Age-related nephropathy and proteinuria in rats with intact kidneys exposed to diets with different protein content

Tubulointerstitial damage and glomerular sclerosis are findings commonly observed in the experimental models of adriamycin and puromycin aminonucleoside nephrosis. It has been suggested that in such models proteinuria might be an important mediator of tubulo-interstitial damage which in turn may determine the progression of the disease favoring the development of glomerulosclerosis. The objective of the present investigation was to establish the temporal relationship between proteinuria, tubulo-interstitial damage and glomerulosclerosis in aging rats with intact kidneys exposed to diets with different protein content. There were six groups of rats studied. Animals of groups 1, 5, and 6 (N = 10) were fed diets containing 20, 35, and 6% protein, respectively, for 20 months and sacrificed at the end of the experimental period. Rats in groups 3 and 4 (N = 6) exhibited marked and mild proteinuria, respectively, after 14 months of maintenance on standard diet, and followed for two additional months after the onset of proteinuria with the aim of evaluating the pattern of renal damage after a relatively short period of proteinuria. Rats in group 2 (N = 10) were fed standard diet and sacrificed before (5 months) and at the onset of proteinuria (10 months). Protein excretion and plasma creatinine were measured for each animal every month. Pathologic examination was performed by light and electron microscopy. At the onset of proteinuria neither renal structural nor functional abnormalities were detected. After 20 months, rats fed standard diet developed tubulo-interstitial damage (score: 1.29 +/- 1.05) and focal glomerular sclerosis (percentage of glomeruli with focal segmental glomerular sclerosis: 16.70 +/- 16.40). A significant correlation was found between the degree of tubulo-interstitial damage and the percentage of glomeruli with focal glomerular sclerosis (r = 0.99, p less than 0.01). Development of tubulo-interstitial damage and focal glomerular sclerosis were correlated with heavy and sustained proteinuria. The high protein diet significantly worsened proteinuria (at month 20: 247.08 +/- 101.73 mg/day), tubulo-interstitial changes (score: 1.99 +/- 0.70), focal glomerular sclerosis (percentage of glomeruli with focal segmental glomerular sclerosis: 21.50 +/- 9.44) and was associated with deteriorating renal function (at month 20, plasma creatinine: 1.20 +/- 0.50 mg/dl).(ABSTRACT TRUNCATED AT 400 WORDS)     

Focal segmental glomerular hyalinosis and/or sclerosis in rats with congenital unilateral hydronephrosis.

Focal segmental glomerular hyalinosis and/or sclerosis (FSHS) was observed in five Wistar-Imamichi rats with congenital unilateral hydronephrosis (CUH rats). Marked proteinuria (164.9 +/- 138.4 mg/day) was observed in the CUH rats. Immunoperoxidase staining for IgM, C3 and IgG was positive in the glomeruli, showing in a focal, segmental pattern that corresponded to the areas of FSHS seen by light microscopy. These glomerular findings were extremely similar to those of human focal glomerular sclerosis (FGS). FSHS was found to be common to both the hydronephrotic kidney and the contralateral kidney without hydronephrosis. Morphometry revealed that the glomerular area of the juxtamedullary glomeruli was greater than that of superficial glomeruli in control rats (11,037 micron2 vs. 6,847 microns2). On the other hand, glomerular hypertrophy was observed in non-sclerotic glomeruli of CUH rats (superficial glomeruli; 12,477-16,123 microns2, juxtamedullary glomeruli; 14,635-18,418 microns2). Also, a decreased in the number of glomeruli within the range 1.8-4.1 per unit area (1 mm2) was seen in CUH rats compared with control rats (mean 4.4). These results suggest that the increased rate of development of FSHS is based on hyperfiltration in the remaining functional nephrons.     

Glomerular epithelial cell function and pathology following extreme ablation of renal mass.

