Electron Microscopic Morphometric Analysis Research Articles

Propionic acid affects the synaptic architecture of rat hippocampus and prefrontal cortex

It is well documented that propionic acid (PPA) produces behavioral, morphological, molecular and immune responses in rats that are characteristic of autism spectrum disorder in humans. However, whether PPA affects the ultrastructure and synaptic architecture of regions of autistic brain has not been adequately addressed. Earlier we show that single intraperitoneal (IP) injection of PPA (175 mg/kg) produces superficial changes in the spatial memory and learning of adolescent male Wistar rats. However, in neurons, synapses and glial cells of hippocampal CA1 area and medial prefrontal cortex transient (mainly) or enduring alterations were detected. In this study, we used electron microscopic morphometric analysis to test the effect of PPA on different structural parameters of axodendritic synapses of the hippocampus and prefrontal cortex. The animals were treated with a single IP injection of PPA (175 mg/kg). The length and width of synaptic active zone, the area of presynaptic and postsynaptic mitochondria, the distance between presynaptic mitochondria and the synapse active zone, the distance between postsynaptic mitochondria and postsynaptic density and the depth and opening diameter of neuronal porosome complex were evaluated. Our results show that synaptic mitochondria of the hippocampus and prefrontal cortex are the most vulnerable to PPA treatment: in both regions, the area of postsynaptic mitochondria were increased. In general, our results show that even small dose of PPA, which produces only superficial effects on spatial memory and learning is able to alter the synapse architecture in brain regions involved in cognition and autism pathogenesis. Therefore, the microbiome may be involved in the control of neurotransmission in these regions.

Immunohistochemical and electron microscopic morphometric image analysis of hepatocellular carcinoma in association of HCV infection

ABSTRACTEarly detection of hepatocellular carcinoma (HCC) is crucial for successful therapy. The present work examined the value of ultrastructural morphometric image analysis of hepatocyte nuclei in patients with chronic hepatitis C virus (HCV) versus HCC cases with chronic HCV and the corresponding surgical tumor-free safe margins (TFMs), to highlight any early predictive signs of neoplastic cellular transformation. This work also performed an immunohistochemical assessment of cytokeratin 19 (CK19) and Ki-67-positive cells to visualize any associated proliferative activity in the examined groups.The results showed significant decrease in the hepatocyte nuclear surface areas in the HCC and TFMs versus those in the HCV cases. The hepatocyte nucleolar surface area was significantly increased in the HCC cases versus that in the HCV cases. This increase was associated with a significant increase in Ki-67-positive cells in the HCC cases compared to those in the other groups. Conversely, the mean number of CK 19-positive cells was significantly reduced in the HCC cases compared to the cell numbers in TFMs and HCV cases with severe hepatic fibrosis. Liver progenitor cells (LPCs) were discerned in the reactive ductules and canaliculo-ductular junctions that characterized TFMs. LPCs were sporadically distributed in the liver lobules and reactive bile ductules in the HCC samples.In conclusion, CK 19 represents an important marker for distinguishing between dysplastic and malignant liver nodules. Electron microscopic morphometric image analysis may be considered as adjunct factor for assessing hepatocyte malignant transformation. Wider scale studies are needed to authenticate these results.

The relationship of retinal vessel diameter to changes in diabetic nephropathy structural variables in patients with type 1 diabetes

We examined whether retinal vessel diameter in persons with type 1 diabetes mellitus is associated with changes in subclinical anatomical and functional indicators of diabetic nephropathy. Persons with type 1 diabetes mellitus had gradable fundus photographs and renal biopsy data at baseline and 5-year follow-up (n = 234). Retinal arteriolar and venular diameters were measured at baseline and follow-up. Central retinal arteriole equivalent (CRAE) and central retinal venule equivalent (CRVE) were computed. Baseline and 5-year follow-up renal structural variables were assessed by masked electron microscopic morphometric analyses from percutaneous renal biopsy specimens. Variables assessed included: mesangial fractional volume, glomerular basement membrane width, mesangial matrix fractional volume and glomerular basement membrane width composite glomerulopathy index. While controlling for other covariates, baseline CRAE was positively associated with change in the glomerulopathy index over the 5-year period. Change in CRAE was inversely related to a change in mesangial matrix fractional volume and abnormal mesangial matrix fractional volume, while change in CRVE was directly related to change in the volume fraction of cortex that was interstitium [Vv((Int/cortex))] over the 5-year period. Baseline CRAE or CRVE or changes in these diameters were not related to changes in other anatomical or functional renal endpoints. Independently of other factors, baseline CRAE correlated with changes in glomerulopathy index, a composite measure of extracellular matrix accumulation in the mesangium and glomerular basement membrane. A narrowing of the CRAE was related to mesangial matrix accumulation. Changes in CRVE were related to changes in Vv((Int/cortex),) a measure of interstitial expansion in persons with type 1 diabetes mellitus.

