Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment

Summary A quantitative morphological study of the mesangial regions has been performed on the kidneys of two groups of insulin treated diabetic rats 6 months after the induction of diabetes. In one group reasonably good control of plasma glucose levels (182±20 (SD) mg/l00ml at 0800 h; 95±35 mg/100 ml at 2300 h) was achieved. This group showed no mesangial changes when compared to a non-diabetic control group. In the second diabetic group poor control was intended (plasma glucose 452±41mg/100 ml and 555±86mg/100 ml respectively). The following differences were noted when this group was compared to the non-diabetic controls and to the rats in which the blood glucose was well-controlled: 1. Increase in total mesangial volume per glomerulus by 42% and 38% (2p = 0.025 and 0.037); 2. Increase in the total amount of basement membrane-like material (BMLM) per glomerulus by 30 and 27% (2p = 0.030 and 0.046); 3. Increase in the total mesangial cell volume per glomerulus by 46 and 43% (2p = 0.033 and 0.048); 4. Increase in volume of electron dense material in the BMLM by about 200% compared to both groups (2p = 0.001 and 0.0003). The study has shown that the mesangial regions also are involved in the diabetic glomerulopathy of experimental diabetes. The morphological changes including increased amounts of basement membrane material are prevented by proper glycaemic control.     

Prevention of diabetic glomerulopathy in streptozotocin diabetic rats by insulin treatment

Summary A single antibody radioimmunoassay has been used to measure albumin excretion in 3 groups of female Wistar rats. Two groups had Streptozotocin diabetes and were treated daily with insulin for 6 months. In one of the diabetic groups good glycaemic control was attempted and throughout the 6 months plasma glucose levels were fairly close to normal (92 ± 33 mg/100 ml at 2300 h and 186 ± 9 mg/100 ml at 0800 h). In the other diabetic group poor control was intended and the group had consistent high plasma glucose levels (576 ± 89 mg/100 ml and 460 ± 43 mg/100 ml). The third group was a non-diabetic control group. — Albumin excretion was measured on two occasions: before the induction of diabetes and after 6 months of diabetes. The geometric mean albumin excretion increased from 0.38 to 2.56 mg/24 h in the 18 non-diabetic controls. In the 20 diabetic rats in good control the geometric mean albumin excretion increased from 0.37 to 1.58 mg/ 24 h (NS compared with controls) and in the group of 22 rats in poor control albumin excretion increased from 0.35 to 6.54 mg/24 h. — The increase in albumin excretion in rats in poor control differed significantly both from that of the non-diabetic controls (2p = 0.023) and from that of the well-controlled diabetic rats (2p = 0.00011).     

Early glomerulopathy is present in young,Type 1 (insulin-dependent) diabetic patients with microalbuminuria

Summary Increased urinary albumin excretion, microalbuminuria, may be the first sign of early diabetic nephropathy. We examined glomeruli by morphometric methods in 17 patients with Type 1 (insulin-dependent) diabetes mellitus and microalbuminuria. The median age was 19 (range 18–29) years, duration of diabetes 12 (8–15) years, mean blood pressure 93 (87–115) mm Hg, glomerular filtration rate 132 (101–209) ml·min⁻¹·1.73 m²⁻², albumin excretion rate (mean over 1 year) 32 (15–194) μg/min. Reference data were obtained from 11 healthy kidney donors. Mesangial volume estimates were obtained by serial sectioning in three total profiles in each of three glomeruli in diabetic patients. Basement membrane thickness and matrix volume fraction were estimated from one level per glomerulus. Two matrix parameters, matrix star volume and matrix thickness, were estimated. Interstitial volume fraction in cortex was measured by light microscopy. The morphological parameters were significantly increased in the diabetic group compared to the control group, basement membrane thickness (mean with 95% confidence intervals) was 595 nm (549–641 nm) vs 305 nm (287–325 nm),p=0.0001; mesangial volume fraction 0.22 (0.21–0.23) vs 0.19 (0.18–0.21),p=0.04, and matrix volume fraction 0.13 (0.12–0.13 vs 0.09 (0.08–0.10),p=0.001. Also matrix star volume and thickness, interstitial volume fraction and mean capillary diameter were significantly increased. The intra-individual variation among glomeruli expressed as coefficient of variation was 7.4% vs 9% (basement membrane thickness) and 11.7% vs 25% (mesangial volume fraction) in the diabetic and the control group, respectively. Increment of basement membrane thickness and matrix volume fraction per year were significantly correlated with mean 1-year HbA_lc (r=0.55 andr=0.51, respectively). We conclude that microalbuminuria in Type 1 diabetes is associated with increased basement membrane thickness and also mesangial matrix expansion. This increment seems to correlate with glycaemic control.     