The role of the pathologic features and dysfunction of glomerular epithelial cells (GECs) in the pathogenesis of glomerular scarring was studied in the remnant kidney model (RK) (1 and 5/6 nephrectomy) in rats. Three weeks after surgery serum creatinine was greater in the RK than either sham-operation controls (SHAM) or spontaneously hypertensive rats (SHRs). Blood pressure was higher in the RK (181 +/- 26 mm Hg) than in SHAM (129 +/- 17, P less than 0.05) but not SHR (195 +/- 15, P less than 0.05). GEC endocytosis, assessed by protamine heparin aggregate (PHA) disappearance (10), was not different from that in SHAM. Glomerular damage was greater in RK (glomerular damage index, 30 +/- 18) than in SHAM animals (4 +/- 3, P less than 0.05) and SHR (0, P less than 0.05), and 2 of 11 RK animals had fibrinoid necrosis and thrombosis of arterioles and glomeruli. Segmental sclerosis occurred in only 1 RK animal (0.6% of glomeruli). Six weeks after surgery serum creatinine and urinary protein excretion remained higher in the RK than in the SHAM animals. Blood pressure was higher in RK (158 +/- 34 mm Hg) than in SHAM animals (144 +/- 24), but the difference was not significant. PHA disappeared from the glomerulus at a slower rate in RK than in SHAM animals (outside the 95% confidence limits of SHAM). Glomerular pathology was more widespread in RK than in SHAM animals (glomerular damage index, 73 +/- 62 versus 3 +/- 8, P less than 0.05), and 4 of 11 animals had acute hypertensive injury in arterioles and glomeruli. Segmental glomerular sclerosis was only seen in the animals with necrotic glomeruli. GEC dysfunction is not demonstrable until long after proteinuria and hypertension are established, and it only occurs in the context of severe, acute glomerular injury when the epithelial cells separate from the capillary wall and undergo severe degenerative changes and necrosis. The acute glomerular and vascular lesions in the RK model are morphologically similar to malignant nephrosclerosis in humans. Segmental glomerular sclerosis occurs only after proteinuria is well established in the context of severe glomerular injury, and it appears to represent, at least partially, progression of more proximate glomerular capillary injury.     

Impairment and recovery of the clipped kidney in two kidney, one clip hypertensive rats during and after antihypertensive therapy

Earlier experiments have shown that in sodium depleted hypertensive rats with bilaterally constricted renal arteries the arterial pressure normalized after blockade of the renin-angiotensin system; simultaneously acute renal failure occurred. In hypertensive rats with unilateral renal artery stenosis an impaired excretory function of the clipped kidney can be expected, but may not be detectable by conventional tests of renal function. Male Wistar rats with chronic two kidney, one clip hypertension were fed a low sodium diet. After 7 days the rats were treated with vehicle, with the vasodilator dihydralazine, or with the angiotension converting enzyme inhibitor MK 421 for 2 weeks. During the 14-day treatment period a continuous blood pressure reduction was achieved in dihydralazine and MK 421 treated rats. Overall excretory kidney function (plasma creatinine concentration) was well maintained in all three groups until the end of the antihypertensive drug treatment. At the end of drug therapy mean glomerular filtration rates of the left clipped kidneys were significantly lower in both treated groups compared to hypertensive controls, and mean glomerular filtration rate of the left clipped kidneys of dihydralazine treated rats was significantly higher than in MK 421 treated rats: controls (N = 6) 1.03 +/- 0.03, dihydralazine-group (N = 10) 0.28 +/- 0.07, MK 421-group (N = 9) 0.03 +/- 0.01 ml/min. Renal blood flows were comparable in both treated groups. Only the left kidneys of rats treated with MK 421 showed a prominent tubular atrophy. Seven days after declipping of the left renal artery and right nephrectomy a considerable restitution of the tubular structure had occurred in the MK 421-group. The recovery of tubular epithelial cells was paralleled by a rise in glomerular filtration rate: MK 421 group (N = 7) 1.25 +/- 0.08 ml/min. Thus, the clipped kidney in two kidney, one clip hypertensive rats showed functional and morphological signs of impairment when systemic arterial pressure was reduced to the normal range. The alterations of the clipped kidney were most pronounced in rats with renin-angiotensin system-blockade.     

Rare glomerular capillary regeneration and subsequent capillary regression with endothelial cell apoptosis in progressive glomerulonephritis.