Renal structure as an indicator for development of albuminuria in normo- and microalbuminuric type 2 diabetic patients

Baseline glomerular structure in microalbuminuric (MA) and proteinuric Caucasian type 2 diabetic patients predicted progressive glomerular filtration rate decline while baseline urinary albumin excretion (UAE) did not. Little is known about whether or not renal structure at the early stages of diabetic nephropathy (DN) in type 2 diabetic patients can predict further functional development of DN. Baseline renal structure and function and follow-up data of renal function were examined in 17 type 2 diabetic patients (11 men, 45 ± 7 (mean ± S.D.) years old) with known diabetes duration 11 ± 8 years without definable renal disease other than DN. Six patients showed normoalbuminuria (NA), 11 microalbuminuria (MA), and were followed up for 6.4 ± 1.8 years after the baseline renal biopsy. Light and electron microscopic morphometric analyses provided quantitative glomerular and tubulointerstitial structural changes. No statistically significant difference was observed in hemoglobin A1c (HbA1c) values or mean blood pressure (MBP) between baseline and follow-up, even though the number of patients placed on antihypertensive drugs increased from 3 to 7. Follow-up UAE was not significantly different from the baseline UAE although 13 of 17 cases showed an increase. Baseline UAE did not correlate with the follow-up UAE or morphometric measures. Glomerular basement membrane width and volume fraction of the mesangium and mesangial matrix positively correlated with follow-up UAE. In NA and MA Japanese type 2 diabetic patients, baseline renal structural measures are more reliable indicators for the development of UAE than baseline UAE.

Detailed glomerular ultrastructure in Japanese type 2 diabetic patients by the quick-freezing and deep-etching method

Mesangial expansion and glomerular basement membrane (GBM) thickening did not correlate with urinary albumin excretion (UAE) in type 2 diabetic patients in our previous studies; therefore, it was necessary to elucidate more detailed ultrastructural changes in the early stages of diabetic nephropathy (DN) in type 2 diabetic patients. The quick-freezing and deep-etching (QF–DE) method allows us to examine three-dimensional ultrastructures of human renal glomeruli in vivo at high resolution. The QF–DE method was applied to six type 2 diabetic patients without definable renal diseases other than DN. Four patients were normoalbuminuric (NA) and the other two were microalbuminuria (MA). Three control specimens were the normal parts from nephrectomies due to renal cell carcinomas. Electron microscopic morphometric analyses provided quantitative glomerular structural changes. Replica membranes were prepared by the QF–DE method, and diameters of mesh structures at the GBM and mesangial matrix (MM) were measured on electron micrographs as previously described. By the QF–DE method, both the GBM middle layer and MM were composed of polygonal meshwork structures. The mesh pores of the GBM and MM were more enlarged and irregular in shape in NA diabetic patients than those of the controls, and these ultrastructural changes became more obvious in MA patients. The mesh diameters of the GBM and MM in the diabetic patients were also larger than those of the controls. Such a mesh diameter of the GBM was well correlated with the amount of UAE, while the mesh diameter of MM showed a slight correlation with UAE. Although there were small number of subjects in the present study, the detailed ultrastructural changes in NA and MA type 2 diabetic patients, which had not been disclosed by conventional electron microscopy, were revealed by the QF–DE method. Increased mesh diameters of GBM might be related with the increase of UAE.

