International Urology and Nephrology 7 (2), pp. 157--170 (1975)

Peripheral Small Vessel Involvement in Chronic Nephritides P. T. SCARPELLI, A. PIERI, M. RIzzo, M. BOCCUNI Department of Medicine and Urology, University of Florence, Italy (Received Iuly 16, 1974)

Small cutaneous vessels, obtained by ear lobe biopsies, were studied in 14 patients with various chronic nephrifides and in 10 normal controls. The capillaries in the group of patients with nephritis were found to undergo two main changes: thickening of the adventitia reticularis and perivascular cellular infiltration in an inverse ratio. This infiltration was seen to be made up of mononuclear cells and an increased n u m b e r of mast cells in various stages of degranulation. Changes in the basement membrane as seen by electron microscopy are not constant. All of the above changes were absent in the controls and are similar to what has been described in previous studies in b o t h experimental and spontaneous pathologic conditions, such as experimental hypertension, diabetes mellitus, scleroderma, rheumatoid arthritis, etc. Small vessel involvement in chronic nephritides could be part of a process of diffuse microvascular damage that includes the kidneys or it may be related to hypertension or to the biochemical changes which follow uremic and pre-uremic states.

Small blood vessel involvement is an important feature in many pathologic conditions even though attempts to establish morphological specificity have, so far, failed. In myxoedema, thickening of the basement membrane, due to increased PAS positive material has been reported in skin and myocardial capillaries [2, 16] and in pulmonary small vessels and veins [17]. In rheumatoid arthritis, involvement of small vessels has been found in skin and muscle [4, 11, 13]. Deposits of PAS positive material, fragmentation of the basement membrane and marked macrophagic activity of connective tissue cells are common findings in the cutaneous small vessels of patients with systemic L. E. [10, 18, 19, 20, 24]. In scleroderma, perivascular inflammatory infiltration with deposits of PAS positive material has been described. Similar findings have been reported in muscle capillaries of patients with polymyositis [9]. Deposits of fibrin, perivascular leucocyte infiltration and occasional necrosis with thrombosis have been found in small vessels (mostly arterioles) in allergic vasculitis [14, 25]. Since 1950, small vessel involvement has been recognized as a characteristic feature in diabetes mellitus. These changes can be distinguished from arteriosclerosis by certain pathological features and histochemical techniques [3, 8]. Since then a large number of studies have supported the view that diabetic 5

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microangiopathy might be characterized by an elementary, generalized lesion with peculiar evolution in certain organs such as the kidney and retina, which have highly specialized functions. The histopathological pattern of diabetic microangiopathy is, however, not specific [26] and is comparable to the changes found in the other conditions such as thickening of the walls of small vessels (precapillaries, capillaries, arterioles, venules) due to widening of the adventitia and/or the basement membrane 1 and/or perivascular cellular infiltration with a large population of mast cells in various degrees of degranulation [21, 22, 23, 27, 28]. Whether or not endothelial proliferation occurs is still controversial. Since these changes tend to be generalized, skin and muscle, which are more suitable for anatomoclinical studies, can be considered sufficiently probative for detection of small vessel involvement. The present study deals with small vessel involvement in the skin of patients with chronic nephritis and renal insufficiency. Materials and methods

The study includes two groups of individuals: 14 patients with chronic nephritis and varying degrees of renal insufficiency and 10 normal controls, matched by sex and age. Patients were admitted to the Department of Internal Medicine of the University of Florence and selected according to the usual clinical, radiological and laboratory examinations and tests. Specimens of skin were obtained by ear lobe biopsy. Under local anesthesia (0.5 ml of 1 7o solution of procaine hydrochloride, injected in the facial root of the ear lobe) biopsy samples were taken by a Keyes cutaneous punch No. 2. The material was fixed in Bairati's or Bouin's fluids and embedded in paraffin. Sections were stained with hematoxylin and eosin, by the PAS method, toluidine blue and the Unna-Pappenheim method. A second specimen was fixed in OsO4 in buffered phosphate according to Millonig and embedded in Epon. Sections obtained by ultramicrotomy of Porter-Blum were con(rasted by uranyl acetate and Pb citrate and studied by electron microscopy (Siemens Elmiskop I). Results