Glomerulonephritis (GN) leading to glomerular sclerosis remains an important cause of renal failure. The glomerulus is a capillary network, but endothelial and vascular reactions during progressive GN are not well understood. We have, therefore, examined the morphological alterations of glomerular capillary network and endothelial cells during the progression of damaged glomeruli to glomerular sclerosis. A progressive model of anti-glomerular basement membrane (GBM) GN was induced in Wistar-Kyoto (WKY) rats with a single injection of anti-rat GBM antibody. Severe necrotizing glomerular injuries were observed between day 5 and week 3 with a reduction in the number of total glomerular endothelial cells and total glomerular capillary lumina per glomerular cross sections. In necrotizing lesions, the glomerular endothelial cells were lost with the destruction of the glomerular capillary network. Moreover, angiogenic capillary repair with proliferation of endothelial cells was rare in severely damaged regions of glomeruli. Subsequently, mesangial hypercellularity and marked mesangial matrix accumulation occurred with absence of the development of a capillary network, and the necrotizing lesions progressed to sclerotic scars until 8 weeks. Although active necrotizing lesions could not be seen in damaged glomeruli between week 4 and week 8, the number of apoptotic endothelial cells gradually increased in the glomerular capillaries (0.10 +/- 0.01 apoptotic endothelial cells/glomerular cross section at week 8 versus 0.00 +/- 0.00 control cells (mean +/- SEM; P < 0.05) with the progression of glomerular sclerosis. Whereas the number of apoptotic endothelial cells increased in the damaged glomeruli, the number of total glomerular endothelial cells decreased (9.3 +/- 3.0 cells/glomerular cross section at week 8 versus 24.8 +/- 3.0 cells in control (mean +/- SD); P < 0.001) with regression of glomerular capillaries (3.6 +/- 2.5 capillary lumina/glomerular cross section at week 8 versus 35.0 +/- 5.0 capillary lumina in control (mean +/- SD); P < 0.001). Finally, glomerular endothelial cells could not be detected in the sclerotic lesions in progressive anti-GBM GN in WKY rats. These data indicate that the destruction of the capillary network of glomeruli and subsequent incomplete angiogenic capillary repair leads to glomerular sclerosis in progressive GN. Endothelial cell apoptosis with glomerular capillary regression may also contribute to the development of glomerular sclerosis. Injury of the glomerular capillary network with endothelial cell damage, including apoptosis and subsequent incomplete capillary repair, plays an important role in the progression of glomerular sclerosis during anti-GBM GN in WKY rats.     

Glomerular sclerosis in Wistar rats: analysis of its variable occurrence after unilateral nephrectomy.

Individual differences in susceptibility to the development of focal and segmental glomerular hyalinosis and sclerosis (FSGHS) were studied in rats after unilateral nephrectomy. A total of 20 male Wistar rats underwent unilateral nephrectomy at 3 months of age. Glomerular number in the removed kidney ranged from 17,000 to 32,700 with a mean value of 25,997 (n = 19). At death 30 weeks later, the incidence of glomeruli with FSGHS in the remaining kidney varied from 0 to 20% with a mean of 4.4%. However, the percentage of glomeruli with FSGHS and the degree of proteinuria did not correlate either with glomerular number per kidney (left and right kidneys were shown to be equivalent for glomerular number) or with glomerular volume on killing. The occurrence of FSGHS correlated significantly with mean protein excretion (P less than 0.01) and serum cholesterol levels (P less than 0.01). In conclusion, in the rat a relatively low number of nephrons in the kidneys does not increase susceptibility to the development of FSGHS.     

Glomerular sclerosis in Wistar rats: analysis of its variable occurrence after unilateral nephrectomy

Individual differences in susceptibility to the development of focal and segmental glomerular hyalinosis and sclerosis (FSGHS) were studied in rats after unilateral nephrectomy. A total of 20 male Wistar rats underwent unilateral nephrectomy at 3 months of age. Glomerular number in the removed kidney ranged from 17,000 to 32,700 with a mean value of 25,997 (n = 19). At death 30 weeks later, the incidence of glomeruli with FSGHS in the remaining kidney varied from 0 to 20% with a mean of 4.4%. However, the percentage of glomeruli with FSGHS and the degree of proteinuria did not correlate either with glomerular number per kidney (left and right kidneys were shown to be equivalent for glomerular number) or with glomerular volume on killing. The occurrence of FSGHS correlated significantly with mean protein excretion (P less than 0.01) and serum cholesterol levels (P less than 0.01). In conclusion, in the rat a relatively low number of nephrons in the kidneys does not increase susceptibility to the development of FSGHS.     