Diabetic nephropathy: An update on renal structure

Abstract The renal lesions underlying renal dysfunction differ in type 1 and type 2 diabetes, although the clinical manifestations of diabetic nephropathy, proteinuria, decreased glomerular filtration rate and increasing blood pressure are similar. Indeed, in type 1 diabetes, although also tubular, interstitial and arteriolar lesions are present, the most important structural changes involve the glomerulus, while several type 2 diabetic patients, despite the presence of microalbuminuria or proteinuria, have normal glomerular structure with or without tubulo-interstitial and arteriolar abnormalities. The clinical manifestations of diabetic nephropathy are strongly related with the structural changes, especially with the degree of mesangial expansion in both type 1 and type 2 diabetes. In the last years it has been demonstrated that also changes in the structure and number of podocytes may be involved in the progression of diabetic nephropathy. Previous studies using light and electron microscopic morphometric analysis described the renal structural changes and the renal structural–functional relationships, documenting the contribution of kidney biopsy studies in understanding the pathogenesis of diabetic nephropathy and in identifying the patients with higher risk to progress to end stage renal disease. Finally this review will summarize studies in pancreas transplant recipients demonstrating that the lesions of diabetic nephropathy in humans are reversible, if normoglycemia is achieved and maintained for a long period of time.

Histopathology of Diabetic Nephropathy

The clinical manifestations of diabetic nephropathy, proteinuria, increased blood pressure, and decreased glomerular filtration rate, are similar in type 1 and type 2 diabetes; however, the renal lesions underlying renal dysfunction in the 2 conditions may differ. Indeed, although tubular, interstitial, and arteriolar lesions are ultimately present in type 1 diabetes, as the disease progresses, the most important structural changes involve the glomerulus. In contrast, a substantial subset of type 2 diabetic patients, despite the presence of microalbuminuria or proteinuria, have normal glomerular structure with or without tubulointerstitial and/or arteriolar abnormalities. The clinical manifestations of diabetic nephropathy are strongly related with the structural changes, especially with the degree of mesangial expansion in both type 1 and type 2 diabetes. However, several other important structural changes are involved. Previous studies, using light and electron microscopic morphometric analysis, have described the renal structural changes and the structural-functional relationships of diabetic nephropathy. This review focuses on these topics, emphasizing the contribution of research kidney biopsy studies to the understanding of the pathogenesis of diabetic nephropathy and the identification of patients with a higher risk of progression to end-stage renal disease. Finally, evidence is presented that the reversal of established lesions of diabetic nephropathy is possible.

Acute-Phase Markers of Inflammation and Glomerular Structure in Patients with Type 2 Diabetes

Type 2 diabetes is frequently associated with an inflammatory status; the relationships between low-grade inflammation and diabetic nephropathy are still unclear. The aim of this study was to evaluate the relationships between acute-phase markers of inflammation, glomerular structure, and albumin excretion rate (AER) in type 2 diabetes. In 74 patients with type 2 diabetes (23 normoalbuminuric, 30 microalbuminuric, and 21 proteinuric) fibrinogen, serum amyloid A protein (SAA), C-reactive protein (CRP), and IL-6 were determined. AER was measured on three 24-h urine collections; GFR was measured by 51Cr EDTA plasma clearance. A kidney biopsy was performed, and mesangial fractional volume [Vv(mes/glom)] and glomerular basement membrane (GBM) width were estimated by electron microscopic morphometric analysis. CRP, fibrinogen, SAA, and IL-6 differed among groups, with proteinuric patients having the highest levels. SAA and fibrinogen correlated with AER (P < 0.03 and P < 0.001, respectively). GBM width and Vv(mes/glom) increased from normoalbuminuric to proteinuric patients [P < 0.005 normoalbuminuric and microalbuminuric versus proteinuric for GBM, P < 0.01 normoalbuminuric versus proteinuric for Vv(mes/glom)]. In patients with increased GBM width (> 396 nm), CRP, SAA, and IL-6 were higher than in patients with normal GBM width (P < 0.003, P < 0.004, and P < 0.0004, respectively). GBM width was directly correlated with fibrinogen (r = 0.33, P < 0.002) and IL-6 (r = 0.25 P < 0.05). In conclusion, this study demonstrates that acute-phase markers of inflammation are associated with nephropathy status and GBM thickening, suggesting a role for inflammation in the pathogenesis of diabetic glomerulopathy.

