Nonimmunologic mechanisms of glomerular injury.

From the above discussion it is clear that many factors have been invoked in the pathogenesis of progressive glomerular injury. Those which are most important include increased PGC, coagulation, serum lipid abnormalities, and hypertrophy. Although many hemodynamic alterations have been identified, increased PGC was noted most constantly. Furthermore, the loss of autoregulatory capability which was observed in some models with progressive glomerulosclerosis usually resulted in increased PGC. Increased PGC has been associated with augmented dietary protein and is seen in the Munich-Wistar rat made diabetic. Such an increase in PGC could cause direct mechanical injury to endothelial and epithelial cells, as well as be responsible for increased mesangial traffic of macromolecules with the potential for stimulating cellular proliferation and mesangial matrix increase. Additional support for the importance of increased PGC is provided by the protective effect of decreasing PGC with CEI therapy and anemia, and by the enhanced autoregulatory capability in both the Milan and Okamoto hypertensive rats. The significance of coagulation factors is confirmed by the formation of platelet and fibrin thrombi in the development of the glomerular lesions. The sequence of glomerular injury suggests that endothelial damage occurs with subsequent formation of platelet aggregates as a response to this injury. Formation of platelet aggregates may be associated with the production of substances potentially injurious to the endothelial cells. Although blocking the appearance of such thrombi by administration of heparin or thromboxane synthetase inhibitor prevents glomerular injury, the blood pressure lowering effect of these agents complicates the interpretation of the studies. Serum lipid abnormalities are also important factors in the progression of nonimmunologic glomerular injury. Such abnormalities are observed with increased dietary phosphorus or lipid, in the obese Zucker rat, and in rats with diabetes mellitus. Reduction in serum cholesterol by administration of clofibric acid or mevinolin diminishes glomerular injury independent of alterations in glomerular hemodynamics. The possible link between increased serum lipids and augmentation of glomerular injury is at present indirect. The importance of hypertrophy as a contributing factor to the progression of nonimmunologic glomerular injury is suggested by several lines of evidence. Hypertrophy, with increase in glomerular size and caliber of capillary loops, may amplify the effect of increased PGC by further intensifying the tension and mechanical stress on all elements of the capillary wall.(ABSTRACT TRUNCATED AT 400 WORDS)

Clip-ablation. A model of experimental hypertension in the rat.

A model of experimental hypertension has been developed in which the features of the two kidney-one clip and renal ablation models are combined in a single rat. It has been designated the clip-ablation model. It is produced by the placement of a silver clip with an opening of 0.13 mm on a branch of the left renal artery supplying one-third of that kidney, followed by right nephrectomy. In this way the effects of renin-dependent hypertension may be studied in glomeruli which are undergoing compensatory changes in response to reduction in renal mass. Clip-ablation rats were compared to rats with 1 1/3 reduction of renal mass at various intervals up to 28 days after operation. Systolic blood pressure rose with increasing time after operation in both groups with the increase being greater in the clip-ablation rats (177 +/- 10 mm Hg) as compared to the ablation rats (153 +/- 6 mm Hg) at 28 days. Plasma renin activity was increased slightly at 3 days in the ablation rats but had returned to normal levels by 28 days. By contrast, the plasma renin activity rose throughout the experimental period in the clip-ablation rats and attained a level of 32.3 +/- 8.0 ng/ml/hour by 28 days. Urine protein was significantly higher than normal only in the 28-day clip ablation rats (71.2 +/- 23.9 mg/24 hour). Glomerular damage index (GDI), a measure of glomerular injury, increased with longer intervals from operation in both experimental groups, paralleling the rise in systolic blood pressure. Beginning on day 14 and onward the GDI was always numerically higher in the clip-ablation rats than in the ablation rats. Stepwise multiple regression analysis indicated that the strongest predictor of GDI was the change in blood pressure. Plasma renin activity had an additional independent effect on GDI in the clip-ablation rats. The finding of more glomerular damage in the clip-ablation rats than in those with simple removal of comparable amounts of renal tissue is in contrast to the lesser amount of damage found in the two kidney-one clip model when compared with the model of removal of renal substance. It is likely that the compensatory hemodynamic changes in response to reduction in renal mass in the glomeruli of clip-ablation rats make them more vulnerable to injury when exposed to a renin-angiotensin induced hypertension.