The effects of high-fat diet on the renal structure and morphometric parametric of kidneys in rats

To characterize the kidney in a high-fat-induced obesity model, we examined the renal structure of adult Sprague-Dawley rats fed a control diet or a high-fat diet for 3 months. Ten adult female Sprague-Dawley rats were fed a diet consisting highly of fat (30%) for a period of 3 months. Ten control rats were maintained with standard rat chow. All animals were weighed every 10 days for 3 months. At the end of the experiment, the naso-anal length of the anaesthetized rats was measured to calculate body mass index, and subsequently whole kidneys of intracardially formalin-perfused animals were removed. Quantitative features of the kidney were analysed with the Cavalieri and physical dissector methods applied to serial paraffin sections. Kidney samples were also examined histologically. The body mass indices of the control and treatment groups were 4.528 +/- 0.242 and 5.876 +/- 0.318 kg m(-2), respectively. The difference between the body mass indices of the two groups was statistically significant (P < 0.01, Mann-Whitney U-test), suggesting that the animals fed with a high-fat diet may be overweight. Stereological examination of the kidneys revealed differences in kidney weight, total kidney volume, volume of cortex, medulla, glomeruli, proximal and distal tubules, and numerical density of glomeruli and glomerular height in the treatment group compared with the control group. Light microscopic investigation showed a dilatation in blood vessels and Bowman's space, mononuclear cell infiltration, degeneration in nephrons, including glomerulosclerosis and tubular defects, and an increase in the connective tissue in the kidneys in the treatment group. We concluded that a fatty diet is responsible for the rats' obesity and may lead to renal deformities as a result of histopathological changes such as dilatation, tubular defects, inflammation and connective tissue enlargement of the kidney.     

Effect of hyperlipidemia on glomerular sclerosis in unilateral nephrectomized rats

The development of focal segmental glomerular sclerosis (FSGS) and its relation to hypertriglycemia were studied in unilateral nephrectomized rats. Group A (n = 6), fed standard rat chow supplemented with 20% beef tallow and 0.6% cholic acid for 25 weeks, showed evidence of hypertriglycemia (109.4 +/- 4.3 mg/dl). Group B (n = 7) was given the same rat chow as group A, but they did not have high serum levels of TG (66.4 +/- 2.3 mg/dl). Group C (n = 6) were the controls and their serum TG levels were 53.0 +/- 3.8 mg/dl. The incidence of FSGS and body weight was significantly higher in group A than in groups B (p less than 0.01) and C (p less than 0.05). In all three groups, rats with over a 4% FSGS revealed significantly high serum TG levels, proteinuria, and body weight, as compared with rats with less than 1% of FSGS. The serum cholesterol levels did not correlate with the incidence of FSGS. We tentatively conclude that hypertriglycemia induced by a diet rich in saturated fatty acid may play an important role in the production and progression of FSGS.     

Focal Segmental Glomerular Hyalinosis and/or Sclerosis in Rats with Congenital Unilateral Hydronephrosis

Focal segmental glomerular hyalinosis and/or sclerosis (FSHS) was observed in five Wistar-Imamichi rats with congenital unilateral hydronephrosis (CUH rats). Marked proteinuria (164.9+138.4mg/day) was observed in the CUH rats. Immunoperoxidase staining for IgM, C3 and IgG was positive in the glomerull, showing in a focal, segmental pattern that corresponded to the areas of FSHS seen by light microscopy. These glomerular findings were extremely similar to those of human focal glomerular sclerosis (FGS). FSHS was found to be common to both the hydronephrotic kidney and the contralateral kidney without hydronephrosis. Morphometry revealed that the glomerular area of the juxtamedullary glomeruli was greater than that of superficial glomeruli in control rats (11,037 μm² vs. 6,847 μm²). On the other hand, glomerular hypertrophy was observed in non-sclerotic glomeruli of CUH rats (superficial glomeruli; 12,477–16,123 μm², juxtamedullary glomeruli; 14,635–18,418 μm²). Also, a decreased in the number of glomeruli within the range 1.8-4.1 per unit area (1 mm²) was seen in CUH rats compared with control rats (mean 4.4). These results suggest that the increased rate of development of FSHS is based on hyperfiltration in the remaining functional nephrons. Acta Pathol Jpn 41: 653–660, 1991.     