The number of glomeruli in Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients

Summary The number of glomeruli per kidney in Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients was estimated by an unbiased stereological method: the fractionator. No significant differences were observed between Type 1 and Type 2 diabetic patients without severe diabetic glomerulopathy and non-diabetic patients. Diabetic patients with proteinuria who were in the early stages of diabetic nephropathy also had a normal number of glomeruli. On the other hand, a subgroup classified as Type 1 diabetic patients with severe diabetic glomerulopathy had significantly less glomeruli compared with Type 1 diabetic patients with mild or no glomerulopathy. A probable explanation is that Type 1 diabetic patients lose glomeruli in relation to the progression of diabetic glomerulopathy. A more theoretical alternative is, however, that development of diabetic glomerulopathy is facilitated by a low number of glomeruli.Presented in part at the first meeting of the European Diabetic Nephropathy Study Group, Pisa, Italy, April 1988, and at the 23rd annual meeting of the Scandinavian Society for the Study of Diabetes, Bergen, Norway, May 1988     

Increase in circulating basement membrane antigens in diabetic rats and effects of insulin treatment

Summary The serum concentrations of two recently discovered antigens derived from basement membranes (7-S collagen and laminin P2) were assayed in streptozotocin-diabetic rats as possible indicators of basement membrane metabolism. The concentrations of both increased significantly after 8 weeks of diabetes, and that of 7-S collagen at least remained elevated up to 24 weeks. Treatment with insulin, which did not correct the metabolic disturbances, inhibited the increase in the concentration of 7-S collagen in serum, but did not completely normalize that of laminin P2.     

Kidney transplantation in Type 1 (insulin-dependent) diabetic patients

Summary The development of diabetic glomerulopathy in kidneys transplanted to diabetic patients was estimated in transplant biopsies and evaluated in relation to suspected clinical risk factors for diabetic nephropathy. Surgical biopsies were taken at baseline and at 24–36 months post-transplantation in 16 Type 1 (insulin-dependent) diabetic patients and 8 non-diabetic control subjects with a glomerular filtration rate more than 30 ml·min^– at follow-up. Immunosuppressive therapy included cyclosporine in all but one case. Stereological methods were used to assess basement membrane thickness, volume fraction of mesangium per glomerulus, and volume fraction of matrix per mesangium. The volume fraction of interstitial tissue per cortex was estimated by light microscopy. After 2 years the basement membrane thickness had increased by 55 nm (SD 58 nm) in the diabetic group. This change was significantly different from that of 2 nm (SD 37 nm) in control subjects (p=0.02). Mesangial volume fraction increased significantly by 0.04 (SD 0.03) in diabetic patients, and this change was significantly different from that of -0.01 (SD 0.04) in non-diabetic patients (p=0.009). No change was detectable in the matrix expressed as fraction of mesangial volume. An increase in interstitial volume fraction from baseline to 2 years was observed, but was significant only in the diabetic group (p=0.04). The changes in structural parameters did not correlate with mean values during follow-up of glycated haemoglobin or estimated protein intake, nor was any pattern discernible in the relationship to graft tissue types. The observed increase in basement membrane thickness corresponds to that observed in native kidneys during the first years of diabetes, whereas an increase in mesangial volume fraction — using a different protocol — was not observed in the early phase of the natural development. Absence of correlation with the various risk factors may reflect an irrelevance of these variables within the current range, or their influence may be offset by stronger mechanisms in the transplant situation, and therefore does not appear in this relatively small series.     

