Charles C.C. O'Morchoe

Transport of protein across lymphatic endothelium in the rat kidney

Abstract The pathways involved in protein transport across the lymphatic endothelium of the rat renal cortex were studied ultrastructurally. Qualitative and quantitative analyses were made on tissue from control animals (saline injected) and from animals injected intravenously with horseradish peroxidase (HRP) or ferritin. Three types of contacts between adjacent lymphatic endothelial cells were seen in control and experimental tissue. These were end to end, overlapping, and interdigitating. Only one instance of an “open” junction was seen in control tissue. No significant difference was noted in the proportions or widths of these three types of cell contact after the administration of HRP or ferritin. Neither was there any increase in the occurrence of open junctions. HRP was frequently seen in the normal intercellular spaces between adjacent endothelial cells. Coated and uncoated vesicles (diameter 80 to 300 nm) were identified in lymphatic endothelium in both control and experimental tissue and were quantified according to their position in the cell and according to their staining intensity. The volume density of small vesicles (diameter 80 to 100 nm) increased significantly from 0.028 in the control group to 0.049 after HRP injection. Likewise the numerical density rose from 16/μm 3 to 22/μm 3 . However, the relative positions of these vesicles did not alter significantly following a protein load. There was a significant increase in the staining intensity of vesicles after the administration of HRP. For example, in control tissue none of the small vesicles was intensely stained in contrast to 8% after HRP injection. Ferritin particles were identified in large intracytoplasmic vesicles (diameter 150 to 500 nm) but not traversing intercellular spaces. It was concluded that (1) the tracer molecules used in this study enter renal lymphatics rapidly after intravenous injection; (2) the major visible routes for transport of the tracer are intracytoplasmic vesicles and normal (as opposed to open) intercellular spaces; and (3) the presence of HRP in the interstitium stimulates an increase in the number of intracytoplasmic vesicles in lymphatic endothelium.

Renal lymphatic ultrastructure and translymphatic transport

Abstract Information on the mechanisms of transport across renal lymphatic endothelium in the dog was derived by qualitative and quantitative ultrastructural analyses. Interlobular and intralobular renal lymphatics were studied in dogs with ureteric obstruction and lymphatic ligation, in dogs with lymphatic ligation only, and in dogs with unimpeded lymph and urine flow. Minor differences were detected in the ultrastructural features of lymphatic endothelium among the three groups of animals and between the two types of lymphatics studied within each group. Approximately 5% of the endothelial intracytoplasmic volume was occupied by vesicles, the majority of which were of the small uncoated variety. On an average 65% of these vesicles lay within the cytoplasm, and of the remainder, most were associated with the luminal border. Interendothelial contacts were of three varieties—end-to-end, overlapping, and complexly interdigitating. One-third of these lacked any specialized junctional complexes, whereas about 60% of the identifiable complexes revealed a fascia occludens. The remainder (approximately 10%) possessed fascia adhaerentes. Only 2 examples, out of 240 contacts studied, could be described as open junctions (greater than 50 nm). The conclusions drawn from this study were that (1) interlobular and intralobular renal lymphatics subserve similar functions in lymph formation, (2) at least in the absence of local irritation or trauma, open junctions do not play a significant role in translymphatic transport of fluid and protein, and (3) for poorly diffusible substances the primary pathways into the lymphatic lumen are intravesicular transport and the normal intercellular channels between contiguous cells.

The organization of endocytotic vesicles in lymphatic endothelium

The lymphatic endothelium from renal hilar lymphatics in the rat was subjected to qualitative and quantitative ultrastructural analysis with emphasis on the extent and disposition of its vesicular component. The uncoated endocytotic vesicles had an average maximum diameter of 0.073 micron and occupied 7% of the cytoplasm. There were approximately 21 vesicles in each cubic micrometer of cytoplasm. In standard electron microscopic preparations of glutaraldehyde-fixed endothelium 50% of the vesicles appeared to lie free within the cytoplasm. The remainder were seen to touch or open onto the luminal or abluminal surface of the endothelium. The degree to which intracytoplasmic endocytotic vesicles remained discrete or communicated with the plasma membrane was examined using tannic acid and ruthenium red. These substances specifically bind to charged molecules on the cell surface and identify membranes continuous with it. When this technique was applied to aldehyde-fixed tissue nearly 90% of the vesicles that were apparently free within the cell could be shown to retain a connection with the surface, approximately equal numbers communicating with either the luminal or abluminal surface. At least 15% of these vesicles existed as intercommunicating clusters. These results suggest that many vesicles are not simple discrete units, but rather form parts of chains that reach either luminal or abluminal surface. Thus, apparently discrete vesicles may be parts of vesicular chains cut in cross section. The possible relation between chemical fixation and this plan of vesicular organization is discussed and it is concluded that while chemical fixation may result in an overestimation of the numbers of intercommunicating vesicles, the qualitative aspects of vesicle disposition seem largely unaffected. Although the functional significance of our observations has yet to be determined, should this plan of vesicular organization apply to initial lymphatics as well, the concept of vesicular transport solely by random movement of discrete vesicles across lymphatic endothelium should be modified.