Vascular endothelial growth factor enhances glomerular capillary repair and accelerates resolution of experimentally induced glomerulonephritis

Vascular endothelial growth factor (VEGF) regulates angiogenesis through endothelial cell proliferation and plays an important role in capillary repair in damaged glomeruli. We tested the hypothesis that VEGF might be beneficial in rats with severe glomerular injury in glomerulonephritis (GN) based on its angiogenic and vascular remodeling properties. Acute GN with severe glomerular destruction was induced in rats by injection of anti-Thy-1.1 antibody (day 0) and Habu-snake venom (day 1). Rats were intraperitoneally injected with recombinant human VEGF(165) (10 microg/100 g body wt/day) or vehicle from day 2 to day 9, and monitored changes in glomerular capillaries, development of glomerular inflammation, and progression to glomerular sclerosis after acute glomerular destruction in both groups. Rats that received anti-Thy-1.1 antibody and Habu-snake venom showed severe mesangiolysis and marked destruction of capillary network on day 2. VEGF was expressed on glomerular epithelial cells, proliferating mesangial cells, and some infiltrating leukocytes, and VEGF(165) protein levels increased in damaged glomeruli during day 5 to day 7. Normal, damaged, and regenerating glomerular endothelial cells expressed VEGF receptor flk-1. However, endothelial cell proliferation and capillary repair was rare in vehicle-treated rats with severe glomerular damage, which progressed to global sclerosis and chronic renal failure by week 8. In contrast, in the VEGF-treated group, VEGF(165) significantly enhanced endothelial cell proliferation and capillary repair in glomeruli by day 9 (proliferating endothelial cells: VEGF(165), 4.3 +/- 1.1; control, 2.2 +/- 0.9 cells on day 7, P < 0.001; and glomerular capillaries: VEGF(165), 24.6 +/- 4.8; control, 16.9 +/- 3.4 capillaries on day 7, P < 0.01). Thereafter, damaged glomeruli gradually recovered after development of capillary network by week 8, and significant improvement of renal function was evident in the VEGF-treated group during week 8 (creatinine: VEGF(165), 0.3 +/- 0.1; control, 2.6 +/- 0.9 mg/dl, P < 0.001; proteinuria: VEGF(165), 54 +/- 15; control, 318 +/- 60 mg/day, P < 0.001). We conclude that the beneficial effect of VEGF(165) in severe glomerular injury in GN emphasizes the importance of capillary repair in the resolution of GN, and may allow the design of new therapeutic strategies against severe GN.     

Intraglomerular thrombotic tendency and glomerular ADPase. Unilateral impairment of ADPase elicits a proaggregatory microenvironment in experimental glomerulonephritis

It has been proposed, predominantly from ex vivo studies, that glomerular ADPase may function as an antithrombotic principle within the rat kidney. Therefore, intraglomerular platelet aggregation was studied in vivo in rats after impairment of glomerular ADPase activity using local X-irradiation (20 Gy). Biochemical assays in suspensions of glomeruli obtained from rats 24 hours after local X-irradiation (group I) demonstrated a significant reduction in ADPase activity as compared to sham treated rats (group II; p less than 0.01). Cytochemical observations at the ultrastructural level showed that this reduction in glomerular enzyme activity represents in particular ADPase activity detectable in the basement membrane. Following X-irradiation, intraglomerular platelet aggregation was quantitatively studied in two groups of rats. Both groups received X-irradiation of the left kidney (20 Gy). Twenty-four hours after X-irradiation, animals received an intravenous injection of either 0.5 ml of saline (group III; N = 6) or 0.5 ml of heterologous nephrotoxic serum (NTS; group IV; N = 6). Subsequently, 24 hours after this injection, platelet aggregation in left kidneys was compared with aggregation in contralateral non-X-irradiated kidneys. The results showed that while X-irradiation per se did not induce intraglomerular platelet aggregation as compared with the contralateral kidney (0.20 +/- 0.08% versus 0.17 +/- 0.06% platelet aggregation/glomerulus), a significant increase in platelet aggregation could be demonstrated in X-irradiated kidneys in the early phase of nephrotoxic serum nephritis as compared with the contralateral nephritic kidney (2.45 +/- 0.66% versus 1.37 +/- 0.35% platelet aggregation per glomerulus; p less than 0.005). A potential effect of altered influx of inflammatory cells after X-irradiation could be excluded since no difference in H2O2 producing cells was observed between left and right kidneys. Thus, while ADPase impairment by X-irradiation does not induce platelet aggregation per se, it is clear that in proaggregatory conditions, like in NTS nephritis, the thrombotic tendency, due to decreased glomerular ADPase, is enhanced. These results demonstrate the functional significance of glomerular ADPase activity as an antithrombotic principle following platelet activation in vivo.     