Kidney function and glomerulopathy over 8 years in young patients with Type I (insulin-dependent) diabetes mellitus and microalbuminuria

Aims/hypothesis: We aimed to investigate prospectively the interrelation between kidney function and glomerular morphological changes over 8 years in young patients with Type I (insulin-dependent) diabetes mellitus and microalbuminuria. Methods: Kidney biopsies were taken at baseline and after 8 years in 18 subjects who were 20 years of age (19–29 mean and range), had duration of diabetes for 11 years (7–18), and who had an albumin excretion rate of 45 μg/min (15–194). The glomerular ultrastructural parameters were analysed using stereological methods. Results: At the end of the study three patients had an increased albumin excretion rate of more than 25 % a year, two of whom developed overt nephropathy. Glomerular filtration rate declined 2.3 ml/min · 1.73 m^–2· yr^–1. Glomerular volume, volume fractions of matrix and mesangium, and basement membrane thickness showed an increase over the 8 years. Multiple regression analysis showed that mean 8-years HbA_{1 c}, matrix volume fraction_baseline and basement membrane thickness BMT_baseline accounted for 70 % of the variation in AER at the end of the study. Mesangial volume fraction_baseline, glomerular filtration fraction_baseline, and mean 8-year HbA_{1 c} accounted for 73 % of the change in glomerular filtration rate from baseline. Smoking was strongly associated with the glomerular filtration rate at baseline (r = 0.65). When glomerular filtration rate_baseline was omitted from the equation, smoking was the only significant parameter linked to the change in glomerular filtration rate from the baseline. Conclusion/interpretation: In patients who had diabetes for 20 years, long-term hyperglycaemia and glomerulopathy found 8 years prior to the study, and possibly smoking, affected renal function (i. e. albumin excretion rate and glomerular filtration rate). [Diabetologia (2002) 45: 253–261] Received: 16 July 2001 and in revised form: 18 October 2001     

Improvement of blood glucose control in IDDM patients retards the progression of morphological changes in early diabetic nephropathy

Summary We investigated in a randomized, prospective study the influence of improved blood glucose control during 2–3 years in young insulin-dependent diabetic (IDDM) patients with microalbuminuria, which is indicative of early nephropathy. Patients were randomized either to intensive treatment by continuous subcutaneous insulin infusion (CSII) (n =9) or CT (n =9). Kidney biopsies were taken at baseline and after 26–34 months. End points were structural changes in the glomeruli. Sensitive, quantitative, morphometric methods were used. The blood glucose control improved significantly (p =0.01) during the study in the CSII-group as glycated haemoglobin (HbA_{1 c}) fell from 10.1 % ([95 % CI] 8.9–11.3) to 8.6 % (7.9–9.2), but not in the CT-group, 10.1 % (8.3–11.9) vs 9.7 % (8.7–10.8). Mean HbA_{1 c} during the study period was significantly lower in the CSII-group than in the CT-group, 8.7 % (8.1–9.3) vs 9.9 % (8.5–11.3), p =0.04. Basement membrane thickness (BMT) increased in both groups, most (CT vs CSII, p =0.03) in the CT-group: 140 nm (50–230) vs CSII: 56 nm (27–86). In the CT-group only an increase was seen in matrix/mesangial volume fraction (p =0.006) and matrix star volume (p =0.04). Furthermore, a positive correlation between mean HbA_{1 c} during the study and change from baseline in BMT (r =0.70, p =0.001) and matrix/glomerular volume fraction (r =0.33, p =0.09, NS) was demonstrated. Albumin excretion rate correlated significantly to BMT and most of the matrix parameters. The present study shows that during a period of only 2.5 years, a close relationship between the level of mean blood glucose and progression of glomerular morphological changes in early diabetic nephropathy can be demonstrated. [Diabetologia (1994) 37: 483–490] Received: 24 August 1993 and in revised form: 23 November 1993     

Thickening of glomerular basement membrane in spontaneously diabetic rats

Summary The glomerular basement membrane of spontaneously diabetic rats was investigated by quantitative analysis using electron microscopy, with special reference to the effect of ageing. Constant agerelated increase in the width of basement membrane was ascertained both in diabetic and control rats, and the mean values of basement membrane thickness were always higher in the spontaneously diabetic rats than in normal control rats. Significant thickening of glomerular basement membrane was found in the diabetic rats at 12 weeks of age, while younger diabetic rats had no definite increase. The difference in basement membrane thickness between diabetic and normal control rats became larger with increasing age.     

