S. McLennan

Decreased matrix degradation in diabetic nephropathy: effects of ACE inhibition on the expression and activities of matrix metalloproteinases.

Aims/hypothesisExtracellular matrix accumulation is thought to be involved in the pathogenesis of diabetic nephropathy. Increased matrix synthesis has been well documented but the effects of diabetes on degradative pathways, particularly in the in vivo setting, have not been fully explored. Furthermore, the effect of renoprotective therapies on matrix accumulation through these pathways has not been examined. We investigated the degradative pathway of type IV collagen and the effects of ACE inhibition in experimental diabetic nephropathy. MethodsDiabetes was induced in 16 rats by administrating streptozocin; 8 of the diabetic rats were allocated at random to receive the ACE inhibitor perindopril (2 mg/l) in their drinking water and 8 age and weight matched rats served as controls. Gene expression of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) was measured by RT-PCR and type IV collagen content by immunohistochemistry. MMP activities were determined by degradation of a radiolabelled substrate and by zymography. ResultsSix months of diabetes was associated with a decrease in mRNA and enzymatic activity of MMP-9 (21 % and 51 % respectively, p < 0.05 vs control) and a 51 % increase in TIMP-1 mRNA (p < 0.05 vs control). By contrast, MMP-2 mRNA was increased but its activity decreased (43 % and 43 % respectively, p < 0.05 vs control). Total degradative capacity of kidney tissue from diabetic rats was also lower (Control: 48 ± 7 %, Diabetic: 33 ± 6 %, p < 0.05). Activation of latent MMPs with amino-phenylmercuric acetate increased matrix degradation by two-fold. However the relative decrease associated with experimental diabetes still remained. All diabetes-associated changes in MMP and TIMP mRNA and activities were attenuated by perindopril treatment in association with reduced type IV collagen accumulation. Conclusions/interpretationThese results indicate that the impairment of matrix degradation contributes to matrix accumulation in diabetic nephropathy and that the beneficial effects of ACE inhibition could in part be mediated by modulation of changes in matrix degradative pathways. [Diabetologia (2002) 45: 268–275]

Abnormalities of ascorbic acid metabolism and diabetic control: differences between diabetic patients and diabetic rats.

Ascorbic acid is required in the synthesis of collagen and is also an important anti-oxidant. In a previous study, plasma ascorbic acid concentration was found to be decreased in diabetic patients but there was no relationship with blood glucose level. In the current study of diabetic patients, both plasma ascorbic acid and its urinary excretion correlated inversely with glycosylated hemoglobin level. Plasma ascorbic acid was also lower in diabetic rats but urinary ascorbic acid was elevated. The divergent trend in urinary ascorbic acid excretion observed in diabetic patients and diabetic rats may be due to difference in the ability of these two species to synthesize ascorbic acid. Difference in renal reabsorption of ascorbic acid may also be a relevant factor. The lower plasma and urinary ascorbic acid levels in diabetic patients with more severe hyperglycaemia indicates that this group of patients is particularly at risk of developing deficiency of this vitamin. As ascorbic acid has many important functions in the body, it may be necessary to supplement this vitamin in patients with chronically poorly controlled diabetes.

Functional and structural abnormalities in the nerves of Type I diabetic baboons: aminoguanidine treatment does not improve nerve function

Aims/hypothesis. To improve understanding of the pathophysiology of diabetic neuropathy and to establish a primate model for experimental studies, we examined nerve changes in baboons with Type I (insulin-dependent) diabetes mellitus. We also examined the effect of aminoguanidine (an inhibitor of the formation of advanced glycation end products) on nerve function.¶Methods. Male baboons (Papio hamadryas) were assigned to four groups; control, diabetic, control and diabetic treated with aminoguanidine. Diabetes was induced with streptozotocin (60 mg/kg, intravenous). Insulin and aminoguanidine (10 mg/kg) were injected subcutaneously daily. Motor and sensory nerve conduction velocity was measured using standard techniques. Autonomic function was examined by measuring heart rate response to positional change. Sural nerve morphometry was analysed in the diabetic group (mean duration 5.5 years) along with their age-matched controls.¶Results. The diabetic groups were smaller in size with a mean HbA1 c of 8.9 ± 1.2 %. The nerve conduction velocity and heart rate response was reduced in the diabetic groups. Morphometric analysis of the diabetic sural nerve showed smaller axon diameter (2.99 ± 0.06 μm vs 3.29 ± 0.06 μm; p < 0.01) accompanied by thinner myelin (1.02 ± 0.02 μm vs 1.15 ± 0.02 μm, p < 0.01) with no change in the axon density. Treatment with aminoguanidine for 3 years had no effect on glycaemic control and did not restore conduction velocity or autonomic dysfunction in the diabetic animals, contrary to the studies in rats.¶Conclusions/interpretation. These results show that the primate is a good model to study diabetic neuropathy and suggest that the accumulation of advanced glycation end products are not an early mechanism of nerve damage in this disorder. [Diabetologia (2000) 43: 110–116]

Urinary Glycosaminoglycan Excretion in NIDDM Subjects: Its Relationship to Albuminura