Comparison of monounsaturated fatty acids and carbohydrates for lowering plasma cholesterol

To examine the effects of dietary fatty acids and carbohydrate on plasma lipids and lipoproteins, 11 patients with a mean plasma total cholesterol level of 251 +/- 10 mg per deciliter were studied on a metabolic ward during three dietary periods, each lasting four weeks. A liquid diet rich in monounsaturated fatty acids ("High-Mono") and a diet low in fat ("Low-Fat") were compared with a diet high in saturated fatty acids ("High-Sat"). The High-Sat and High-Mono diets contained 40 percent of their total calories as fat and 43 percent as carbohydrate; the Low-Fat diet had 20 percent fat and 63 percent carbohydrate. Body weight was kept constant by adjusting total caloric intake. As compared with the High-Sat diet, both the High-Mono and Low-Fat diets lowered plasma total cholesterol (by 13 percent and 8 percent, respectively) and low-density lipoprotein cholesterol (by 21 percent and 15 percent, respectively). As compared with the High-Sat diet, the Low-Fat diet raised triglyceride levels and significantly reduced plasma high-density lipoprotein cholesterol. In contrast, the High-Mono diet had no effect on levels of triglycerides or high-density lipoprotein cholesterol. The ratio of low-density to high-density lipoprotein cholesterol was also significantly lower when the High-Mono diet rather than the Low-Fat diet was followed. Therefore, in short-term studies in which liquid diets are used and body weight is kept constant, a diet rich in monounsaturated fatty acids appears to be at least as effective in lowering plasma cholesterol as a diet low in fat and high in carbohydrate.     

Maturational development of glomerular ultrafiltration in the rat.

To ascertain the cause of low glomerular filtration rate in newborn and immature mammals, we measured glomerular pressures and flows directly in immature (30- to 45-day-old) euvolemic Munich-Wistar rats with surface glomeruli. As with total kidney GFR, single nephron (SN)GFR was found to be significantly lower than in adult rats, on average by 40% when corrected for kidney weight. Equality between efferent oncotic pressure and transglomeruler hydraulic pressure difference (deltaP) was usually achieved in immature rats, indicating that the glomerular capillary ultrafiltration coefficient is not a factor limiting SNGFR and GFR in immature rats. Although the average values for deltaP in immature rats were slightly, albeit significantly, lower than in adults, markedly lower values (79 +/- 5 vs. 136 +/- 10 nl/min per g kidney wt) for glomerular plasma flow rate (QA) proved to be the primary factor responsible for the lower SNGFR and GFR values in immature rats. Considerably higher values for afferent and efferent arteriolar resistances contributed to this low QA state in immature rats.     

Effect of cholesterol on the position of segmental lesions in unilaterally nephrectomized rats.

Different positions of segmental lesions within glomeruli may correspond to different pathogenetic mechanisms. The effect of a high cholesterol diet on the position of lesions had not previously been investigated. This was studied in rats following unilateral nephrectomy, as a change in position would suggest a different mechanism of damage. Thirty-two female WAG/ola rats had unilateral nephrectomy. Half the rats were given a diet supplemented with 4 per cent cholesterol and 1 per cent cholic acid. At death, six at 10 weeks after nephrectomy and the rest at 24 weeks, kidney sections were examined microscopically. There were significantly more segmental lesions in the cholesterol-fed rats than in the controls, and these lesions were almost entirely at the glomerular hilum in both groups. Significantly more glomeruli contained foamy cells in the cholesterol-fed group, both within lesions and away from them. These findings confirmed that in reduced renal mass, segmental lesions are mainly hilar. The diet increases the number of glomeruli affected by lesions, but these are still mainly hilar. Therefore one possibility is that hypercholesterolaemia worsens the hyperfiltration effect on glomeruli. The diet also produces foamy cells scattered throughout the glomeruli but these do not appear to develop into segmental lesions.     

