View source for Paper - The mechanism of kidney development in human embryos as revealed by an early stage in the agenesis of the ureteric buds

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=The Mechanism of Kidney Development in Human Embryos as Revealed by an Early Stage in the Agenesis of the Ureteric Buds=

Peter Gruenwald  Cook County Graduate School Of Medicine, Chicago, Illinois  

One Plate (Six figures)  

==Introduction==
The examination of serial sections of an apparently normal human embryo of 9 mm. has revealed the failure of the kidneys, in this specimen, to develop. Because of the early stage of agenesis, an opportunity was thus presented for studying the role of different embryonic structures in the development of this comparatively frequent malformation.


The embryo in question had been fixed in Bouin’s ﬂuid and embedded in paraffin and no evidence of any anomaly had been suspected by gross inspection. Serial transverse sections of 8,u in thickness were made and stained after the azan modification of Mallory’s connective- tissue stain.


Upon section, the right umbilical artery was found to be abnormally small in diameter both in the umbilical cord and in the anterior abdominal wall; indeed it could not be traced to the aorta, its only visible connection being with an artery of the right leg. The corresponding artery on the other side is normal in size and position.


The right artery was probably undergoing obliteration at the time of death of the embryo. If this process could have gone on to completion, the artery in question might have become completely invisible in later stages and a primary occurrence of only one umbilical artery might have been predicated. This vessel would have differed only in position from that in sireniform monsters in which both umbilical arteries are replaced by a single median vessel.


Among the organs of the urinary tract the mesonephros is wholly normal in structure on each side. Both wolffian ducts are present and of normal width (10 X 45 u) in the mesonephrie region. At the level of the caudal end of the mesonephros the ducts are dilated up to a diameter of about 100 n. It is dillicult to say whether this is still within normal limits, as a slight dilatation always appears at this level. A short distance from the opening into the hind—gut the width of both ducts decreases considerably so that in the most caudal part a patent lumen cannot be seen with certainty on all sections. There is, however, no evidence of an obstruction. No ureteric buds can be found in this embryo. The only structure which possibly might be considered as a vestigial ureteric bud is a small dorsal divertieulum of the left wolﬂian duct originating from the dilated part shortly below the end of the mesonephros (U’ in fig. 2). This formation, however, is hardly longer than wide, its length being about 40 14. No such structure can be seen on the right side.


The metanephrogenie tissue is very clearly visible on both sides. It is continuous with the mesonephros and forms a distinct cell mass on either side of the body. figure 2 gives a view of this mass on the right side; in consequence of the curvature of the embryo the metanephrogenie cell masses of both sides cannot be shown in the same section. Both masses are clearly separated from each other by loose mesenehyma. The questionable vestigial left ureteric bud mentioned above does not reach the nephrogenic tissue. It ends at a distance of about 80 ll from the latter.


This case affords an opportunity to study in a human embryo the appearance of the metanephrogenie tissue when not in contact with a ureteric bud. In chick embryos this condition has been produced experimentally by Boyden (’27, ’32) and G-riinwald ('37, ’38 a) by damaging the growing end of the wolﬂian duct in younger stages. As a result, the ureteric bud failed to develop and the metanephrogenie tissue, distinctly visible on the seventh day, could only be found with difficulty on the twelfth day of incubation, at a time when it should have developed distinct epithelial structures under normal conditions. In these experiments the differences in structure between the metanephrogenic cell masses with and without ureteric bud are quite similar to those to be described here (see below).


In the man, the youngest case hitherto described is a 10mm. embryo (Boyden, ’32). Besides a complete absence of the left ureteric bud, an abnormal contact between the two metanephrogenic masses was described and the question was raised whether this might not have led to the formation of a horseshoe kidney if both ureteric buds had been present. The histological structure of the tissue was not reported in detail because of the poor preservation of the embryo. However, the illustrations demonstrate clearly the great difference in size between the metanephrogenic tissue that is associated with a ureteric bud and that which is without a ureteric bud. Boyden emphasizes that there is no evidence that absence of a kidney is due to an aplasia of the metanephrogenic tissue. Gruber (l. c., p. 269) also deduces this from the fact that in cases of absence of one ureteric bud the other one may grow across the midline and form a kidney with the metanephrogenie tissue of the defective side.


