Reduction of podocytes number in late diabetic alloxan nephropathy: prevention by glycemic control

PURPOSE: To determine podocyte number and GBM thickness in diabetic rats either under glycemic control or without glycemic control at 6 and 12 months after diabetes induction. METHODS: 100 wistar rats weighing 200-300g were divided into 6 groups: Normal group (N6 and N12- 25 rats); Diabetic group (D6 and D12- 25 rats), diabetic treated group ( DT 6 and DT 12- 25 rats) on insulin 1,8- 3,0 IU/Kg associated with acarbose (50mg to 100g of food) daily mixed in chow. Alloxan was injected intravenously in a dose of 42 mg/Kg of weight. Body weight, waterintake, 24-h diuresis, glycemia and glucosuria were determined before induction, 7 and 14 days after induction and monthly thereafter. Treatment started at day 14. Three groups were sacrificed at 6 months (N6,D6, DT6) and 3 groups at 12 months (N12, D12, DT12) with the renal tissue being prepared for electron microscopy. RESULTS: Glycemia in DT6¨and in DT12 was significantly different from that in D6 and D12 rats and similar to that in N6 and N12 animals. The number of podocytes in DT6 was not different from that in N6 and D6 (median = 11); the number of podocytes in DT12 (median = 11) differed from that in D12 (median = 8), but not from that in N12 (median = 11). GBM thickness in D6 (0.18 micrometers) was lower than in D12 (0.29 micrometers); while in DT6 (0.16 micrometers) it was lower than in D6 (0.18 micrometers). In DT12 (0.26 micrometers), it was lower than in D12 (0.29 micrometers). CONCLUSION: The control of hyperglycemia prevented GBM thickening in early and late (12 mo) alloxan diabetic nephropathy and podocyte number reduction.

OBJETIVO: Avaliar o número de podócitos e espessamento da membrana basal glomerular (MBG) em ratos diabéticos com e sem controle glicêmico com 6 e 12 meses da indução. MÉTODOS: 100 ratos Wistar com 200-300g compuseram 6 grupos: Normal (N6, N12 - 25 animais) Diabético (D6,D12 - 25 animais) e diabético tratado com insulina 1,8 a 3,0 U/Kg e acarbose misturada a ração (50g para cada 100g de ração) (DT6 e DT12 - 25 animais). Aloxana foi ministrada via endovenosa na dose de 42mg/Kg. Peso, ingestão hídrica e diurese de 24 horas e glicemia e glicosúria foram determinados antes da inoculação, 7 e 14 dias após e mensalmente. No 14ª dia foi iniciado o tratamento. Três grupos de animais (N6, D6 e DT6) foram sacrificados no 6° mês e três grupos (N12, D12 e DT12), no 12ª mês sendo o tecido renal processado para estudo à microscopia eletrônica. RESULTADOS: A glicemia dos animais DT6 e DT12 diferiram significativamente, dos ratos D6 e D12, e não diferiram dos grupos N6 e N12. O número de podócitos do grupo DT6 não diferiu de N6 e D6 (mediana=11); o número de podócitos de DT12 (mediana=11) diferiu de D12 (mediana=8) e não diferiu de N12 (mediana=11). O espessamento da MBG de D6 (0,18 micrômetros) foi menor que D12 (0,29 micrômetros); de DT6 (0,16 micrômetros) foi menor que D6 (0,18 micrômetros) e de DT12 (0,26 micrômetros) foi menor que D12 (0,29 micrômetros). CONCLUSÃO: O controle da hiperglicemia preveniu o espessamento da MBG na nefropatia diabética aloxânica precoce (6 meses) e tardia (12 meses), e a diminuição do número de podócitos.

Glomerular basement membrane thickness in normal adults and its application to the diagnosis of thin basement membrane disease: an Indian study.

Microscopic hematuria of non-urologic origin warrants ultrastructural study of renal biopsy. Thinning and variations in the texture of glomerular basement membrane (GBM) are difficult to be recognized under light microscope; transmission electron microscope (TEM) therefore plays a vital role in identifying such changes. Ultrastructural morphometry is a valuable diagnostic aid when GBM is suspected of being abnormally thin. In an effort to determine the normal GBM thickness (GBMT) in Indian adults and to determine the cutoff value of GBMT for a diagnosis of thin basement membrane disease (TBMD), we determined GBM thickness in 25 normal adults. Postmortem biopsies of 25 normal adults (16 males and 9 females) aging between 18-58 years were included in the study. GBM thickness was determined through ultrastructural morphometry on accurately enlarged electron micrographs as harmonic mean of 50 orthogonal intercepts across the GBM in each case. Study revealed a mean GBM thickness of 321 nm with a standard deviation (SD) of 28 nm. Mean-2SD (321-56), that is 265 nm, was fixed as cutoff value of GBMT for the diagnosis of TBMD. A systematic split study of control subjects revealed thicker GBM (329+/-38 nm) in higher age group (35-60 years) as compared to GBMT (316+/-21 nm) in lower age group (18-30 years). Males in higher age group also revealed thicker GBM (males: 343+/-39 nm versus females: 300+/-12 nm). Ten patients with non-urologic hematuria and having GBMT<265 nm were diagnosed as cases of TBMD. Patients with TBMD revealed significantly attenuated GBM as compared to age and sex matched controls (214 +/- 40 nm versus 311 +/- 17 nm; p<0.0005). No overlap was observed in the distribution of GBMT in patients of TBMD and age and sex matched controls. Ultrastructural morphometry is the ultimate and appropriate method for diagnosing TBMD.

Ultrastructural morphometry using dual axes tangential scale: a technical revelation.

While performing ultrastructural morphometry, under or over estimation of ultrastructural size could be avoided by using accurate measuring devices. Biological investigators have always relied on conventional linear scale for the baseline measurement of ultrastructural size parameters on electron micrographs to project the dimensions of intracellular organelles or tissue components. Since it was not possible to measure decimal fractions of mm with linear scale, a 'dual axes tangential scale' has been designed for measuring ultrastructural image parameters on electron micrographs with an accuracy of 0.1 mm to minimize the error in finally computed size of ultrastructural component. In an exercise using 'dual axes tangential scale' and 'conventional linear scale', measurement of glomerular basement membrane thickness (GBMT) as orthogonal intercepts across the GBM revealed a 'coefficient of variation' at 4.4% with dual axes tangential scale as compared to 'coefficient of variation' at 10.9% with linear scale, expressing superiority of dual axes tangential scale over linear scale. Use of mathematical formula rather than nomogram has been preferred. However, 'slide guide, ultrastructure size calculator' could also be used for discerning ultrastructural size after measurement with dual axes tangential scale.