Glomerular volume in type 2 (noninsulin-dependent) diabetes estimated by a direct and unbiased stereologic method

Glomerular volume was estimated in 20 type 2 diabetic patients (age 64 +/- 6 years, duration of diabetes 6 +/- 5 years) compared with 14 sex- and age-matched controls, as well as in a group of 11 very long-term type 1 diabetic patients (age 61 +/- 12 years, duration of diabetes 44 +/- 11 years). One whole autopsy kidney was obtained prospectively, and a known fraction (approximately equal to 1/140) was sampled systematically and embedded in plastic (JB-4 glycolmetacrylate), thereby essentially eliminating shrinkage. Sections 15-microns thick were stained with periodic acid-Schiff. Mean glomerular volume was estimated on a random sample of glomeruli using the disector method. Frequency of glomerular occlusion and mean volume of open glomeruli was estimated. Mean glomerular volume was not different between type 2 diabetic patients and controls (5.3 +/- 1.7 M mu3/1.73 m2 versus 5.3 +/- 1.9 M mu3/1.73 m2) nor was total glomerular volume or kidney weight. Frequency of glomerular occlusion was 4.8 +/- 5.7% in controls, 8.9 +/- 7.8% (p = 0.10) in type 2 patients, and 16.8% +/- 20.7 (p less than 0.05) in type 1 patients. In type 2 patients there was a correlation between frequency of glomerular occlusion and mean volume of open glomeruli (r = 0.44, p = 0.05), and the same tendency was seen in type 1 patients (r = 0.49, p = 0.12). By the present method the absolute level of glomerular volume was increased by at least a factor of two compared with previous studies. This illustrates the problems arising from shrinkage of tissue in paraffin and stresses the importance of using an unbiased stereological method. The lack of increase in total glomerular volume is in accordance with clinical findings of lack of glomerular hyperfiltration in type 2 patients, findings in contrast to those in type 1 diabetes. It is suggested that hyperfiltration per se is not the cause of glomerulopathy.     

Messenger RNA Expression for Growth Factors in Glomeruli from Focal Glomerular Sclerosis

Focal glomerular sclerosis was induced in rats by chronic injections of puromycin aminonucleoside (PAN) on Days 0, 27, 34, and 41 and by unilateral nephrectomy on Day 22. Rats were sacrificed on Days 0, 8, and 20 (acute phase) and on Days 48, 60, and 80 (sclerotic phase). The percentage of sclerosing glomeruli was 16.6% on Day 48 and increased significantly to 72.8% on Day 80. We examined glomerular mRNA levels for proliferating cell nuclear antigen (PCNA), platelet-derived growth factor (PDGF)-A and B chains, transforming growth factor (TGF)-β, epidermal growth factor (EGF), insulin-like growth factor (IGF)-I, and basic fibroblast growth factor (bFGF) on Days 0, 8, 20, 48, 60, and 80. Although these growth factor mRNA levels showed little change in glomeruli until Day 20, all growth factor mRNA levels increased in glomeruli during the sclerotic phase of PAN nephrosis as glomerular sclerosis progressed. On Day 80, mRNA levels for PCNA, PDGF-A and B chains, TGF-β, EGF, IGF-I, and bFGF increased 12-, 10-, 12-, 15-, 2-, 2-, and 8-fold, respectively, in the glomeruli of PAN-treated rats with marked glomerular sclerosis when compared with control rats. Unilateral nephrectomy without PAN administration did not cause glomerular sclerosis up to Day 80 and mRNA levels for PCNA, PDGF-A and -B chains, TGF-β, EGF, IGF-I, and bFGF in this group were almost the same as those in the normal sham-operated group. These data suggest that changes in growth factor mRNA levels in glomeruli may contribute to the development of PAN-induced glomerular sclerosis.     

Increased capillary branching contributes to angiotensin type 1 receptor blocker (ARB)-induced regression of sclerosis

Chronic kidney disease is characterized by progressive glomerulosclerosis and tubulointerstitial fibrosis. High-dose angiotensin type 1 receptor blocker (ARB) or angiotensin-converting enzyme inhibitor can induce regression of existing glomerulosclerosis, at least in part by decreasing matrix accumulation. However, the potential mechanisms of remodeling of capillary loops remain obscure. This study aimed to determine whether capillary branching was augmented in glomeruli with ARB-induced regression of sclerosis. Three-dimensional confocal images were assessed by graph theory analysis to explore the topology of the glomerular capillary network. Compared with normal glomeruli, glomeruli of rats with progressive sclerosis were enlarged but had a significantly reduced number of capillary segments and capillary branch points and decreased complexity of the glomerular network. In contrast, in rats with regression of sclerosis induced by ARB, glomerular enlargement was due to a significantly increased number of glomerular capillary segments and capillary branch points and restored complexity of the capillary network. These data support the theory that capillary growth contributes to regression of sclerosis and is mediated at least in part by ARB-induced increased complexity and branching of capillary segments.     