Atherosclerosis in the hypercholesterolemic hare. Comparison of coronary artery lesions induced by dietary cholesterol in the hare and the rabbit.

Atherosclerotic lesions in coronary arteries were compared in 10 hybrid hares and 14 rabbits after induction of hypercholesterolemia, using a cholesterol-enriched diet. All proximal portions of hare coronary arteries contained intimal lesions, often with severe luminal stenosis. These lesions were characterized by the presence of foam cells, smooth muscle cells, and areas of atheronecrosis. Foam cells were also found focally in the media. As part of the intimal changes, iron deposition was present in 65% and calcification was present in 32.5% of proximal segments examined. The proportion of segments with intimal lesions and the intima/media cross-sectional area ratios (I/M ratios) were greatest in proximal segments with stepwise decreases in the mid and distal segments. As area of myocardial infarction was present in one hare. In contrast, 46.5% of proximal segments of rabbit coronary arteries had no intimal lesions and those lesions present had no calcium or iron deposition. No infarction was observed in rabbit hearts. The proportion of segments with lesions and the mean I/M ratios were significantly greater in the hare than the rabbit, with proximal and mid coronary segments showing the most marked differences. The hare appears to develop coronary artery lesions more like those seen in man, with high grade, proximal stenoses occurring uniformly in hypercholesterolemic animals. In contrast, the atherosclerosis developing in rabbit coronary arteries is less uniform and includes involvement of intramyocardial arterioles. The hare offers several advantages as a model of human atherosclerosis.

Clonal mapping of the human aorta. Relationship of monoclonal characteristics, lesion thickness, and age in normal intima and atherosclerotic lesions.

The surfaces of 8 aortas from women heterozygous for G-6-PD isoenzymes were mapped for an examination of the relationships of monoclonality, lesion type, lesion thickness, and age of the patient. The percent B isoenzyme value of samples of normal intima (n = 315), fatty steak (n = 68), or fibrous plaque (n = 64) was used to define monoclonality, expressed as the [Z] score, the number of standard deviations from the mean percent B isoenzyme of samples of underlying media. Intimal thickness increased significantly with type of lesion, such that intima less than fatty streak less than fibrous plaque, and with the age of the patient. The percentage of monoclonal portions also increased with lesion type, such that 1% of samples of normal intima were monoclonal, compared with 4.4% of fatty streaks and 12.5% of fibrous plaques (P less than 0.005). Monoclonality increased with intimal thickness when normal intima, fatty streaks, and fibrous plaques were combined (P = 0.0001). When examined separately, normal intima showed a direct correlation between monoclonality and intimal thickness. In contrast, the monoclonality of fatty streaks was inversely associated with thickness (P = 0.016) and the monoclonality of fibrous plaques not related to thickness. When entered into a multiple regression model, lesion type and age, but not lesion thickness, significantly predicted monoclonality. The lack of association of intimal thickness with monoclonality suggests that it is the type of lesion that determines monoclonality and not merely its thickness. This implies that mechanisms other than clonal selection are responsible for the monoclonal characteristics of human atherosclerotic lesions.

Relation of glomerular injury to preglomerular resistance in experimental hypertension.