Another case, much older however, was reported by Kornfeld in 1925-1926. This 5—cm. embryo has a defect that corresponds exactly to the condition that arises after experimental obstruction of the wolfiian duct. The caudal part of the left mesonephros is missing as well as the wolifian duct, both at this level and caudal thereto. No kidney is present on this side and no trace of the metanephrogenic tissue is visible any longer, as I could confirm myself when Doctor Kornfeld kindly showed me the serial sections of his case.


In order to evaluate the inﬂuence of the woltfian duct on the Inetanephrogenic tissue, the kidneys of the present case have been compared with those of a normal embryo of the same stage, namely with a 9.5-mm. specimen, treated with the same histological technic (figs. 3 and 5). A section of the cranial portion of the left mesonephros is given in figure 5. It shows a portion of the distal enlargement of the ureteric bud (U) in close contact with the metanephrogenic tissue (T). Farther craniad Schreiner’s ‘Zwischenblastem’ is visible (Z), consisting of a small portion of the nephrogenic tissue which is not used for the formation of either mesonephros or metanephros. figure 4 shows a portion of the left metanephrogenic tissue (T) of the abnormal embryo, which extends craniad up to the last tubule of the mesonephros (M). However, in this case the ‘Zwischenblastem’ cannot be differentiated from the metanephrogenic tissue. Both pictures are reproduced with the same magnification. Comparing the metanephrogenic tissues of both embryos, we find differences in number, shape, and arrangement of the cells. The difference in number is quite striking, as can be seen by comparing figures 2 and 3. Already, the metanephrogenic cells of the normal embryo must have undergone a rapid multiplication. The cells of the dense inner layer of the metanephrogenic tissue not only have oblong nuclei but are arranged radially around the ureteric bud. In the defective embryo where the ureteric buds are missing, the cells have round nuclei and are scattered irregularly. Only under low power does their arrangement suggest the presence of indistinct cell cords (fig. 2). There is thus a striking resemblance between the metanephrogenic tissue of this abnormal embryo and the ‘Zwischenblastem’ of normal embryos.


Similar conditions can be seen in the chick embryos used for the above mentioned experiments. In a 7-day embryo with no ureteric bud on the left side the left metanephrogenic tissue forms an indistinctly limited mass of cells with round nuclei differing from the connective tissue by the absence of fibers. In the normal kidney the cells have increased in number and are centered around the ureteric bud. There is a very large number of mitoses in the metanephrogenic tissue of the normal right side contrasting sharply with the rare incidence of cell division on the left side. Twelve-day embryos of the same series of experiments show only small remnants of the metanephrogenic tissue on the side which had been operated upon. A low—power photograph of a section showing the difference between both sides was published previously (Grriinwald, ’37, fig. 5b). figure 6 is a high—power view of another section of the left metanephrogenic tissue of the same embryo. There is a dense cell mass staining deeper with eosin than the surrounding connective tissue. Its nuclei are round in contrast to those of the connective tissue and no fibers can be seen in the nephrogenic cells. Otherwise no distinct border between the two tissues exists. In general the condition of the defective metanephrogenic tissue produced experimentally in the chick embryo is the same as that occurring spontaneously in the human embryo.
==Discussion==

The above comparison of defective and normal human ki.d— neys of the same age indicates clearly that contact with the ureteric bud is the factor that quickly induces multiplication, regular arrangement and changes in the shape of the metanephrogenic cells. The uninﬂuenced metanephrogenic tissue closely resembles in structure the small complex of undifferentiated nephrogenic tissue (‘Zwischenblastem’) which in normal embryos separates the blastema of the kidney from the mesonephros. This morphological similarity may be explained on the basis that these two tissues are developing under the same conditions, i.e., independently from the uretcric bud, in the abnormal embryo. This suggests that the ‘Zwischenblastem’ which disappears completely, is merely that portion of the metanephrogenic tissue which is uninﬂuenced by the ureteric bud.

