fine morphology of regressing human mesonephric nephrons

EXPERIMESTAL AND hfOLECULAR PATHOLOGY 26, 169-183 ( 1977)

Fine Morphology of Regressing Human Mesonephric Nephronsl

CESARE DE MARTINO, LUCIANO ZAMBONI,* AND LIDIA ACCINNI f

institute Regina Elena for Cancer Research, Rome, Italy; *Department of Pathology, Harbor General Hospital, Torrance, California and University of California,

Los Angeles, California; j C.N.R.-Clinica Medica 2, University of Rome, Rome, Italy

Received April 24, 1976, and in revised form August 5, 1976

The changes which characterize the involution of human mesonephric nephrons have been studied by means of light and electron microscopy. The most salient glomerular changes appear to be modifications of the mesangial cells and other components of the capillary wall, resulting in progressive reduction and eventual obliteration of the vascular bed of the glomerulus; tubular changes are essentially char- acterized by severe epithelial cell degeneration and the presence of pleomorphic hmrinal casts. The possible mechanisms responsible for the regression of the human mesonephros are discussed together with an evaluation of the analogies between the glomerular and tubular changes accompanying the regression of mesonephric nephrons and those occurring in the nephrons of the definitive (metanephric) kidney in a wide variety of pathologic conditions.

INTRODUCTION

It is known that the mesonephros (temporary kidney) undergoes regression simultaneously with differentiation of the definitive (metanephric) kidney (Gersh, 1937; Arey, 1965); the period of intrauterine life during which this phenomenon takes place varies in different species. In the human embryo, the mesonephric nephrons begin to degenerate gradually in a cranial-caudal direction around the ninth week of embryonal development (Willis, 1958; Langman, 1967; Bodemer, 1968), and, at the end of the fourth month, the mesonephros becomes totally nonfunctional (Vogh and Cassin, 1970). Some of the structural changes which accompany the involution of the mesonephros have been studied in birds (Von Mihalkovics, 1885; Firket, 1914, 1920; Stampfli, 1950; Haffen, 1951a, b; Moog, 1962, 1965; Tilney, 1964; Volle and Beaumont, 1964; Creager and Hansen, 1965; Wendler, 1965; Russo-Caia, 1966; Salzageber and Weber, 1966)) in sheep ( Davies and Davies, 1950)) in rabbits ( Bernier and Beaumont, 1964)) and in man ( Rainer, 1911; Altschule, 1930). However, while studies on the regression of the avian mesonephors are numerous, the observations of the same process as it occurs in

1 This investigation was supported, in part, by Grants No. 73.00449.04 and 74.00155.06 from the Consiglio Nazionale delle Ricerche and Angelini Research Institute, Rome, Italy.

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0014-4800

170 DE MARTINO, ZAMBONI AND ACCINNI

mammals, especially in humans, are few and have been made exclusively with conventional methods of morphological investigation. This paucity of information has prompted us to study the fine morphologic changes undergone by the human temporary kidney during different stages of involution and to compare them with those occurring in the definitive kidney in a wide variety of pathologic conditions.

MATERIALS AND METHODS

Mesonephroi of a 12-week-old female human embryo obtained from legal abortion were used for this study. The tissue was fixed by immersion, in part, in 1% buffered OsOl for 1 hr and, in part, in picric ‘acid-formaldehyde (Zamboni and De Martino, 1967; Stefanini et al., 1967) for 24 hr; the latter was then post- fixed for 1 hr in 1% buffered 0~0~. After being embedded in Epon 812, the tissue was sectioned for light microscopy (OS-l-pm-thick sections stained with buffered toluidine blue) and for electron microscopy. For the latter method, the sections were stained with lead hydroxide or by a silver methenamine procedure without periodic acid oxidation (De Martin0 and Zamboni, 1967), and they were ex- amined in a Siemens Elmiskop 101 electron microscope.

RESULTS

At the stage of embryonal development considered in this study, the human mesonephros can be distinguished into three portions: a cranial segment extending from the upper tip of the organ to the upper pole of the developing gonad; an intermediate segment adjacent to the gonad; and a caudal portion below the inferior pole of the gonad. Since the regression of the mesonephros proceeds in a cranial-caudal direction, the study of different portions of the same organ allowed us to monitor the sequential phases of the regressive process which were obviously more advanced in the cranial segment than in the caudal portion; for example, at the stage of embryonal development considered in our study, the nephrons of the cranial portion were in advanced stages of regression, whereas those of the intermediate segment were just beginning to undergo involution, and those of the caudal portion appeared to be intact, and, in fact, some were still differentiating.