A semiquantitative glomerular damage index (GDI) was determined for overall (O), superficial (S), and juxtamedullary (JM) glomeruli in four models of experimental hypertension in the rat to assess the severity and distribution of injury in light of present day knowledge of glomerular hemodynamics. After a four week period of similar hypertension, comparison of Group 1 (renal ablation) with Group 2 (aortic ligature) revealed OGDIs of 0.420 +/- 0.064 (SEM) vs. 0.062 +/- 0.019, P less than 0.0001, SGDIs of 0.250 +/- 0.071 vs. 0.035 +/- 0.007, P less than 0.0089, and JGDIs of 0.455 +/- 0.071 vs. 0.155 +/- 0.036, P less than 0.002. Within Group 1 the SGDI and JMGDI were not significantly different but within Group 2 the SGDI was less (P less than 0.005) than the JMGDI. Arterial/arteriolar damage was comparable in both groups. After an eight week period of similar hypertension, comparison of Group 3 (deoxycorticosterone-saline) with Group 4 (stroke-prone spontaneously hypertensive rats) showed OGDIs of 0.301 +/- 0.065 vs. 0.128 +/- 0.023, P less than 0.025, SGDIs of 0.289 +/- 0.096 vs. 0.072 +/- 0.015, P less than 0.044, and JMGDIs of 0.394 +/- 0.083 vs. 0.307 +/- 0.062, NS. Within Group 3 the SGDI and JMGDI were not significantly different, but within Group 4 the SGDI was less (P less than 0.002) than the JMGDI. Vascular damage in the two groups was comparable. Taking into account known physiologic data, the findings are consistent with the idea that increased preglomerular resistance is protective of glomeruli, whereas decreased resistance with increased pressure and/or flow is injurious.(ABSTRACT TRUNCATED AT 250 WORDS)

Glomerular hyalinosis and its relation to hyperfiltration.

Reduction in renal mass in rats results in hyperfiltration of the remnant nephrons, accompanied by injury to the glomeruli and their eventual sclerosis. This study was undertaken in a rat model with 5/6 reduction of renal mass to follow the evolution of glomerular damage, over an 11-week period, with particular emphasis on the widely prevalent, although seldom discussed, lesion of hyalinosis. Light, electron, and immunofluorescence microscopic studies were performed and blood pressure, excretion of urinary albumin, and serum creatinine levels determined. Systolic blood pressure, urinary albumin excretion, and serum creatinine levels were all increased by the third week following operation. Blood pressure and serum creatinine continued to increase throughout the period of study. Glomerular damage was focal and segmental, and glomeruli were equally affected in both the juxtamedullary and outer zones of the cortex. Endothelial injury was noted to be the first indicator of glomerular damage, followed closely by alterations in the epithelial cells. The early hyalinosis lesion was characterized by an accumulation of homogeneous electron-dense material beneath damaged endothelial cells with later encroachment on the capillary lumen resulting in the easily recognizable eosinophilic, periodic acid-Schiff-positive lesion by light microscopy. These alterations were accompanied by complex changes within the mesangium, including both mesangiosclerosis and mesangiolysis. Glomerular hyalinosis, glomerular sclerosis, vascular damage, blood pressure, and albuminuria were ranked in order of severity and the rankings subjected to multiple regression analysis. Significant correlations were present between glomerular sclerosis and hyalinosis, arterial damage and blood pressure, and hyalinosis and urinary albumin excretion. The hyalinosis lesion accompanying the progressive glomerular sclerosis in this model resembles that seen in a number of human conditions. In addition, the correlations of hyalinosis with glomerular sclerosis and albuminuria reflect its association with glomerular injury; it is likely that it will prove to be a reliable marker of hyperfiltration injury.

Effects of acute, angiotensin-induced hypertension on intrarenal arteries in the rat.

A perfusion-fixation and vascular casting technique was used to assess the effects of acute, angiotensin-induced hypertension on the intrarenal arteries and, for comparison, the small arteries of the intestine. The first objective was to establish that the technique accurately preserves postmortem the vascular changes induced by acute hypertension. To do this, the easily accessible intestinal arteries were examined and photographed both in vivo and after fixation and injection of Batson's no. 17 casting resin in a group of angiotensin-treated rats and controls. The second objective was to apply the technique to observe and compare acute hypertensive changes in the intrarenal and intestinal arteries; studies included scanning electron microscopy of vascular casts and transmission electron microscopy of vessel walls using ferritin as a tracer to assess permeability. In the angiotensin-treated rats, casts of both intrarenal and intestinal arteries showed many well-defined zones of constriction and nonconstriction. Transmission electron microscopy of both the smaller intrarenal (interlobular) arteries and intestinal vessels revealed focal smooth muscle rarefaction and abnormal permeability to ferritin, found only in the nonconstricted zones. This study provides new evidence that in the kidney, as in the intestine, acute hypertension produces a characteristic pattern of arterial constriction and nonconstriction, and that hypertensive vascular lesions with accompanying increased permeability occur exclusively in the nonconstricted zones.