Another point of interest is the normal ‘migration’ of the metanephros. Many explanations have been advanced both with respect to the way it happens and with regard to the role played by the two constituents of the kidney. As I pointed out in a critical review of different theories (’38 b), there are many unknown factors which may be concerned in  this change in position. Even at this late date we cannot say with assurance that tl1e metanephrogenic tissue inﬂuences either the intensity or the direction of growth of the ureteric bud. The possibility that it does, has been emphasized especially by Gilman in his discussion of a rare form of dystopic kidney. Another theory, suggested by the course of normal development, is that the ureteric bud and its branches push the metanephrogenic tissue forward and thus accomplish the ascensus. This possibility received favorable consideration in my article of ’38 b, but a study of this defective embryo shows that the distance between the metanephrogenic tissue and the point where the ureteric bud should originate, grows considerably within a short period even in absence of the ureteric bud. Apparently, therefore, this is merely a shift caused by relative differences in growth and therefore should not be ascribed to an action of the ureteric bud. Similarly, the subsequent change in position of the metanephrogenic tissue in respect to the mesonephros can be explained on the basis of the well-known downward transposition of the mesonephros and gonad. Moreover, the kidney by that time is so large compared with the diameter of the ureter that the latter could not possibly displace it by pushing it upward. This, however, does not mean that the ureteric bud is unable to inﬂuence the position of the kidney in early stages and thus to produce dystopia. It might cause, by irregular growth, a suﬂicient dislocation of the whole anlage to amount for the formation of a horse—shoe kidney, but all we can conclude from the present case is that the normal shift of the permanent kidney is due to differences in growth of the surrounding tissue and not to an active power exerted by the ureteric bud. 


Brockmann (’36) reaches the same conclusion although he failed to examine young stages. Starkenstein (’38), after investigating younger stages, maintained that the ‘migration’ of the kidney extends over a longer distance than is usually recognized. He opposes Brockmann’s opinion and supposes an active ascensus, without giving any convincing reasons for it. This rapid independent displacement of the metanephrogenic tissue observed in the present case, has another important bearing on kidney development. It predicates that the normal fusion of the two constituents which is essential for further normal development, can only take place during a short period, for the metanephrogenic tissue is withdrawn from the place where the ureteric bud should join it soon after the two are supposed to come together. It is doubtful whether a normal kidney can develop after that time, if one excepts the possibility that other cells can also be stimulated to kidney development by the ureteric bud. 


In this connection Brown’s report is of special interest. This author investigated the embryonic development of a strain of mice which by earlier exposure to x-ray treatment had acquired high percentages of deformities of the urinary and other tracts. Some of these embryos had an unbranched ureteric bud reaching only to the border of the metanephrogenic tissue. The latter, however, showed no evidence of stimulation. Brown concluded: “It seems absolutely necessary that the ureter bud get into the blastema mass in order to form a functional kidney. Succeeding only in part or just reaching the blastema produces various conditions . . . .” (l.c., p. 147). To be sure, in Brown’s cases the objection can be made that one or both of the renal primordia were rendered genetically inferior by the x-ray damage to the parents, but the anomaly can also be explained very well by retarded outgrowth of the ureteric bud.


Concerning the cause of agenesis in the present case, no evidence is available. Certainly, there is nothing to suggest a causal connection with the other two anomalies, the abortive right umbilical artery and the slight increase in diameter of the wolffian ducts.


Further development would certainly have led to complete absence of both kidneys, although it is possible that the rudimentary diverticulum of the left wolﬂian duct described above, might have formed an abortive ureter.