The fine morphology of the young and intact glomeruli of the human mesonephros has been described elsewhere (De Martin0 and Zamboni, 1966) and need not be elaborated here any further; a portion of one such glomerulus is shown in Fig. 1, mostly for the purpose of comparing it with the structural organization of the regressing glomeruli in the cranial and intermediate portions. In these, the most salient regressive changes appear to consist of progressive reduction followed by total obliteration of the lumina of the capillary vessels. These phenomena seem to be accounted for mainly by changes of the mesangial cells with concomitant alterations of other components of the capillary wall. The mesangial cells appeared very prominent and displayed morphological characteristics of hypertrophy (Fig. 2). Axial regions containing two or more mesangial cells were not at all uncommon (Fig. 2). The hypertrophic mesangial cells appeared to push against the overlying endothelium of the glomerular capillaries, bringing about a marked reduction of the lumina of the vessels (Fig. 2), the

INVOLUTION OF HUMAN EMBRYO MESONEPHROS 171

FIG. I. Capillary loop of a normal mesonephric glomerulus. L: capillary lumen. E: endothelium. VE: visceral epithelium. hl: mesangial cells. s: subendothelial zone. US: urinary space. X7500.

restriction of which appeared to be increased further by marked enlargement of the “subendothelial zone” of the glomerular capillary wall. All mesangial cells were surrounded by variable, but generally large, quantities of an amorphous material of medium electron opacity (mesangial matrix) in the context of which collagen fibrils, isolated or in small bundles, could be evidenced (Figs. 2 and 2a). At more advanced stages of regression, mesangial cells decreased in number


FIG. 2. Capillary loop of a mesonephric glomerulus undergoing regression (compare with Fig. 1). The capillary lumen (L) is almost completely obliterated as a consequence of the presence of hypertrophic mesangia1 cells (M ) pushing against the overlying endothelium (E). Collagen fibrils (arrows) are visible in the subendothelial zone (s) of the capillary walls. Note the disappearance of the foot processes of the visceral epithelium (VE ) . Silver methenamine stain. X 14,000.

and appeared to become dedifferentiated into fibroblast-like cells (Figs. 3 and 4) and to be surrounded by large quantities of the amorphous material described previously which, by being deposited all around the perimeter of the capillaries, brought about concentric thickening of the wall of the vessels and considerable narrowing of their lumina (Figs. 3 and 4). In these areas, the amorphous material appeared to be pervaded by a maze of linear convolutions (Figs. 3 and 4), interpreted by us to represent remnants of the capillary basal laminae, now highly infolded due to progressive reduction of the glomerular vascular bed. In some glomeruli, the mesangial cells were found not only in the axial regions but in the peripheral portions of the capillary walls where they were interposed between the endothelium and visceral epithelium (Fig. 4). In the most advanced stages noted in this study, the glomeruli appeared to be transformed into pre-


FIG. 2a. A mesangial area of a mesonephric glomerulus undergoing regression. Numerous collagen fibrils (arrows) are present in the increased mesangial matrix (asterisks). E: endothelium. VE: visceral epithelium. M: mesangial cell. X42,000.


FIG. 3. Part of a glomerulus in an advanced stage of regression. The lumen (L) of the capillaries is considerably reduced by the large amount of material (asterisks) deposited in the subendothelial zone (s). The cytoplasm of the endothelial cells (E) shows numerous projections extending into the capillary lumen. Note the paucity of the mesangial cells (M), the shrinkage of the capillary walls, and the disappearance of the foot processes of the visceral epithelium (VE ). CE: capsular epithelium. X4500.


dominantly avascular masses that were surrounded by crowded podocytes and contained endothelial and mesangial cells in various stages of degeneration, as well as large deposits of amorphous material which were particularly conspicuous

in the regions close to the vascular pole and in the wall of the glomerular arterioles in their intrahilar portions (Fig. 5).