Glomerular structure and function in proteinuric Type 2 (non-insulin-dependent) diabetic patients

Summary Glomerular ultrastructure was examined in a series of 20 Type 2 (non-insulin-dependent) diabetic patients with proteinuria. Reference was made to data previously obtained in non-diabetic kidney donors and in Type 1 (insulindependent) diabetic patients with similar degrees of proteinuria. The Type 2 diabetic patients demonstrated the changes which characterize the diabetic glomerulopathy seen in Type 1 diabetic patients: basement membrane thickening, and increase in the mesangium and mesangial matrix expressed as fraction of the glomerular volume. Among the Type 2 diabetic patients there was more variation then among the Type 1 diabetic patients, as this group included subjects with normal parameters. The group means and coefficients of variation (=SD/mean) of the glomerulopathy parameters combined in the glomerulopathy index=basement membrane thickness/10+V_v(matrix/glom)·100 were 81 (0.30) and 92 (0.15) in the two diabetic groups, clearly different from the non-diabetic index, 42 (0.16). All Type 2 diabetic patients who also had retinopathy had a glomerulopathy index above the normal range. Similar changes in glomerular composition were seen in the two diabetic groups: with increasing glomerulopathy the volume of matrix dominated over the peripheral basement membrane, and a shift in the ratio of interfaces was seen: mesangial surface towards capillary lumen increased relative to the urinary surface, and peripheral capillary surface comprised less of the total capillary surface. Data indicated marked glomerular hypertrophy, which correlated with the mesangial volume fraction, thus encompassing preserved filtration surface per glomerulus. An inverse correlation obtained between the index of glomerulopathy and current glomerular filtration rate, as well as the ensuing rate of decline in glomerular filtration rate, as well as the ensuing rate of decline in glomerular filtration rate: (index (glomerulopathy) vs rate of decline in glomerular filtration rater=0.84,p<0.0001). No correlation was found between glomerular volume and the ensuing rate of decline in glomerular filtration rate.     

Skeletal growth of fetuses from streptozotocin diabetic rat mothers: in vivo and in vitro studies

Summary For largely unknown reasons severe or moderate diabetes of pregnant rats results in pronounced fetal growth retardation. Therefore, some skeletal growth parameters of fetal rats from streptozotocin diabetic mothers were studied in vivo and in vitro. Two days post conception rats were intravenously injected with 65 mg/kg body weight streptozotocin. On day 20 post conception 8 normal and 8 diabetic rat mothers received 5 Ci 3-H thymidine intraperitoneally. One day later the experiments were terminated. Fetal body weight and body length were significantly (p<0.05–0.001) reduced in the hyperglycaemic rats compared to normal rats, as was the thymidine incorporation into rib cartilage (p<0.02). In the cell culture colony formation from isolated chondrocytes of normal and hyperglycaemic fetuses was determined. Proinsulin, insulin (62.5–250 ng/ml), insulin-like growth factor I and II (6.25–25 ng/ml) significantly (p<0.05–0.001) augmented colony formation in a dose-dependent manner, with the somatomedins being 8 times more effective than proinsulin or insulin. Isolated chondrocytes from hyperglycaemic compared to normal fetuses formed significantly (p<0.05–0.001) fewer colonies in the basal state and in response to all 4 hormones. The results confirm the growth retardation of fetuses from diabetic rat mothers. A reduced responsiveness of chondrocytes from hyperglycaemic fetuses to various growth factors could be demonstrated as compared to cells from normal fetuses.     