The Relationship of Diabetic Retinopathy to Preclinical Diabetic Glomerulopathy Lesions in Type 1 Diabetic Patients

Few epidemiological data exist regarding the correlation of anatomic measures of diabetic retinopathy and nephropathy, especially early in the disease processes. The aim of this study was to examine the association of severity of diabetic retinopathy with histological measures of diabetic nephropathy in normoalbuminuric patients with type 1 diabetes. The study included participants (n = 285) in the Renin-Angiotensin System Study (RASS; a multicenter diabetic nephropathy primary prevention trial) who were aged >/=16 years and had 2-20 years of type 1 diabetes with normal baseline renal function measures. Albumin excretion rate (AER), blood pressure, serum creatinine, and glomerular filtration rate (GFR) were measured using standardized protocols. Diabetic retinopathy was determined by masked grading of 30 degrees color stereoscopic fundus photographs of seven standard fields using the Early Treatment Diabetic Retinopathy Study (ETDRS) severity scale. Baseline renal structural parameters, e.g., fraction of the glomerulus occupied by the mesangium or mesangial fractional volume [Vv(Mes/glom)] and glomerular basement membrane width, were assessed by masked electron microscopic morphometric analyses of research percutaneous renal biopsies. No retinopathy was present in 36%, mild nonproliferative diabetic retinopathy in 53%, moderate to severe nonproliferative diabetic retinopathy in 9%, and proliferative diabetic retinopathy in 2% of the cohort. Retinopathy was not related to AER, blood pressure, serum creatinine, or GFR. All renal anatomical end points were associated with increasing severity of diabetic retinopathy, while controlling for other risk factors. These data demonstrate a significant association between diabetic retinopathy and preclinical morphologic changes of diabetic nephropathy in type 1 diabetic patients.

Quantitave and qualitative changes in vascular endothelial growth factor gene expression in glomeruli of patients with type 2 diabetes.

Vascular endothelial growth factor (VEGF) exists in three main splice variants, characterized by 121, 165 and 189 amino acids (VEGF 121, VEGF 165 and VEGF 189) and acts via two specific receptors: VEGF-R1 or Flt-1 and VEGF-R2 or KDR. VEGF plays an important role in the pathogenesis of diabetic retinopathy. This study examined the relationship between VEGF and its isoforms and the severity of diabetic nephropathy in type 2 diabetes. We evaluated the glomerular gene expression of VEGF and its receptors and studied the relationships with renal functional and structural parameters in type 2 diabetic patients. Glomeruli from 17 kidney biopsies were microdissected; 14 out of 17 biopsies were also subjected to electron microscopic morphometric analysis to estimate glomerular structural parameters. VEGF mRNA was studied by comparative kinetic RT-PCR and real-time RT-PCR in order to identify the three different isoforms and to quantify VEGF, VEGF-R1 and VEGF-R2 mRNA levels. (i) Glomerular VEGF mRNA levels were inversely related to albumin excretion rate (r=-0.66, P=0.004); (ii) both the degree of mesangial and mesangial matrix expansion were inversely related to VEGF 165 mRNA levels (r=-0.73, P=0.005 and r=-0.64, P=0.017), and directly to VEGF 121 mRNA levels (r=0.74, P=0.003 and r=0.73, P=0.004); and (iii) VEGF and VEGF-R2 mRNA levels were directly related (r=0.62, P=0.033). These findings suggested that quantitative and qualitative changes in VEGF expression are present in type 2 diabetic patients with nephropathy and might be involved in the pathogenesis and progression of diabetic glomerulopathy.

Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos.

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

PC-1 amino acid variant Q121 is associated with a lower glomerular filtration rate in type 2 diabetic patients with abnormal albumin excretion rates.

To study the relationships between the PC-1 K121Q variant and diabetic nephropathy (DN) in patients with type 2 diabetes. A total of 125 patients with type 2 diabetes and abnormal albumin excretion rate (AER) (range 20-5416 microg/min) were followed up for 4 years with repeated measurements of glomerular filtration rate (GFR). Genomic DNA was extracted from all patients, and the PC-1 K121Q polymorphism was determined by the PCR AvaII restriction enzyme. A subset of 64 patients underwent a percutaneous kidney biopsy at baseline, and glomerular structure was analyzed by electron microscopic morphometric analysis. At baseline, age (56 +/- 8 vs. 59 +/- 7 years), BMI (28.3 +/- 4.3 vs. 28.6 +/- 3.7 kg/m(2)), known duration of type 2 diabetes (11.1 +/- 7 vs. 11.9 +/- 8 years), and HbA(1c) (8.6 +/- 1.8 vs. 8.4 +/- 1.7%) were similar in K121K (KK, n = 87, 73 men/14 women) and XQ (35 K121Q + 3 Q121Q, n = 38, 27 men/11 women) patients. Baseline GFR was 96 +/- 28 ml. min(-1). 1.73 m(-2) and was related (P = 0.01-0.001) to age, known diabetes duration, and systolic blood pressure. XQ patients had lower GFR (P < 0.05) than KK patients (88 +/- 30 vs. 100 +/- 26 ml. min(-1). 1.73 m(-2)); this difference persisted also after factoring in age and known diabetes duration. The rate of progression of DN was similar in KK and XQ patients: %deltaGFR was 4.1/year (median, range: 22.9-30.6) vs. 4.2/year (9.8-26.7). Morphometric parameters of diabetic glomerulopathy were similar in the two genotype groups. Among patients with type 2 diabetes with abnormal AER, those carrying the Q PC-1 genotype have more severe DN but not a faster GFR decline than KK patients, thus suggesting faster DN development since diabetes diagnosis in XQ patients.