Mesangial and capillary wall changes were also associated with modifications of other components of the glomerulus. Even in the initial stages of involution, the cells of the visceral epithelium appeared to be tall and arranged very close to each other (Figs. 2, 3, and 4); these cells had undergone disappearance of their foot processes over large segments of the capillary wall (Figs. 2, 3, and 4) and had developed large numbers of microvilli which crowded the urinary space (Figs. 2, 3, and 4). The endothelial ceils were increased in thickness, and the fenestrations of their cytoplasm had disappeared (Figs. 2, 3, and 4). The cells of

the parietal epithelium appeared to undergo focal degeneration, shown mostly in the form of pronounced cytoplasmic vacuolization (Fig. 6).

Tubular changes were very prominent and were in the form of a degeneration and necrosis of the epithelial cells which became swollen and vacuolated (Fig. 7) and, eventually, were sloughed off into the lumen. Regression of all subcellular components was also seen with consequent formation of myelin-like figures and large pleomorphic cytosegresomes (Figs. 8 and 9); aggregation of needle-shaped structures (crystals?) in the lumen of the proximal tubules was also noted (Fig. 9 and inset). Casts of granular (Fig. 10) and amorphous (Figs. 11 and 12) material frequently filled the tubular lumen. Prominent calcifications, isolated or confluent, were often present in the intertubular interstitium (Fig. 13 and inset). Macrophages and leukocytes were not seen in the interstitial tissue surrounding the regressing nephrons.

DISCUSSION

An analysis of the observations made in the course of this study should include a discussion of (a) the mechanisms responsible for the degeneration of the mesonephric nephrons, and (b) the patterns by which this process evolves. Discussion concerning the first point can be only speculative, since we have no elements to evaluate the possibility, postulated by Willis ( 1958), that the regression of the human mesonephros may be triggered by the activity of the developing thyroid, a factor which is known to be operative in human embryos in the third month of intrauterine life (Arey, 1965) and which has been postulated and experimentally demonstrated to be responsible for the regression of the pronephros in amphibia (Lynn, 1948, Lynn and Peadon, 1955; Fox, 1966, 1967; Fox and Turner, 1967) and of the mesonephros in birds (Moog, 1962, 1965; Creager and Hansen, 1965). Another possibility, which does not necessarily ex- clude the role of the thyroid, is that the involution of the human mesonephros may be initiated by an ischemic condition resulting from the shift of local circula- tion from the indirect portal type to the adult type (Bodemer, 1968).

INVOLUTION OF HUMAN EMBRYO MESOSEPHROS 17’7

The pattern followed by the regressive process, as seen in our study, is similar to that described in previous studies (Von Mihalkovics, 1885; Rainer, 1911; Firket, 1914; Altschule, 1930; Davies and Davies, 1950; Stampli, 1950; Haffen,

1951 a, b; Moog, 1962, 1965; Bernier and Beaumont, 1964; Tilney, 1964; Volle and Beaumont, 1964; Creager and Hansen, 1965; Wendler, 1965; Russo-Caia, 1966; Salzgeber and Weber, 1966). However, although in the regressing avian mesonephros, macrophages and leukocytes are normally present in the interstitial tissue ( Wendler, 1965; Russo-Caia, 1966), interstitial infiltrates of these cells were not noted in our study.

One of the most important features of the process appeared to be the progressive obliteration of the glomerular vascular bed brought about by hypertrophic changes of the mesangial cells with concomitant deposition of amorphous material and collagen fibrils along the wall of the glomerular capillaries. Our ob-

servations seem to indicate that the deposition of these elements was mediated by the secretory activity of the hypertrophic mesangial cells which, in the process, assumed morphological characteristics typical of connective tissue cells; thus, our observations confirm the notion derived from previous studies (Latta, 1961; Zamboni and De Martino, 1968; Simon and Chatelanat, 1969) that the mesangial cells are capable of collagen fibril synthesis under a variety of experimental and pathologic conditions, These considerations are of interest in that they represent yet another indication of the multipotentiality of the mesangial cells which, additionally, are capable of contractile activity (Goormaghtigh, 1942a, b; Yamada, 1955; Michielsen, 1961; Latta et al., 1962; Michielsen and Creemers, 1967; Zam- boni and De Martino, 1968; Pease, 1968; De Martin0 et al., 1969, 1973; Becker, 1972; Accinni et al., 1975), very likely directed at monitoring the flow of circula- tion in the various districts of the glomerular capillary bed (Zamboni and De Martino, 1968; De Martin0 et al., 1973), and phagocytic activity, aimed at clearing the lumina and walls of glomerular capillaries of a variety of substances (Latta et al., 1960; Farquhar and Palade, 1962; Bartman et al., 1964; Pardo and