Changes in plasma lipids and lipoproteins in overweight men during weight loss through dieting as compared with exercise

We studied separately the influence of two methods for losing fat weight on the levels of plasma lipids and lipoproteins in overweight sedentary men--decreasing energy intake without increasing exercise (diet), and increasing energy expenditure without altering energy intake (exercise, primarily running)--in a one-year randomized controlled trial. As compared with controls (n = 42), dieters (n = 42) had significant loss of total body weight (-7.8 +/- 0.9 kg [mean +/- SE]), fat weight (-5.6 +/- 0.8 kg), and lean (non-fat) weight (-2.1 +/- 0.5 kg) (P less than 0.001 for each variable), and exercisers (n = 47) had significant loss of total body weight (-4.6 +/- 0.8 kg) and fat weight (-3.8 +/- 0.7 kg) (P less than 0.001 for both variables) but not lean weight (-0.7 +/- 0.4 kg). Fat-weight loss did not differ significantly between dieters and exercisers. All subjects were discouraged from altering their diet composition; however, dieters and exercisers had slight reductions in the percentage of kilojoules derived from fat. As compared with the control group, both weight-loss groups had significant increases (P less than 0.01) in plasma concentrations of high-density lipoprotein (HDL) cholesterol (diet vs. exercise, 0.13 +/- 0.03 vs. 0.12 +/- 0.03 mmol per liter), HDL2 cholesterol (0.07 +/- 0.02 vs. 0.07 +/- 0.02 mmol per liter), and HDL3 cholesterol (0.07 +/- 0.02 vs. 0.06 +/- 0.02 mmol per liter) and significant decreases (P less than 0.05) in triglyceride levels (diet vs. exercise, -0.35 +/- 0.14 vs. -0.24 +/- 0.12 mmol per liter). Levels of total and low-density lipoprotein cholesterol were not significantly changed, relative to values in controls. None of these changes were significantly different between dieters and exercisers. Thus, we conclude that fat loss through dieting or exercising produces comparable and favorable changes in plasma lipoprotein concentrations.     

Effects of tissue preparation on glomerular volume and capillary structure in the rat

The effect of tissue preparation on glomerular volume in normal rats was assessed. In group 1 rats (N = 8), kidney tissue was obtained by immersion-fixation of needle biopsy cores and excised slices from the left kidney and by perfusion-fixation of the remaining right kidney at close to ambient arterial pressure. In group 2 rats (N = 8), tissue was obtained by kidney perfusion at a supernormal pressure (approximately 165 mm Hg). Studies in group 1 showed that mean glomerular volume (VG) was not different in biopsy cores (1.07 +/- 0.13 x 10(6) mu 3) and in kidney slices fixed by immersion (0.92 +/- 0.09 x 10(6) mu 3). A significantly higher value for VG (1.51 +/- 0.18 x 10(6) mu 3) was obtained in kidneys perfusion-fixed at close to ambient arterial pressure. Morphometric studies showed that reduced VG in immersion-fixed tissue was associated with lowered values for peripheral capillary wall surface area (225 +/- 21 x 10(3) mu 2 versus 159 +/- 27 x 10(3) mu 2, p less than 0.05) and reduced mean capillary radius (4.5 +/- 6 mu versus 2.7 +/- 3 mu, p less than 0.05) compared with perfusion-fixed tissue. The data suggest that glomerular capillaries contract when tissue is immersion-fixed and shows that values for mean peripheral capillary wall surface area/glomerulus and mean glomerular capillary radius obtained in immersion- and perfusion-fixed tissue cannot be directly compared. Studies in group 2 showed that VG was not altered by perfusion at a supernormal pressure (1.40 +/- 0.16 x 10(6) mu 3) as compared with perfusion at ambient pressure (1.51 +/- 0.18 x 10(6) mu 3). Further studies in group 1, however, showed that values for VG obtained in paraffin-embedded tissue were approximately 40% lower than values for VG obtained in methacrylate-embedded tissue from the same kidneys.