Per K. Christensen

Expression, Localization, and Function of the Thioredoxin System in Diabetic Nephropathy

Excessive reactive oxygen species play a key role in the pathogenesis of diabetic nephropathy, but to what extent these result from increased generation, impaired antioxidant systems, or both is incompletely understood. Here, we report the expression, localization, and activity of the antioxidant thioredoxin and its endogenous inhibitor thioredoxin interacting protein (TxnIP) in vivo and in vitro. In normal human and rat kidneys, expression of TxnIP mRNA and protein was most abundant in the glomeruli and distal nephron (distal convoluted tubule and collecting ducts). In contrast, thioredoxin mRNA and protein localized to the renal cortex, particularly within the proximal tubules and to a lesser extent in the distal nephron. Induction of diabetes in rats increased expression of TxnIP but not thioredoxin mRNA. Kidneys from patients with diabetic nephropathy had significantly higher levels of TxnIP than control kidneys, but thioredoxin expression did not differ. In vitro, high glucose increased TxnIP expression in mesangial, NRK (proximal tubule), and MDCK (distal tubule/collecting duct) cells, and decreased the expression of thioredoxin in mesangial and MDCK cells. Knockdown of TxnIP with small interference RNA suggested that TxnIP mediates the glucose-induced impairment of thioredoxin activity. Knockdown of TxnIP also abrogated both glucose-induced 3H-proline incorporation (a marker of collagen production) and oxidative stress. Taken together, these findings suggest that impaired thiol reductive capacity contributes to the generation of reactive oxygen species in diabetes in a site- and cell-specific manner.

Monocyte Function in IDDM Patients and Healthy Individuals

Interleukin 1β(IL‐1β) and tumour necrosis factor alpha (TNF‐α) may be pathogenetically important in insulin‐dependent diabetes mellitus (IDDM), which is associated with genes of the HLA region. Since a regulatory role of HLA region genes on monokine production may exist, we looked for an association between the monokine and prostaglandin E2 (PGE2) responses of monocytes (Mo) from 20 healthy males (18–50 years) with HLA‐DR types relevant for IDDM susceptibility and resistance (DR 1,2, DR 1,3, DR 1,4, DR 3,4). Monokine assays were established and evaluated and the secretions of IL‐1β, TNF‐α, and PGE2 measured in Mo cultures (2 h, 6 h, 20 h) prepared by endotoxin‐free techniques and stimulated by low‐dose E. coli lipopolysaccharides (LPS). There were no significant associations between Mo responses and HLA‐DR phenotype. Likewise, Mo from DR2 (n=5) and DR4 (n= 5) homozygous healthy males demonstrated no significant differences in monokine and PGE2 responses of Mo.

Autoregulation of glomerular filtration rate during spironolactone treatment in hypertensive patients with type 1 diabetes: a randomized crossover trial

BACKGROUND Autoregulation of GFR, i.e. maintenance of relative constancy of GFR despite variations in mean arterial pressure (MAP) >80 mmHg, is impaired in diabetic kidney disease; furthermore, some antihypertensive drugs may jeopardize autoregulation. The aim of our study was to establish if spironolactone affects the ability to autoregulate GFR. METHODS Sixteen hypertensive type 1 diabetic patients with persistent normoalbuminuria (presumed normal autoregulation) completed this randomized, double-masked, crossover trial. After a 4-week wash-out period, patients received spironolactone 25 mg o.d. and matched placebo for 4 weeks in random order. After each treatment period, the ability to autoregulate GFR was determined by measuring GFR ((51)Cr-EDTA clearance) before (basal) and after acute blood pressure reduction by intravenous injection of clonidine. RESULTS During placebo, the mean (SE) basal GFR was 115 (5) ml/min/1.73 m(2) and the BP was 146 (4)/81 (2) mmHg corresponding to a MAP of 103 (2) mmHg. Spironolactone did not significantly reduce GFR or BP. Injection of clonidine induced a significant reduction in the MAP of 17 (2) and 19 (1) mmHg during placebo and spironolactone treatment, respectively, and an overall reduction in GFR of 11 and 15 ml/min/1.73 m(2) (both comparisons NS between treatment periods). Signs of impaired autoregulation were present in nine patients during placebo and in nine patients during spironolactone treatment. Relative changes in GFR on placebo treatment correlated with diabetes duration (R = 0.67, P < 0.01) but were not related to duration of hypertension, baseline BP, GFR, HbA1c or to changes in BP. CONCLUSION Spironolactone did not change the overall ability to autoregulate GFR in 16 hypertensive type 1 diabetic patients with normoalbuminuria. Our data are suggestive that the ability to autoregulate GFR is gradually impaired with increasing diabetes duration, a phenomenon not previously described in normoalbuminuric patients.

Autoregulation of Glomerular Filtration Rate in Patients with Diabetes

The close relationship between elevated blood pressure and diabetic nephropathy are documented both in Type 1 and Type 2 diabetic patients. Approximately 75–85% of diabetic patients with nephropathy are hypertensive [1–3].

The Thioredoxin System in Diabetic Nephropathy

32 Hyperhexosemia-Induced Vasoactive Factors and Extracellular Matrix Protein Production in Kidneys are Mediated via NFB. BRADLEY J. DE SOUZA, JANE CHIU, BIAO FENG, BIJU GEORGE AND SUBRATA CHAKRABARTI, Department of Pathology, University of Western Ontario, London, ON Increase in reactive oxygen species production, in diabetes, is critical in the initiation of the cellular responses by activating redox-sensitive transcription factor, nuclear factor kappa B (NFB). NFB activation may lead to cellular structural and functional alteration via altered expression of cytokines. We investigated the role of NFB in the development of early changes in diabetic nephropathy We used a hyperhexosemic, normoinsulinimic model of chronic diabetic complication to avoid potential problems associated with systemic insulin therapy needed for longterm diabetic animals. Hence, NFB(p50) knockout mice and their wild-type controls were fed with a 30% galactose diet for 2 months. Renal tissues were analyzed. Galactose-fed animals showed increased in serum reducing sugars, without any alterations of body weight. Wild type galactose-fed animals demonstrated increase in the mRNA expression of ET-1 and TGF1. Furthermore ECM proteins, regulated by ET-1 and TGF 1, such as fibronectin and collagen1 4 were upregulated. Interestingly, poly ADP ribose polymerase mRNA expression was not altered. Such alterations were prevented in the kidneys of galactose-fed knockout animals. These results demonstrate that early changes in diabetic nephropathy may be mediated through NFB-dependent signaling pathways. Understanding these mechanisms is important in identifying potential novel treatment targets. Supported by CDA