Decidual cell reaction induced by prostaglandin F2? in the mature ophorectomized rat

SummarySprague-Dawley rats were oöphorectomized and after a 2–3 week recovery period were given daily injections of progesterone (2.0 mg/0.1 ml) for six consecutive days. On the fourth day of progesterone treatment 0.2 mg of estradiol 17β was given in addition and the right uterine cornua were subjected to one of five experimental maneuvers. On the sixth day of progesterone treatment the uterine cornua were weighed and processed for light and electron microscopy. The weights of all left cornua (84.6 +- 3.7 mg) and the right cornua of PBS-injected (93.3 +- 11.5 mg) and sham operated uteri (83.6 +- 19.8 mg) were comparable. A significant increase (p<0.001) in weight was found in cornua that received PGF2α (144 +- 6.7 mg), PGF2α with mild local trauma (scratch) (146 +- 28.0 mg), and scratch alone (162 +- 12.7 mg). The majority of cornua treated by scratch alone, or by PGF2α with or without scratch, showed a decidual cell reaction by light microscopy and had a significantly higher mitotic index than those treated with saline, or by sham operation. When specimens were evaluated for the presence of the OCR, the highest rank was found in tissues treated by scratch alone or by PGF2α with or without scratch. Morphometric evaluation by light microscopy indicated that the extent of decidualization in PGF2α-treated tissue was comparable to that of scratchtreated tissue. Ultrastructural observation of PGF2α-treated tissue revealed that decidual cells closely resembled those treated with scratch. However, electron microscopic morphometry revealed that cells that responded to PGF2α had higher volume and surface densities of organelles associated with metabolic activity than did cells responding to scratch alone. These results demonstrate that locally administered PGF2α can initiate, in the hormonally prepared mature oöphorectomized rat, a DCR comparable to that induced by local trauma.

Concentration of renin in the renal interstitium, as reflected in lymph.

The relationship between renin activity in renal venous plasma and in renal interstitial fluid, as reflected in the hilar lymph, was observed. Control measurements in 28 dogs demonstrated that renin levels in the hilar lymph were 3.5 +/-- (SEM) 0.4 times higher than in renal venous plasma and 6.1 +/- 1.0 times higher than in arterial plasma. Renin activity was increased to varying levels by raising ureteric pressure and by the administration of different doses of furosemide. Under all conditions, the changes in renin activity in renal venous plasma and in the interstitium, as reflected in the hilar lymph, were in parallel although often marked in the hilar lymph. Thus, the study shows that under conditions of increased renin release, the interstitial renin activity rises in consort with increases in arteriovenous plasma differences. This increase in interstitial renin activity occurred even under circumstances when renal blood flow is known to increase, thereby suggesting that angiotensin generated in the interstitium may have little effect on the cortical arteriolar caliber.

Lymphatic transport pathways during volume expansion

Abstract Morphological evidence on lymphatic transport was derived qualitatively and quantitatively in control and volume-expanded rats in order to identify any changes in transendothelial pathways which accompany enhanced lymph formation. Volume expansion was induced by intravenous mannitol and by intraperitoneal hypertonic or hypotonic saline (10% of body wt). Horseradish peroxidase and ferritin were used as macromolecular tracers. Volume expansion had little effect upon intraendothelial cytoplasmic components. Intracytoplasmic vesicles contained tracer molecules in control and experimental groups and quantitative analysis revealed only one significant difference between the groups—a reduction in vesicular volume and numerical densities in animals receiving mannitol. The major changes between control and experimental animals were in the presence of attenuated areas of cytoplasm, widening of intercellular pathways, and the presence of open regions in the expanded group. In these respects there was little qualitative difference among the three experimental groups, although some quantitative differences were recorded. It was concluded that vesicular transport plays a significant role in translymphatic protein movement and that when the interstitial fluid load to lymphatics is increased the excess fluid preferentially enters between adjacent endothelial cells or diffuses across areas of extreme attenuation. Open regions or junctions were considered to play little if any role in lymph formation under control conditions, but to achieve increasing importance with increases in fluid movement.