Diabetic nephropathy: renal structural studies in type 1 and type 2 diabetic patients

The clinical manifestations of diabetic nephropathy, proteinuria, decreasing glomerular filtration rate and increasing blood pressure are similar in type 1 and type 2 diabetes, while the renal lesions differ. Indeed, in type 1 diabetes, although also tubular, interstitial and arteriolar lesions are present, the most important structural changes involve the glomerulus, in contrast several type 2 diabetic patients, despite the presence of microalbuminuria or proteinuria, have normal glomerular structure with or without tubulo-interstitial and arteriolar abnormalities. The renal functional parameters are strongly related with the structural changes, especially with the degree of mesangial expansion in both type 1 and type 2 diabetes. Previous studies using light and electron microscopic morphometric analyses described the renal structural changes and the structural–functional relationships of diabetic nephropathy. This review focuses on these topics, documenting the contribution of renal biopsy studies in understanding the pathogenesis of diabetic nephropathy and in identifying the patients with higher risk to progress to end-stage renal disease.

Is podocyte injury relevant in diabetic nephropathy? Studies in patients with type 2 diabetes.

Podocyte structural changes have been suggested to be involved in the pathogenesis of albuminuria in diabetes. We evaluated podocytes density, number, and structure in 67 white patients with type 2 diabetes: 21 normoalbuminuric (NA), 23 microalbuminuric (MA), and 23 proteinuric (P). Kidney function and biopsy studies were performed; 20 kidney donors served as control subjects. Electron microscopic morphometric analysis was used to estimate numerical density of podocytes per glomerulus [Nv(epi/glom)], filtration slit length density per glomerulus (FSLv/glom), and foot process width (FPW). The number of podocytes per glomerulus (Epi N/glom) was obtained by multiplying Nv(epi/glom) by mean glomerular volume. Nv(epi/glom) was significantly decreased in all type 2 diabetic groups compared with control subjects and was lower in MA and P than in NA (P < 0.0001, ANOVA). Epi N/glom was lower in MA and P patients compared with control subjects (P < 0.002, ANOVA); however, there were no significant differences among the type 2 diabetic groups. In addition, MA and P had decreased FSLv/glom and increased FPW compared with NA (P < 0.005 for both, ANOVA). The albumin excretion rate was inversely related to Nv(epi/glom) and FSLv/glom and directly to FPW (P < 0.0005 for all), whereas there was no correlation with Epi N/glom. In conclusion, changes in podocyte structure and density occur since the early stages of diabetic nephropathy and might contribute to increasing albuminuria in type 2 diabetic patients. These findings also suggest that in white type 2 diabetic patients, the density of podocytes may be functionally more relevant than the absolute number.

Morphometric and Ultrastructural Characteristics of the Mitochondrial System of Cardiomyocytes in the Rat Left Ventricle under Conditions of Hypergravity

For the first time, electron-microscopic morphometric analysis of the mitochondrial system in cardiomyocytes of the left ventricle was performed in rats exposed to hypergravity (2 g). After five days of exposure, the number of long mitochondria sharply increased in the interfibrillar zone of cardiomyocytes. The numbers of intermitochondrial junctions (IMJs) were increased in all zones of mitochondria localization. The ultrastructure and numerical density of mitochondria remained within the normal limits. The same picture was revealed on day 19 (the end of exposure), but the numbers of IMJs in the perinuclear and subsarcolemmal pericapillary zones were lower than on day 5. One months after the end of 19-day exposure at 2 g, the test parameters of the mitochondrial system did not return to the norm. Apparently, this is why repeated exposure to hypergravity (2 g for five days after 30-day rest) failed to evoke a similar response from the mitochondrial system of cardiomyocytes.