Shapiro, 1966). Also of importance is the observation that the glomerular changes noted in this

study are similar to those occurring in the definitive kidney as a result of a wide

FIG. 4. Mesonephric glomerulus in advanced stages regression. The capillary lumen (L) is

almost obliterated as a consequence of the deposition of large amounts of amorphous material

all around the vessel wall. This material is pervaded by a maze of linear convolutions con-

sidered to represent infolded remnants of the capillary basal lamina. A mesangial fibroblast- like cell (M) is interposed between the capillary endothelium and the visceral epithelium

( VE ) . The visceral epithelial cells are tall and close to each other; there is total disappearance

of their foot processes and the presence of numerous microvillosities which protrude into the

urinary space. (US). L: capillary lumen. X12,000.

FIG. 5. Light micrograph showing a regressing mesonephric glomerulus with its vascular pole

and adjacent proximal tubules (PT). Note the avascular appearance of the glomerulus and the large areas of hyalinization in proximity of the hilus. A: Glomerular arteriole; OS-l-p-thick

section, toluidine blue stain. X 1000.

17s DE MARTINO, ZAMBONI AND ACCINNI

FIG. 6. Focal degeneration of cells of the parietal epithelium (arrows) in a regressing mesonephric glomerulus; 0.5-I-*m-thick section, toluidine blue stain. X 1000.

FIG. 7. Focal degeneration of epithelial cells of a proximal tubule. Notice the vacuolization of the cytoplasm of the degenerating cell (Cl), the swelling of the organelles, and the decreased number of microvilli in the brush holder. G: normal tubular cell. x6500.

FIG. 8. Autophagic vacuole ( AV) in the apical cytoplasm of an epithelial cell of the proximal tubule and atrophy of the brush border microvilli (arrows). L: tubular lumen. X13,000.


FIG. 9. Lumen of a proximal tubule obliterated by compact masses of cellular debris, often including needle-shaped structures (arrow) as shown at higher magnification in the inset. X 16,000. Inset, X94,000.

FIG. 10. Granular cast obliterating the lumen of a proximal tubule; 0.5-l-pm-thick section, toluidine blue stain. X1500.

variety of glomerulopathies. For example, increased thickmess of the capillary wall, proliferation and hypertrophy of mesangial cells, migration of these cells into the peripheral portions of the capillary wall, increased secretion and deposi-


FIGS. 11 and 12. Casts of amorphous material in the lumina of collecting tubules. Fig. 11. Toluidine blue stain. 0.5-I-pm-thick section. X1500. Fig. 12. X10,000.

tion of mesangial matrix, and synthesis and deposition of collagen fibrils are well known changes characterizing a broad spectrum of proliferative, membranous, and membrano-proliferative glomerulonephritides <and leading, in the most severe of these, to fibrotic obliteration of the glomerulus (Simon and Chatelanat, 1969; Heptinstall, 1974).

The changes noted during the various stages of the mesonephric nephrons in the cells of the glomerular visceral epithelium, i.e., disappearance of foot processes and development of microvilli, and in the cells of the proximal, distal, and collecting tubules, i.e., tubular casts, disappearance of brush border, cell necrosis and calcifications, are also of the type known to occur in a wide variety of pathological conditions affecting the definitive kidney (Chatelant and Simon, 1969; Simon and Chatelanat, 1969; Heptinstall, 1974).

In conclusion, the present observations seem to be of importance in that they demonstrate the existence of close similarities between the morphological changes observed in the mesonephros during its involution and those of the metanephros during the course of several renal diseases, notwithstanding the fact that those occurring in the mesonephros result from a physiological process of involution, whereas those occurring in the nephrons of the definitive kidney are usually the consequence of pathological insults.

ACKNOWLEDGMENTS

The excellent technical assistance of Miss Laura Vassallo and Mr. Mario Termine is deeply appreciated.


FIG. 13. Confluent and isolated calcifications are frequently seen around the periphery of the proximal tubules; the calcification indicated by the arrow is shown at high magnification in the inset. X9500. Inset, X22,000.


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fine morphology of regressing human mesonephric nephrons

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