An ultrastructural study of the transport pathways across arcuate, interlobar, hilar, and capsular lymphatics in the dog kidney

Abstract The ultrastructurally visible transport pathways across arcuate, interlobar, hilar, and capsular lymphatics were studied in canine kidneys with unimpeded lymph and urine flow. The four types of vessels were lined by a nonfenestrated endothelium resting on a discontinuous basal lamina and contained valves. Stereological techniques were applied to determine the volume and numerical densities for cytoplasmic vesicles (80–90 nm in diameter). Values for those parameters increased sequentially in the direction of lymph flow from the kidney. Thus, vesicular volume density rose from 6% of endothelial cytoplasm in arcuate lymphatics to 14% in hilar vessels, and the numerical density increased from 11 vesicles/μm 3 to 33 vesicles/μm 3 . Most of the vesicles lay apparently free within the cytoplasm, the remainder were evenly dispersed along the luminal and abluminal cell borders. Three varieties of interendothelial cell contacts were present: end-to-end, overlap, and interdigitation. Specialized junctional complexes associated with these contacts were either fasciae occludentes or fasciae adhaerentes, although in many instances neither were present. In general, arcuate and interlobar lymphatics had more cell contacts which lacked junctional specializations than did hilar and capsular lymphatic vessels. Only one example out of 676 cell contacts could be classified as “open” (greater than 30-nm gap along the entire contact length). It was concluded that (1) the four types of lymphatic vessels in the present study have ultrastructural features of lymph-forming (as opposed to simple collecting) vessels, (2) the ultrastructurally visible transport pathways in the four types of vessels studied are through “normal” (5- 30-nm wide) intercellular channels and via cytoplasmic vesicles, (3) arcuate and interlobar lymphatics have a structure which is consistent with the formation of lymph from medullary interstitial fluid, (4) hilar and capsular lymphatics may have a role in extrarenal lymph formation, and (5) “open” contacts are not involved normally in translymphatic transport in the kidney.

Renal contribution to thoracic duct lymph in dogs

1. The renal contribution to thoracic duct lymph was measured in seventeen anaesthetized fasting dogs by measurement of thoracic duct flow before and after renal arterial occlusion.

Lymphatic endothelial cell inclusion bodies

Paracrystalline, compact tubular, and myeloid inclusion bodies were observed in endothelial cells from canine renal, mesenteric, and thoracic duct lymphatics. Paracrystalline inclusions were membrane bound and were composed of filaments organized into closely packed hexagons (18 nm across). Compact tubular inclusions were confined to cisternae of endoplasmic reticulum and the nuclear envelope, and their component elements (24 nm in diameter) were more loosely aligned than in the paracrystalline type. Myeloid bodies had a limiting membrane and were composed of membranous whorls (10 nm thick) which were stacked frequently into lamellae. Paracrystalline and myeloid inclusion bodies showed acid phosphatase activity which suggested some lysosomal association but the functional role of compact tubular inclusions was not elucidated.

The rate of translymphatic endothelial fluid movement in the canine kidney

Abstract The rate of fluid movement across the lymphatic endothelial wall, as measured in microliters per minute per square centimeter of endothelium, is estimated from published and unpublished data on the canine kidney. Published values for total renal lymph flow, derived by several different techniques, are reviewed and a mean value of 0.3 ml/min/100 g is taken as a reasonable working estimate. Morphologically the intrarenal lymphatic system is considered to consist of four types of lymphatics—intralobular, interlobular, arcuate, and interlobar—and the endothelial surface area of these is calculated by geometric formulas. Thus each of the four types of lymphatics is considered as a single cylinder having a radius equal to the mean radius of that type of lymphatic and a length equal to the total length of all lymphatics of that type. The convex surface area ( A c ) of interlobular and intralobular vessels is calculated by the formula A c = 2v r , where the radius ( r ) is measured in tissue sections and the volume ( v ) is derived from published volume density data. A c for arcuate and interlobar lymphatics is estimated by the formula A c = n (2 πrl ), where n is the number and r is the radius of these lymphatics, as obtained from tissue sections, and l is the average length of the associated arteries. By these methods, values of A c for intralobular, interlobular, arcuate, and interlobar lymphatics of 59, 87, 34, and 15 cm 2 , respectively, are calculated. All four types are considered, on ultrastructural evidence, to be vessels that participate in the transport of lymph. By dividing total renal lymph flow by the total lymphatic endothelial surface an approximate value of 1 μl/min/cm 2 is obtained for the average rate of fluid transfer across lymphatic endothelial cells in the control dog kidney.