt1 expression in a diabetic milieu or high glucose condition Rabbit polyclonal to AMHR2. has been reported in various cells16 17 We examined Sirt1 expression in PT and glomeruli after streptozotocin (STZ) treatment. By immunostaining we detected Sirt1 expression in both PT and glomeruli before STZ treatment. Eight weeks after STZ treatment Sirt1 levels were decreased in PTs but remained unchanged in glomeruli. Twenty-four weeks after STZ treatment Sirt1 levels in glomeruli were also decreased (Fig. 1a). Laser micro-dissection followed by RT-PCR revealed that mRNA expression decreased in PT before glomeruli in diabetic mice (Fig. 1b Supplementary Fig. 1a b). These observations indicated that molecular alterations in PT occurred at a very early stage in diabetes before the increase in albuminuria as documented in several previous reports18-20. To delineate the significance of this switch in PT we examined the effects of Sirt1 overexpression in PT-specific transgenic (TG) mice. First we confirmed using an antibody to FLAG that TG mice overexpressed Sirt1 in PT but not in glomeruli (Supplementary Fig. 1c). Immunoblotting immunostaining and laser micro-dissection followed by RT-PCR all showed that the reduction in Sirt1 expression Pneumocandin B0 in wild-type (WT) mice at 24 weeks after STZ treatment was prevented in TG mice in PTs and glomeruli (Fig. 1c and Supplementary Fig. 1a b). Plasma glucose concentrations increased in STZ-treated diabetic mice 4 weeks after treatment in both WT and TG mice and the increase was sustained until 24 weeks. We sacrificed mice 24 weeks after STZ treatment Supplementary Fig. 1d). Plasma glucose concentrations did not differ between WT and TG mice at either 8 or 24 weeks (Supplementary Fig. 1d). Body weights blood urea nitrogen and creatinine concentrations creatinine clearance and kidney weights did not differ between WT and TG mice Supplementary Fig. 1e-i). Eight weeks after STZ treatment urinary albumin excretion was unchanged (Fig. 1d) although Pneumocandin B0 24 weeks after treatment urinary albumin excretion was.