A) Light microscopy 1. Capillaries: two main histological changes were found in the group of patients with nephritis: thickening of the adventitia reticularis (which represents the basement membrane as seen by light microscopy) and perivascular cellular Our studies on small vessel involvement in diabetes mellitus [23, 28] convinced us that the adventitia reticularis (according to Volterra [29, 30, 31] and Allara [ 1 ]), as seen by light microscopy, should be considered as separate from basement membrane (lamella basale according to Fawcett) as seen by electron microscopy, of which the former can be independently involved. International Urology and Nephrology 7, 1975

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Fig. I. Two main histological findings of cutaneous microvascular involvement in chronic nephritis as seen by light microscopy, a) Thickening and splitting of adventitia reticularis. PAS, • 650. b) Perivascular cellular infiltration. PAS, • 350 5*

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infiltration (Fig. la, b). As in diabetic microangiopathy the two main pathological features are present in the same specimen but in an inverse ratio: the sections with cellular infiltration showing less thickening of the vascular wall and vice versa. Neither thickening of the wall nor cellular infiltration was evident in the majority of normal controls. The occasional thickening of the wall of the capillaries, present in a small number of aged controls, cannot be considered significant since cellular perivascular infiltration was not found. Perivascular cellular infiltration is produced by various types of cells, generally mononucleates (monocytes, lymphocytes, fibroblast~ ) and mostly by a great number of mast cells in various phases of degranulation. Degranulation is sometimes so active that mast cells have almost completely lost their characteristic metachromasia when stained with toluidine blue. In such cases a superficial examination, especially at low magnification, can miss a number of cells. A more careful study will, however, allow the detection of a residual metachromasia and the presence of granules, scattered throughout the ground substance around the cells. 2. Arterioles: in 9 of the 14 cases with chronic nephritis no changes in the arteriolar wail were found. In 3 of these 9 cases, materiaI obtained was considered insufficient to give an accurate evaluation and was therefore discarded. In the 6 remaining cases with nephritis the arteriolar wall was involved by a process which is generally indicated as "plasmatic vasculosis". Such changes are characterized by a loss of muscular fibrocells, thickening of the wall, homogenization of perivascular connective tissue. The irregular thickening of the wall is due to subendothelial accumulation of eosinophilic material; lymphocytes, polymorphonuclear leucocytes and monocytes adhere loosely to the endothelial surface and eccentric thickening of the media is visible under low magnification. Adventitial connective tissue cells and mast cells are more numerous than normal. Some arterioles seemed completely fibrotic with almost complete obliteration of their lumen. No significant changes were found in the arterioles of the controls. B) Electron microscopy In each case the endothelial cells present similar features. The nuclei have an irregular shape, and this nuclear distortion has recently been seen by Majno et al. [ 15] and was interpreted by them as a possible sign of cellular contraction. Mitochondria are more frequently seen than in the control cases, and the cells have a rich endoplasmic reticulum. Vacuoles of various shapes and sizes are also frequently observed, some of which seem to originate from folding of the plasmatic membrane. The lumenal surface is quite irregular due to endothelial protrusions. Ultrapynocytosis seems reduced (Fig. 2). With regard to the basement membrane one notices that the perivascular connective tissue is increased and collagen synthesis appears to be more abundant than normal. In some zones the formation of fibres is prolific and a number of fibres with a periodic structure are seen gathered in bundles, arranged in a disorderly manner (Fig. 3). Fibres around adventitial cells are immersed in a ground substance which has the same electronic International Urology and Nephrology 7, 1975

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Fig. 2. Chronic glomerulonephritis (16 years old girl). Very marked thickening of adventitia reticularis with a normal basement m e m b r a n e (bm). The endothelial nucleus (N) shows an irregular contour. Endothelial protrusion (p) into the capillary lumen is present. • 13 000 International Urology and Nephrology 7, 1975

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Fig. 3. Chronic glomerulonephritis (44 years old woman). Detail of the capillary adventitia reticularis. Numerous thin bundles of collagen fibrils can be seen (f). The arrow points to an area of spfitting of the basement membrane. The nucleus (N) and the capillary lumen (L) are also seen

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Fig. 4. Chronic glomerulonephritis (44 years old woman). Early phase of adventitial fibrosis. Sparse collagen fibers dispersed in a moderately electron-dense ground substance International Urology and Nephrology 7, 1975