Effects of cigarette smoking on glomerular structure and function in type 2 diabetic patients.

Prospective studies have established smoking as an independent risk factor for diabetic nephropathy, suggesting an adverse effect of smoking on glomerular structure and function. To test this hypothesis, this study evaluated GFR, metabolic profile, and smoking habits in 96 patients with type 2 diabetes and abnormal albumin excretion rate (AER). All patients underwent percutaneous kidney biopsy: mesangial fractional volume [Vv (mes/glom)] and glomerular basement membrane (GBM) width were estimated by electron microscopic morphometric analysis; interstitial fibrosis was estimated semiquantitatively by light microscopy. Forty-eight patients were smokers. Compared with nonsmokers, smokers had higher values of HbA(1c) (P = 0.002), AER (P = 0.026), GFR (P = 0.004), and GBM width (P = 0.002); moreover, GFR was higher in current smokers than in former smokers (P = 0.001), and GBM width was related to heavy smoking (F = 5.4; P = 0.006). Multiple linear regression analyses revealed that HbA(1c) was associated with fasting blood glucose (beta coef = 0.52; P < 0.001), smoking habit (beta coef = 0.31; P < 0.001), insulin therapy (beta coef = 0.22; P = 0.012), and male gender (beta coef = -0.20; P = 0.020); AER was related to Vv (mes/glom) (beta coef = 0.32; P = 0.003), GBM width (beta coef = 0.28; P = 0.016), and interaction between smoking habit and HbA(1c) (beta coef = 0.24; P = 0.040). GFR was negatively correlated with Vv (mes/glom) (beta coef = -0.57; P < 0.001) and age (beta coef = -0.29; P = 0.001) and positively correlated with GBM width (beta coef = 0.27; P = 0.012), heavy current smoking (beta coef = 0.24; P = 0.028), and HbA(1c) (beta coef = 0.28; P = 0.040); GBM width was explained by Vv (mes/glom) (beta coef = 0.53; P < 0.001), interaction between heavy smoking and HbA(1c) levels (beta coef = 0.25; P = 0.003), and diabetes duration (beta coef = 0.23; P = 0.010). Smoking habit did not affect the index of interstitial fibrosis. In conclusion, cigarette smoking affects glomerular structure and function in type 2 diabetes and may be an important factor for the onset and progression of diabetic nephropathy.

Glomerular structural changes and structural-functional relationships at early stage of diabetic nephropathy in Japanese type 2 diabetic patients.

Details of renal structural changes and structural-functional relationships at the early stage of diabetic nephropathy (DN) in type 2 diabetes are not well known. The present review focuses on these topics from previous studies using light and electron microscopic morphometric analysis. Glomerular hypertrophy, one of the histological changes in DN, is present in type 2 as well as in type 1 diabetic patients. However, mechanisms of increased glomerular size might be different from those in type 1 diabetes. Other typical glomerular changes, glomerular basement membrane thickening and mesangial expansion, are present in normoalbuminuric type 2 diabetic patients as a group. However, these parameters are similar between normo- and microalbuminuric patients. Renal structural-functional relationships cannot be seen in type 2 diabetic patients and therefore urinary albumin might not be a reliable indicator for glomerular structural changes in type 2 diabetic patients. Although previous reports showed reversibility of advanced diabetic glomerulosclerosis in type 1 diabetes by 10 years of strict glycemic control, there is no report regarding histological reversibility by therapeutic interventions in type 2 diabetic patients. In addition, it is unclear whether DN lesions are concordant or discordant with diabetic retinopathy grade in type 2 diabetes. From this information, renal structural changes or structural-functional relationships in type 2 diabetic patients might be heterogeneous and different from those in type 1 diabetic patients. Careful longitudinal study of renal structure and function including serial renal biopsy at the early stage of DN in type 2 diabetes is necessary.

Two-dimensional mapping of the endogenous proteins of the rat hepatocyte Golgi complex cleared of proteins in transit.