Cholesterol-induced atherosclerosis. Clonal characteristics of arterial lesions in the hybrid hare.

Utilizing the observation that a majority of human atherosclerotic fibrous plaques show monoclonal characteristics, we carried out this study to determine the clonal characteristics of cholesterol-induced atherosclerosis in the hybrid hare. If this is a valid model for human atherosclerosis, the lesions produced in the aorta should be monoclonal. Glucose-6-phosphate dehydrogenase (G-6-PD) was used as an X-linked cellular marker in the female hybrid hare (Lepus timidus X Lepus europaeus), which is heterozygous for electrophoretically separable isoenzymes of G-6-PD. Hares were fed cholesterol over either a 6-month or a 16-month period, and the easily dissectable lesions in the aorta and common iliac arteries were assayed for isoenzyme activity at these times. Of the 93 lesions assayed, all had polyclonal characteristics except a single monoclonal lesion found in an animal fed cholesterol over a 16-month period. Hares fed over the 16-month period showed lesions with isoenzyme patterns having a significantly higher contribution of L. timidus isoenzyme than those found in underlying media. This suggested that a selection of cells with the L. timidus X-chromosome had taken place, but the degree of this selection was not great enough to allow any of the lesions to be defined as monoclonal.

The clonal characteristics of human aortic intima. Comparison with fatty streaks and normal media.

The clonal characteristics of normal-appearing but thickened aortic intima were studied by the use of the isoenzymes of glucose-6-phosphate dehydrogenase (G-6-PD) as cellular markers in females heterozygous for this X-linked enzyme. Isoenzyme patterns of 133 samples of intima were compared with those of 237 samples of underlying media and with those of 58 fatty streaks dissected from the same aortas. The proportion of samples of intima and fatty streaks with monoclonal or intermediate characteristics was the same, but both had more monoclonal or intermediate samples than underlying media (P less than 0.05). However, samples of intima showed a central clustering tendency of isoenzyme values similar to that of underlying media, while values from fatty streaks showed a bimodal distribution suggesting the presence of cell populations in the process of becoming monoclonal. The data suggest that clonal proliferation may begin in normal-appearing intima but that it progresses through a fatty streak stage before proceeding to the monoclonal fibrous plaque.

The effects of heparin treatment on vascular permeability and vessel wall damage in acute hypertension in the rat.

The experiment was designed to test whether the ability of heparin to prevent vascular "fibrinoid" necrosis in severe hypertension is related to its effects on vascular permeability. Hypertension was produced by infusion of angiotensin II into rats for 1 or 4 hours; some rats were treated with heparin and others not. Native ferritin was used as a tracer, and small arteries of the intestine were examined by light and electron microscopy. Rats not subjected to angiotensin II were used as controls. Half were treated with heparin, and half were not. Arteries from normotensive animals showed no signs of permeability to ferritin, damage to the vessel wall, or irregularities in caliber. Vessels from hypertensive rats had alternating zones of constriction and dilatation. Ferritin penetration and vessel wall damage were found only in dilated zones; these were focal in the 1-hour experiments and more extensive in the 4-hour experiments. No differences were observed between heparin-treated and non-heparin-treated rats with respect to permeability to ferritin or to vessel wall damage. However, in the 4-hour experiments, non-heparin-treated animals had occasional fibrinlike deposits at sites of severe medial damage; these were never found in heparin-treated animals. The findings suggest that although heparin does not appear to affect vascular permeability or medial damage during acute hypertension, it may prevent polymerization of fibrin in damaged vessel walls--presumably a result of the drug's anticoagulant properties.

Endothelial cell activity in experimental hypertension. Effects of hemodynamic factors.