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density as the basement membrane. Fibres outside of the adventitial cells have a larger diameter as compared to the ones interspersed between the adventitial cells and the endothelial cells (Figs 2, 3). The fibrotic process and the cellular infiltration are in an inverse ratio as also seen by light microscopy. The adventitia can be thinner or thicker in various sections. The basement membrane in our series shows no significant changes. In rare instances it is thicker and tends to be stratified or split (Fig. 3). We have obtained similar results in skin specimens obtained from diabetics or patients with other pathologic conditions. In some instances, electron microscopy seems to confirm the early onset of changes in the adventitia as seen by light microscopy: the pericapillar connective tissue is increased but fibrosis is evidently at an early stage since the fibres are thin and not collected into bundles, the adventitia is multistratified and made up of material with an electronic density identical to that of the basement membrane (Fig. 4). Mast cells, together with connective tissue cells, lymphocytes, and monocytes are an essential part of the infiltrate. Some mast cells are full of granules, others have few granules, some are empty (Fig. 5a, b). In two cases, granules had the typical structure described in normal human mast cells; while in the other cases, typical granules are mixed with clear granules which have lost their scroll-like structure (Fig. 6a, b).

Discussion

Our findings, obtained in skin specimens, support the view that peripheral small vessels undergo structural changes in patients with chronic nephritis and renal insufficiency. Capillaries are more intensively and consistently involved even though occasional changes (4 out of 14 cases) were found in arterioles. Histologic changes have a focal distribution in that in different sections of the same case two distinct pictures can be found: a) perivascular cellular infiltration (mostly made up of mast cells in the active phase of degranulation) with thin adventitia; b) thickening of the adventitia with increased PAS positivity and rare cells around the wall. In this second instance, electron microscopy shows an increased synthesis of collagen with formation of a number of periodic fibres, immersed in a substance which has an electron density similar to that of the basement membrane. The basement membrane, as seen by electron microscopy, is generally comparable to that of the controls and thickening and stratification, when present, are extremely localized. Such data emphasize the necessity of distinguishing the basement membrane by light microscopy (adventitia reticularis) from the one seen at E. M. since the pathological changes of these two structures can be dissociated. International Urology and Nephrology 7, 1975

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The two histological features could be chronologically related and represent merely two different steps of the same process which could be conceived of as starting with a cellular infiltration around a thin vessel wall and proceeding toward total fibrosis through a thickening of the wall. Such a hypothesis can explain the variety of morphological features found in different vessels of the same subject and sometimes in different portions of the same vessel. The role of the mast cells in this process is still to be proved. The fact that mast cells undergo various degrees of degranulation might mean that they are in an active functional phase which could affect small vessels in two opposite ways: a) by participating in the process of fibrosis; b) by affecting the wall permeability of vessels already fibrotic by freeing various substances, such as heparin, histamine, etc. Similar histologic features, with the same inverse ratio of wall thickening and perivascular cellular infiltration, were found by us in several pathologic conditions [26], such as diabetes mellitus (skin and conjunctiva), S. L. E. and scleroderma (skin). Therefore, the submicroscopic features of these microvascular lesions do not seem to be, by themselves, sufficiently distinctive, as claimed by Norton [18], to be considered specific for any one disease entity. The fact that these microvascular changes tend to be generalized brings up the usual question of whether they are to be considered part of a generalized microangiopathy on which the disease might be based, or as complication of the underlying disease. In this respect it is quite difficult to interpret our data. Nevertheless, three possibilities seem to arise. 1. Peripheral small vessel involvement could be part of a chronic process of diffuse microvascular changes which has, in the kidneys, a characteristic histological pattern with its typical clinical manifestations. If this hypothesis could be proved, a common pathogenesis becomes acceptable for both renal and peripheral small vessel involvement. At present, an immunological mechanism seems to play a main role in chronic nephritis in two possible ways : either by antibasement membrane antibody formation or by antigen-antibody complex deposition [6]. Some experimental data, as discussed at length by Kniker [12], seem to suggest the possibility of an immunogenic vasculitis as the basis for the peripheral vascular involvement. 2. Peripheral small vessel involvement in nephritis with chronic renal insufficiency could be related to hypertension. In fact, most of our patients had high B.P. values even though the degree of vessel involvement was not proportionately related to these values. Such a hypothesis is quite acceptable for the arteriolar changes, which are similar to the ones found in experimental hypertension. Byrom [5] in his extensive studies on experimental hypertension and later Gardner and Matthews [7] have described, primarily in arterioles, all the events from reversible modification of vascular permeability (plasmatic vasculosis) to fibrinoid necrosis. Similar changes have also been found in a number of other pathologic conditions [7]. A persistently sustained increase of B. P. exists at the arteriolar level in hypertension but whether or not this occurs at the capillary level is still controInternational Urology and Nephrology 7, 1975