Epalrestat prevents the decrease in gastric mucosal blood flow and protects the gastric mucosa in streptozotocin diabetic rats

We have recently reported that steady-state gastric mucosal blood flow (GMBF) is decreased in streptozotocin (STZ) diabetic rats, and that their GMBF response to burn-stress is impaired, probably via a nitric oxide (NO)-mediated mechanism. Accordingly, this study was designed to investigate the relation of aldose reductase (AR) and NO synthase to the regulation of GMBF during chronic hyperglycemia. STZ rats were treated with or without chronic oral administration of an AR inhibitor, epalrestat (EPA) and/or an NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME). GMBF was measured by laser-Doppler velocimetry (LDV). In the STZ rats, GMBF after a 24-h fasting period was decreased significantly 4 weeks after the onset of diabetes and this was accompanied by an increase in the gastric ulcer index (UI) (a measure of the length of gastric erosions and ulcers). Chronic oral administration of EPA to the STZ rats dose-dependently inhibited the increased UI and the decreased GMBF after the fasting stress, whereas chronic oral administration of L-NAME further increased the UI and further decreased the GMBF. EPA administered in combination with L-NAME to the STZ rats reduced the effects of L-NAME, but the effects did not reach significance. These results suggest that EPA protects the gastric mucosa of diabetic rats, by preventing the decrease in GMBF that is, at least in part, caused by NO-related mechanisms. (Received Mar. 3, 1998; accepted Aug. 28, 1998)     

An alteration in internodal myelin membrane structure in large sciatic nerve fibres in rats with acute streptozotocin diabetes and impaired nerve conduction velocity

Summary Replicas of the freeze-fracture surfaces of the internodal myelin membranes of large sciatic nerve fibres from normal and diabetic rats were compared by quantitative electron microscopy. The internodal myelin of the diabetics was examined 14 days after streptozotocin (70 mg/kg IV) induced persistent hyperglycaemia, conditions under which sciatic motor nerve conduction velocity (MNCV) is consistently decreased by 20%. The number of intramembranous particles per unit area of both the P-face and the E-face of the internodal myelin membrane was significantly decreased in the diabetics. This alteration in the structure of the internodal myelin membrane was not found in large sciatic nerve fibres from diabetic rats treated with insulin from day 3 through 14, or from diabetic rats fed a diet containing 1% myoinositol; these are conditions under which the development of decreased sciatic MNCV is prevented or ameliorated. An alteration in internodal myelin structure occurs in acute streptozotocin diabetes which may explain the associated decreased sciatic MNCV.The authors wish to thank Mrs. Sarah Aquino and Marthe Sidler, Misses Irina Barinov and Isabelle Bernard, and Mr. Patrice Fruleux for their expert assistance.     

Modification of the glomerular basement membrane in sucrose-fed and streptozotocin-diabetic rats

Summary Rats were fed on diets containing either sucrose or starch as the carbohydrate component (55%) for eight months. Diabetes was induced in animals of both groups by injecting streptozotocin (50 mg/kg body weight). Diabetic rats failed to gain weight, had enlarged kidneys, polyuria and elevated blood glucose levels. Starch and sucrose fed rats gained weight normally and had normal blood glucose levels. Sucrose fed rats had enlarged kidneys. Regional thickening of the glomerular basement membrane was present in sucrose-fed and diabetic rats but not in starch-fed controls. Glomerular basement membrane isolated from pooled kidney cortices from rats in the different experimental groups were analysed for amino acid, disaccharide and hexosamine content. Hydroxylysine (9 to 20%), hydroxyproline (21 to 24%), disaccharide (27%) and hexosamine (26%) were increased in membranes insolated from the three experimental groups, compared with starch-fed non-diabetic controls. An increase in low molecular weight components of the glomerular basement membrane of sucrose-fed and diabetic rats was observed using electrophoresis in sodium dodecyl sulphate. Significantly higher (p<0.001) glucosyltransferase activity was present in kidney supernatants prepared from sucrose-fed (1050±60 nmol/2 h/kidney) compared to starch-fed rats (510±40 nmol/2 h/kidney). Sucrose feeding induces changes similar to those found in diabetes and the induction of diabetes made little difference over the feeding of sucrose alone.     

Ultrastructure of heart muscle in short-term diabetic rats: influence of insulin treatment

Summary The ultrastructure of myocardium was examined in short-term diabetic rats. Morphometric analysis showed the volume of myocytic mitochondria, sarcoplasmic reticulum and lipid droplets to be significantly increased compared with those of control animals. Further measurements of mitochondria and sarcoplasmic reticulum indicated that the augmentation of these compartments was accountable by the enlargement of pre-existing mitochondria, which were swollen, and of pre-existing tubules of sarcoplasmic reticulum, the lumen of which was dilated. After insulin treatment the morphological changes were returned to normal which indicates that they were not due to the toxic effect of streptozotocin but were caused by the diabetic state per se. This suggestion is further supported by the finding that experimentally induced metabolic acidosis without diabetes did not cause any morphologically detectable changes in the heart muscle. It is concluded that short-term diabetes in the rat causes mitochondrial swelling, dilatation of sarcoplasmic reticulum and accumulation of lipid in cardiac myocytes, and that these changes are preventable with insulin treatment. We suggest that insulin may have an important role in the maintenance of metabolism in heart muscle.     