The cause of the increased endothelial cell proliferative activity found in experimental hypertension in the rat is not clear. In this experiment the part played by altered vascular hemodynamics was explored in a model in which part of the aorta was subjected to a high blood pressure and the other not. Hypertension was produced by the application of a tight ligature to the aorta between the levels of the two renal arteries, the mechanism being interference with the blood supply to the more distally placed kidney. Because the aorta is constricted, the segment above the ligature is subjected to high blood pressure and the segment below it to lowered pressure, although both segments are exposed to humoral factors such as renin and angiotensin II. Aortic endothelial cells were labeled with tritiated thymidine and cell replication and cell density studies carried out using en face monolayer preparations of endothelium. It was found that endothelial cell replication was considerably increased at 7 days following induction of hypertension in segments of aorta above the ligature but not in the segment below the ligature. The increased activity was short-lived and had returned to normal levels at 4 weeks. Cell density was increased above the ligature at both 1 and 4 weeks following induction of hypertension. The changes are consistent with the idea that the increased cell replication and consequent increased cell density above the ligature were initiated by the increased stretching of the aortic wall attending the development of hypertension.

Aortic endothelial cell activity in high renin and normal renin models of hypertension in the rat.

To determine whether renin or angiotensin might have an effect on endothelial cell activity, we studied cell replication and cell density in two models of hypertension in the rat. The first was a model in which the plasma renin activity was high during the three time periods studied (1, 2, and 4 weeks after renal artery constriction) and the second, a model with elevated plasma renin activity only during the initial period (1 weeks). Aortic endothelial cells were labeled with tritiated thymidine period to killing, and en face endothelial cell monolayers were prepared. The ratio of labeled cells to total cells (thymidine index) and the ratio of total number of cells to number of fields observed (cell density) were calculated for the whole aorta, segments of the aorta, and zones within the segments. In both hypertensive models, an elevation of the thymidine index (indicative of increased endothelial cell replication) was observed at 1 week but not at subsequent times when the levels had returned to those of the controls. Cell density was increased at all time periods studied and like the thymidine index showed no significant differences between the two hypertensive models. Although renin or angiotensin (or other humoral factors) could have been responsible for the increased thymidine index after 1 weeks, it is considered that hypertension itself it a more likely cause, taking into account the decreased indices at later time periods when one model has high renin levels and the other does not. The increased cell density at all time periods studied in both models is regarded as an adaptive reaction to the increased blood pressure.

Clonal characteristics of cutaneous scars and implications for atherogenesis.

Atherosclerotic plaques in man have monoclonal characteristics. One possible explanation for this observation is that clonal selection occurs as cells proliferate at sites of intimal injury. To test whether such clonal selection could occur during the formation of an intimal "scar", the clonal characteristics of human cutaneous scars were studied with the use of glucose-6-phosphate dehydrogenase (G-6-PD) as a cellular marker. Scars from 7 black women heterozygous for the A and B isoenzymes of G-6-PD were divided into 870 portions consisting of contiguous normal skin, scar margin, outer scar, and inner scar. Portions were further divided into surface and deeper portions of scar. In 4 of the 7 cases, significant differences were observed between the mean percentage of total activity in the B isoenzyme band in portions of scar and portions of skin. Significant differences between surface and deeper portions of both skin and scar were also observed in 5 cases. The variance of isoenzyme values from portions of scars was not significantly greater than that within portions of skin. It is concluded that the results are consistent with clonal selection occurring during the healing of skin wounds. However, the magnitude of the difference (no greater than a 5.7% B isoenzyme difference) is far too small to even partially explain the marked differences in isoenzyme patterns observed between atherosclerotic plaques and uninvolved aortic wall. The results do not support the idea that the monoclonality of human atherosclerotic plaques is due to clonal selection following the healing of an intimal injury.

The effects of heparin treatment on hypertension and vascular lesions in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats (SPSHRs) were used to test the theory that heparin treatment may prevent the development of "malignant" hypertension and fibrinoid vascular lesions in the kidney. Subcutaneous injections of heparin (100 units/100 g body weight) were given every 8 hours over a 5-week period to 12 young (10-week-old) SPSHRs. A control group of 12 SPSHRs was injected with saline. Both heparin-treated and control animals showed an incremental rise in mean systolic pressure, but the pressure was significantly lower (P less than 0.05) in the heparin-treated animals during weeks 1-4 of treatment. There were significantly fewer fibrinoid vascular lesions (P less than 0.03) in the heparin-treated group. In 7 additional heparin-treated and 7 control SPSHRs plasma and blood volumes were determined for assessment of the effects of heparin treatment. There was no significant difference in total blood volume or plasma volume between the two groups, but heparin-treated animals had lower hematocrit levels. In 8 SPSHRs direct arterial pressures were recorded for 1 hour after a single heparin injection, and no acute changes in blood pressure were observed. The results suggest that heparin treatment prevents the development of severe fibrinoid vascular lesions and also attenuates the rate of the rise in systolic blood pressure; moreover, this reduction in blood pressure is not caused by a significant reduction in blood volume or an acute hypotensive effect of heparin.