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Versial, since there B. P. is likely to show c o n s t a n t fluctuations in various districts n o t necessarily b o u n d to systemic pressure [32]. 3. Peripheral small vessel i n v o l v e m e n t could also be related to complex biochemical changes which follow uremic or pre-uremic states, such as a n o m a l i e s in extraceUular electrolyte c o n c e n t r a t i o n , urea a n d creatinine levels, or some other, so far u n k n o w n substances.

References 1. Allara, E.: Su alcune particolarifft ultrastrutturali dell'endotelio dei capillari sanguigni. Mon. Zool. It. 72, Supp. 22 (1964). 2. Baker, E. M., Hamilton, J. D.: Capillary changes in myxoedema. Lab. Invest. 6, 218 (1957). 3. ]31umenthal, H. T., Alex, M., Goldenberg, S.: A study of lesions of the intramural coronary artery branches in diabetes mellitus. Arch. Path. 70, 27 (1960). 4. ]3unim, J. J., Sokoloff, L.: The occurrence and significance of vascular lesions in rheumatoid arthritis. Tr. Ass. Am. Phys. 70, 318 (1957). 5. Byrom, F. B. : The Hypertensive Vascular Crisis. An Experimental Study. W. Heineman Med. Books Ltd., London 1969. 6. Dixon, F. J. : The pathogenesis of glomerulonephritis. Am. J. Med. 44, 493 (1968). 7. Gardner, D. L., Matthews, M. A.: Ultrastructure of the wall of small arteries in early experimental rat hypertension. J. Path. 97, 51 (1969). 8. Goldenberg, S., Alex, M., Joshi, R. A., Blumenthal, H. T.: Nonatheromatous peripheral 'vascular disease of the lower extremity in diabetes mellitus. Diabetes. 8, 261 (1959). 9. Gonzales-Angulo, A., Fraga, A., Mintz, G. : Submicroscopic alterations in capillaries of skeletal muscles in polymyositis. Am. J. Med. 45, 873 (1968). 10. Guiraudon, C., Saporta, L , Delbarre, F.: Les 16sions vasculaires dans la scl6roderrnie; in F. Delbarre "La scl6rodermie." Masson, Paris 1972, p. 263. 11. Hollingsworth, J. W. : Local and Systemic Complications of Rheumatoid Arthritis. W. B. Saunders Co., Philadelphia 1968. 12. Kniker, W. T." Immunogenic Vasculitis: Is It Part of Diabetes Mellitus? in "Vascular Complications of Diabetes Mellitus", ed. by S. J. Kirnura and W. M. CaygiU. C. V. Mosby Co., Saint Louis 1967. 13. Kulka, F. cit. Hollingsworth. 14. McCombs, R. P., Patterson, J. P., MacMahon, H. E.: Syndromes associated with "allergic" vasculitis. New En#L J. Med. 255, 251 (1956). 15. Majno, G., Shea, S. M., Leventhal, M.: Endothelial contraction induced by histaminetype mediators. An electron microscopic study. J. Cell. Biol. 42, 647 (1969). 16. McFadden, P. M., Berenson, G. S.: Basement membrane changes in myocardial and skeletal muscle capillaries in myxedema. Circulation 45, 808 (1972). 17. Naeye, R. L. : Capillary and venous lesions in myxederna. Lab. Invest. 12, 465 (1963). 18. Norton, W. L.: Comparison of the microangiopathy of systemic lupus erythematosus, dermatomyositis, scleroderma, and diabetes mellitus. Lab. lnvest. 22, 301 (1970). 19. Norton, W. L., Nardo, J. M.: Vascular disease in progressive systemic sclerosis (scleroderma). Ann. Int. Med. 73, 317 (1970). 20. Panja, R. K., Sengupta, K. P., Aikat, ]3. K.: Vascular changes in the cutaneous lesions of lupus erythematosus and scleroderma. Brit. J. Derm. 78, 43 (1966). 21. Peri, A., Scarpelli, P. T.: Small blood vessel involvement in diabetes mellitus: light microscope studies of specimens obtained by ear lobe biopsy. Commun. 1a Riunoone International Urology and Nephrology 7, 1975

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