The discovery of additional endogenous Golgi proteins will lead to significant new insights into Golgi function. To this end, stacked Golgi fractions (SGFs) were isolated from rat liver before (CTL SGF) and after molecules in transit through the Golgi were cleared by pre-treatment with cycloheximide (CHX SGF). Electron microscopic (EM) morphometric and biochemical analyses showed that the in vivo stacked morphology is retained, that > 90% of the elements can be positively identified as Golgi stacks and cisternae, and that transmembrane protein markers of the Golgi complex are enriched 300- to 800-fold over starting postnuclear supernatant (PNS). High-resolution two-dimensional (2-D) gel mapping has been carried out on the CTL PNS, CTL SII (an intermediate fraction), CTL SGF, CHX SGF, CHX SGF - high pH supernatant, and CHX SGF - high pH pellet. This analysis, coupled with immunoblotting and alignment of the 2-D gels with master gels, has allowed the identification of a number of known proteins and the preliminary characterization of the most abundant 173 Golgi-specific proteins. These 173 proteins have been placed into three categories: cargo, cytosolic Golgi-associated, and resident Golgi proteins. These categories are tentative and will be modified as more data are acquired from immunoblotting and protein sequencing.

Progressive alveolar septal injury in primates exposed to 60% oxygen for 14 days

Moderate exposures to hyperoxia are becoming increasingly common in clinical medicine as advancing technology allows O2 to be more effectively delivered to nonintubated patients. The sensitivity of the lung to injury by a subchronic exposure to 60% O2 was investigated, using baboons and serial lobar biopsies. Because results obtained from different regions of the lung were compared, the alveolar architecture of different lung lobes of three controls was studied, with the use of electron microscopic morphometric analyses, to assess possible lobar differences in volume, surface, and numerical densities of cells and tissues. In animals exposed to 60% O2, the same techniques were used to assess specific tissue changes in the epithelial, interstitial, and endothelial compartments of the alveolar septa. All six lobes of the normal baboon lung were found to be identical with respect to alveolar architecture. Thus, for gases of low reactivity and given in high concentrations, such as O2, cross-comparisons between different lobes are appropriate. Exposure to 60% O2 was found to cause proliferation of alveolar type II epithelium, suggesting a low-grade, chronic epithelial injury. Animals allowed to recover for 8 wk in room air showed progressive changes in the alveolar interstitium, involving increases in both cells and matrix. Because sequential lobar resections were done, animals were exposed both to 60% O2 and to the effects of general anesthesia and thoracotomies. The exposure to 60% O2 for 2 wk in this experimental setting leads to an alveolar septal injury that includes a progressive interstitial fibrotic response.

Antioxidants protect podocyte foot processes in puromycin aminonucleoside-treated rats.

Whether a reduction in urinary protein excretion in rats coadministered puromycin aminonucleoside and antioxidants was associated with a reduction in alterations to glomerular epithelial cell (podocyte) ultrastructure was examined. Daily urinary protein excretion was measured in rats that received a single i.v. injection of saline or puromycin aminonucleoside with or without coadministration of antioxidants. The coadministration of alpha-tocopherol/ascorbic acid, dimethyl thiourea, or superoxide dismutase to puromycin aminonucleoside-treated rats reduced proteinuria by approximately 90, 40, and 60%, respectively, over the 18-day period studied. For a second group of rats, daily urinary protein excretion was measured and kidneys were processed for light microscopy and transmission and scanning electron microscopy 4, 5, and 10 days after injection. Transmission electron microscopic morphometric analysis of glomeruli from puromycin aminonucleoside-treated rats coadministered antioxidants revealed significantly reduced foot process effacement on Days, 4, 5, and 10 compared with rats that received puromycin aminonucleoside alone. Thus, at Day 10, puromycin aminonucleoside-treated rats coadministered alpha-tocopherol/ascorbic acid, dimethyl thiourea, or superoxide dismutase contained 90, 74, and 88% (P < 0.01 in all cases) more glomerular epithelial cell filtration slits per unit length of glomerular basement membrane than rats treated with puromycin aminonucleoside alone. In contrast, by scanning electron microscopy, the antioxidants were found to provide no protection against the changes occurring in glomerular epithelial cell bodies and major processes. These results provide further evidence of a role for reactive oxygen species in puromycin aminonucleoside nephrosis and indicate that the antioxidants provide protection against the changes occurring in glomerular epithelial cell foot processes.