The failure of furosemide-induced salt and water loss to convert benign to malignant hypertension in the rat.

The concept has been advanced that malignant hypertension is precipitated in the rat with renal hypertension by a sudden loss of sodium in the urine. In order to test this hypothesis modest degrees of hypertension were produced in Holtzman rats by the application of a silver clip to one renal artery, not touching the opposite kidney. When the systolic blood pressure reached a level between 160 and 180 mm Hg, loss of sodium and water was induced by the administration of furosemide, given either orally over a 7-day period, or by 3 intramuscular injections over a 24-hour period. Sodium and water balance studies, blood pressure determinations, histologic assessment of blood vessels in the nonclipped kidney, and measurement of activity of the juxtaglomerular apparatus were carried out in these 2 groups and appropriate control animals. It was found that in spite of a considerable natriuresis and diuresis in furosemide-treated animals, there was neither a significant increase in the blood pressure nor development of more severe vascular lesions in the nonclipped kidney than in the kidneys of control animals.

Evidence for two populations of fatty streaks with different roles in the atherogenic process.

Isoenzymes of glucose-6-phosphate dehydrogenase, used as cellular markers, showed that a minority of fatty streaks have clonal characteristics intermediate between those of normal arterial wall and those of fibrous plaques, suggesting that such streaks are evolving into fibrous plaques. "Intermediate" fatty streaks are found to cluster in patients in the middle-age years, suggesting that few fatty streaks evolve into plaques in the advanced years. It is suggested that two populations of fatty streaks exist: one destined, perhaps by its location or risk factors, to develop into fibrous plaques; and a second which remains as fatty streaks and never goes on to become fibrous plaques. This hypothesis helps to explain some of the evidence disclaiming the fatty streak as a forerunner of the atherosclerotic plaque.

The effect of clonidine on tubular obstruction in postischemic acute renal failure in the rabbit demonstrated by microradiography and microdissection.

Acute renal failure was produced in vasopression-pretreated rabbits by clamping the left renal pedicle for one hour and removing the opposite kidney. Treatment with clonidine, as antihypertensive drug that blunts the kidney's response to vasopressin, resulted in significantly higher creatinine clearance and urine flow rate in the first 6 hours after unclamping. Clonidine (30 microgram/kg given intravenously 30 minutes before unclamping) also significantly lessened the number of hyaline casts in outer medullary tubules and inner medullary loops of Henle 6 hours after unclamping and reduced the number of abnormal tubular contours in microadiograms produced by infusing barium sulfate into the renal artery at sufficient pressure to rupture glomerular capillaries, causing an escape of contrast material into the tubules. The spaces consistently observed between the ends of barium columns and hyaline casts in microdissection studies and the great lengths of the hyaline casts suggest that hyaline casts obstruct the flow of tubular fluid. Clonidine treatment resulted in fewer, shorter, and thinner hyaline casts. These results indicate that tubular obstruction by hyaline casts plays an important role in early postischemic acute renal failure, and that clonidine's beneficial effect is due in part to a reduction in cast formation.

Clonal characteristics of experimentally induced "atherosclerotic" lesions in the hybrid hare.

The female hybrid hare (Lepus timidus x Lepus europaeus) is heterozygous for electrophoretically separable, X-linked isoenzymes of glucose-6-phosphate dehydrogenase. The isoenzymes of this animal have been used as cellular markers in the study of the clonal origins of experimentally induced atherosclerotic lesions. Aortic lesions produced in the hybrid hare by feeding cholesterol and injuring the aortic wall with a catheter have been shown to have polyclonal characteristics and in this way are fundamentally different from atherosclerotic fibrous plaques in man.