Nephropathy is a serious microvascular complication of diabetes mellitus which is preceded by a period of microalbuminura. Increased loss of proteoglycan (PG) from glomerular basement (GBM) has been postulated to alter glomerular charge selectivity which contributes to urinary loss of albumin. In this study we measured the excretion of urinary glycosaminoglycans (GAG), the degradation products of PG, in 82 non‐insulin‐dependent (NIDDM) (Type 2) diabetic and 34 non‐diabetic subjects. We found that diabetic subjects had a significantly higher GAG urinary excretion rate compared to non‐diabetic subjects (12.54 ± 5.67 vs 8.80 ± 3.99 μg glucuronic acid min−1, p = 0.0001). Categorizing for albuminuric status shows that the diabetic normo‐, micro‐ and macroalbuminuric groups have a higher GAG excretion rate than non‐diabetic subjects. Heparan sulphate (HS) GAG urinary excretion was measured in 25 samples from diabetic subjects and 18 non‐diabetic subjects. Diabetic subjects excreted more HS GAG than controls both as a rate or as a percentage of total GAG (3.70 ± 1.94 vs 2.38 ± 1.48 μg glucosamine min−1, p = 0.02; 31.6 % ± 12.5 vs 23.1 % ± 10.4, p = 0.02). Categorizing for albuminuric status shows that micro‐ and macro‐albuminuric groups have a significantly higher HS GAG excretion rate than non‐diabetic subjects. We conclude that, as in IDDM, excretion of GAG and HS GAG is higher in NIDDM and may precede the development of microalbuminuria.

Non-enzymatic Glycosylation of Tissue Protein in Diabetes in the Rat

SummaryNon-enzymatic glycosylation of tissue and haemolysate proteins has been studied in normal and diabetic rats by reduction with tritiated sodium borohydride (NaB3H4) alone or in combination with chromatography on m-aminophenyl-boronic acid coupled to Biogel P-6. With NaB3H4 reduction alone, there was a linear relationship between plasma glucose and tritium incorporation into haemolysate protein. However, increased non-enzymatic glycosylation of tissue protein could not be demonstrated with NaB3H4 reduction alone. Tritiated glycosylated amino acids could be selectively removed by maminophenylboronic acid immobilized on Biogel P-6, then eluted by acidification and the radioactivity in the acidic peak used to estimate non-enzymatic glycosylation. Using the combined techniques, an increase in non-enzymatic glycosylation was observed in heart, kidney and liver obtained from rats with diabetes of 18 weeks duration.

The effects of aminoguanidine on renal changes in a baboon model of Type 1 diabetes

BACKGROUND The efficacy of aminoguanidine (AG) on primary prevention of diabetic nephropathy was investigated in a nonhuman primate model of Type 1 diabetes over a period of 4 years. METHODS Adolescent male baboons (Papio hamadryas) were assigned to four groups: control, diabetic, and control and diabetic treated with AG. Diabetes was induced with streptozocin (60 mg/kg) and treated with insulin to maintain a mean HbA1c level of about 9%. AG was given subcutaneously (10 mg/kg) each day. All animals had annual renal biopsies and 24-h urine collections for measurements of glomerular basement membrane (GBM) thickness, fractional mesangium volume (FMV), albumin excretion rate (AER), and creatinine clearance. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were also determined. RESULT The diabetic animals had increased GBM after 2 years of diabetes, but there was no increase in FMV over the study period. AG prevented the thickening of GBM at the 3- and 4-year time points. AG and diabetes synergistically increased the GFR. All diabetic animals developed increased albuminuria during the study although lower than the conventionally accepted microalbuminuria range. AG was not able to prevent this and, in fact, led to the nondiabetic animals also developing albuminuria. CONCLUSION This is the first study to investigate the early use of AG in ameliorating renal damage in a primate model of Type 1 diabetes. The structural and functional changes in the kidney of these animals resemble those seen in the early stages of the human disease. AG was able to significantly reduce the thickening of GBM due to diabetes. This may suggest a potential role for this in primary prevention of diabetic nephropathy in the future.

Reduction of leucocyte proteolytic enzyme activity in diabetic patients with microalbuminuria and proteinuria : its possible role in diabetic nephropathy

Mesangium enlargement and glomerular basement membrane thickening are cardinal features of diabetic nephropathy. The reasons for these changes are uncertain but decreased degradation of extracellular matrix may play a role. Mesangium degradation can be modulated by factors intrinsic to the kidney or by factors in the circulation. In this study the capacity of leucocyte proteolytic enzymes to degrade mesangium matrix materials was investigated. Leucocytes were obtained from 57 patients with NIDDM (age 58.3 ± 8.8 years, duration 9.4 ± 7.3 years, body mass index (BMI) 30 ± 6 kg m−2, HbA1c 7.7 ± 2.0 %) and 21 control subjects (age 55.1 ± 14.6 years, BMI 25 ± 4 kg m−2). Leucocyte lysates from control and NIDDM subjects with normal AER degraded matrix to the same extent (40.6 ± 8.2 % vs 42.9 ± 13.5 %) while lysates from patients with microalbuminuria and proteinuria were less able to degrade matrix (33.0 ± 14.2 % and 26.1 ± 12.7 %, respectively). There was a significant inverse correlation between matrix degradation and AER (r = − 0.49) and multiple regression analysis showed that AER was the most important factor determining degradation rate (R2 = 0.24). Degree of metabolic control, age, and blood pressure were not significant factors. The major enzyme(s) responsible for the matrix degradation was identified as metalloproteinase(s). We conclude that leucocytes from diabetic patients with abnormal albumin excretion have a decreased proteolytic capacity to degrade extracellular matrix. This may play a role in the glomerular basement membrane thickening and mesangium expansion which occurs in diabetic nephropathy.