==Summary==
# The study of an early stage of aplasia of the ureteric buds in a 9—mm. human embryo shows that normally this structure inﬂuences the metanephrogenic tissue Very soon after it comes in contact with it, thereby inducing the metanephrogenic cells to multiply and to assume the shape and arrangement typical for the normal internal layer of the metanephrogenic tissue.
# The normal change in position of the permanent kidney is due to shifts by growth of the surrounding tissue and not to a pushing activity of the ureter.
# It is probably of great importance that the ureteric bud grows out of the Wolffian duct exactly at the correct time to warrant a normal development.
# An early stage of obliteration of the right umbilical artery was noticed in the same embryo.

==Literature Cited==

BOYDEN, E. A. 1927 Experimental obstruction of the mesonephric ducts. Proc. Soc. Exp. Biol. Med., VOl. 24, pp. 572-576. 

1932 Congenital absence of the kidney. An interpretation based on a 10—mm. human embryo exhibiting unilateral renal agenesis. Anat. Rec., vol. 52, pp. 325-350. 

BROCKMANN, A. W. 1936 Form— und Lagcentwicklung der Niere. Morphol. Jahrb., vol. 77, pp. 605-665. 

BROWN, A. L. 1931 An analysis of the developing metanephros in mouse embryos with abnormal kidneys. Am. J. Anat., vol. 47, pp. 117-172. 

GILMAN, M. B. 1927 A note on a rare congenital pelvic kidney. Anat. Rcc., vol. 36, pp, 149-154. GRUBER, Gr. B. 1927 Missbildungen der Harnorgane. In: Schwalbe and Gruber, Die Morphologie der Missbildungen des Menschen und der Tiere. fischer, Jena. 

GRUNWALD, P. 1937 Zur Entwicklungsmechanik des Urogenitalsystems beim Huhn. Arch. Entw.-mechan., vol. 136, pp. 786-813. 

1938a Entwicklungsmechanische Untersuchungen iiber die Genese einiger Fehlbildungen des Urogenitalsystems. Beitr. pathol. Anat., Vol. 100, pp. 309-322. 

1938b Embryologische Beitrage zur Kasuistik und Grenese der Nierendystopien. Virchow’s Arch., vol. 303, pp. 47-59. 

KORNFELD, W’. 1925-1926 Ueber einen Fall von einseitigem Nierenmangel bei einem menschlichen Embryo aus dem 3. Embryonalmonate. Anat. Anz., Vol. 60, pp. 497-515. 

STARKENSTEIN, W. 1938 Ueber die Anlage und die Wanderung der Nachniere beim Menschen. Morphol. Jahrb., vol. 81, pp. 8-20. 

==Plates==
1 Section of the proximal part of the umbilical cord of the maldeveloped 9-mm. human embryo, showing the small diameter of the right umbilical artery (R) and the large left umbilical artery (L). C, coelomic cavity.


2 Frontal section of the same embryo, showing the right metanephrogenic tissue (T) which is not differentiating in the absence of the stimulating ureterie bud. C, abdominal cavity (left side); L, left umbilical artery; U’, rudimentary left ureterie bud (“?) ; W, left wolffian duet. Arrow indicates position of mid line.


3 Section of a normal 9.5-mm. human embryo, corresponding to figure 2. C, abdominal cavity; L, left umbilical artery; T, left metanephrogenie tissue; U, left ureterie bud; W, left wolffian duet; Z, ‘Zwisehenblastem.’ Same magnification as in figure 2.


4 High power photomierograph of the left metanephrogenie tissue of the same embryo as in figures 1 and 2. M, last mesonephric tubule; '1‘, metanephrogenie tissue.


5 High power photomierograph of a part of the normal kidney shown in figure 3. T, metanephrogenie tissue; U, ureterie bud; Z, ‘Zvvisehenblastem.’ Same magnification as in figure 4. ’  6 High power photomicrograph of the remnant of the left metanephrogenic tissue of chick embryo 438 (10 days a11d 20 hours of incubation), following destruction of the left wolﬂian duct on the second day of incubation and the consequent failure of the left ureteric bud.


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