High-performance liquid chromatography analysis of circulating insulins distinguishes between endogenous insulin production (a potential pitfall with streptozotocin diabetic rats) and islet xenograft function

Summary Porcine islets of Langerhans were microencapsulated according to the alginate-polylysine procedure, and implanted into the peritoneal cavity of 15 streptozotocin-induced (70 mg/kg) diabetic rats (6000 microencapsulated islets per rat). In four animals, a sustained decrease in plasma glucose level below 8.3 mmol/l was observed for up to nine months. However, it was possible to recover microcapsules from the peritoneal cavity of only one rat, and they were found to be damaged and containing no detectable tissue. When insulin in the plasma of three of these animals was analysed by reversed phase high-performance liquid chromatography, only rat insulins I and II, but not porcine insulin was detectable, indicating unambiguously that at the time of analysis, the correction of diabetes in these animals was due to the function of the recipient's own pancreas rather than the continued, long-term, function of the implanted porcine islets. These data confirm that in this model of diabetes, function of the host pancreas can resume following islet transplantation, leading in turn to the potential for a major bias in the interpretation of the data. In the case of an islet xenograft, when the donor's and recipient's insulins can be separated by highperformance liquid chromatography, this non-invasive analytical method should prove useful for identifying the source of insulin in the circulation, and thus the relative functional status of the endogenous and transplanted islets.     

Glucose and insulin changes following a renoportal shunt in streptozotocin diabetic rats with pancreatic islet isografts under the kidney capsule

Summary The effect on glucose metabolism of altering the site of the venous drainage of an isograft of isolated adult islets implanted beneath the renal capsule, from the systemic circulation to the portal circulation was determined in streptozotocin-induced diabetic rats. Reversal of diabetes was accomplished by the transplantation of 1000–1200 isolated islets beneath the left kidney capsule. The rate of fall of the glucose concentration (as expressed by the K value) was found to be significantly decreased in transplanted animals (1.7 ± 0.5%/min; mean ± SD) compared with normal animals (2.4 ± 0.5%/min). Draining the left renal vein into the portal circulation restored the K value to that of normal animals (2.5 ± 0.4%). However the fasting glucose concentration was significantly higher and the basal insulin levels lower in both normal and transplanted rats with a renoportal shunt.     

The cortical distribution pattern of diabetic glomerulopathy

Summary In advanced diabetic nephropathy, all nephrons are affected. However, glomerulopathy shows some variation in severity. The aim of this study was to see whether the distribution of glomerular lesions bears any relationship to known renal anatomical/functional organization. Autopsy material from 12 long-term Type 1 (insulin-dependent) diabetic patients was collected retrospectively. Sections presenting the whole depth of cortex were subdivided into 3 zones of equal width. Two parameters were estimated in the deep and the superficial zones separately: (1) in open glomeruli, the volume of red-stained (PAS-positive) material as a fraction of the total glomerular volume, and (2) the number of occluded glomeruli as a fraction of the total number. The results show that the volume fraction of red-stained (PAS-positive) material, as well as the frequency of occluded glomeruli, are similar in the superficial and the deep zone. In addition, the spatial distribution of occluded glomeruli was tested for randomness. The occluded glomeruli are not randomly distributed, but show clustering. Furthermore, they tend to be located in columns perpendicular to the kidney surface, a tendency which cannot be explained by the general tendency for glomeruli to show such an arrangement. We conclude that it is unlikely that the combined structural and functional differences that exist between the superficial and deep glomeruli play a major role in the development of diabetic glomerulopathy. The kidney topology influences the process of glomerular occlusion. Several a priori, plausible mechanisms for the process are discussed.