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From the Departments of Obstetrics,*
Histoembryology,
and
Nephrology¶
and Institut National de la
Santé et de la Recherche Médicale Unité
423,
Hôpital Necker-Enfants Malades,
Université René Descartes; the Department of
Pathology,§
Hôpital Cochin; and the
Department of Histoembryology,||
Hôpital Saint
Antoine, Paris, France
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Abstract |
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Introduction |
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Over the past decade, the survival rate of TTS babies has increased owing to improved neonatal resuscitation and to prenatal therapy based on amniodrainage,1-3 coagulation of the intertwin placental shunts,4-6 selective feticide,7-9 or amniotomy.10 However, TTS still carries a perinatal mortality as high as 50% with a short-term neurological morbidity of 5 to 18%,2,5,8 underscoring the need for further research.
Previous studies suggest that TTS has a complex pathophysiology that is derived primarily from an unbalanced blood flow from the donor to the recipient through arteriovenous placental anastomoses. In the recipient twin, polyuria is thought to result from hypervolemia11,12 and could be partly mediated by the activation of atrial–natriuretic-factor production.13 In addition, a recent case report suggested that systemic hypertension may occur in the recipient during the fetal stage.14 In contrast, the diuresis of the donor decreases, possibly as a consequence of hypovolemia. Therefore, the regulation of fetal volemia and diuresis could be central to the pathophysiology of TTS .
We speculated that the activation of the renin angiotensin system (RAS) in the donor could be one of the noxious adaptive changes in TTS. To test this hypothesis, we conducted a retrospective postmortem study in 21 monochorionic twin pairs who died from TTS and in 39 controls. The morphology of the fetal kidneys and the level of renin expression were studied as markers of the potential involvement of the RAS in the cascade of hemodynamic and metabolic changes induced by the syndrome.
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Materials and Methods |
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Gestational age at clinical presentation ranged from 17 to 28 menstrual
weeks and, at delivery, from 19 to 30 weeks. There were 13 terminations
of pregnancy, 3 miscarriages at 
24 weeks, 4 premature deliveries with
neonatal deaths of both twins, and 1 premature delivery with
intrauterine death of the donor and neonatal death of the recipient.
The prenatal diagnosis of TTS was based on the occurrence of major
polyhydramnios in one twin of a monochorionic diamniotic pregnancy.
Nine recipients were hydropic, and eight had cardiomyopathy without
hydrops. The donor had oligohydramnios in 20 cases, 7 of which
presented with a stuck-twin syndrome (Table 1)
. The diagnosis of a
monochorionic-diamniotic twin pregnancy was confirmed by a gross
examination of the placenta and by a histological study of the
intertwin membrane. In all cases, postmortem findings confirmed the
diagnosis of TTS, the donor being growth retarded with a small heart
and kidneys while the recipient twin was larger, with an enlarged heart
and kidneys (Table 2)
. There were no
associated malformations.
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Morphology
Both kidneys of each twin were fixed in 10% buffered formalin. Frontal corticomedullary sections were taken through the hilus and embedded in paraplast. Sections (4 µm thick) were stained with trichrome-light green, trichrome-safran, or periodic acid/Schiff reagent.
Immunohistochemistry
Immunostaining was performed on formalin-fixed paraffin-embedded tissues by the avidin-biotin method and with the Universal Immunostaining Streptavidin-Peroxidase Kit (Immunotech, Marseille, France). Good conservation of the kidneys allowed their study in 19 twin pairs. Deparaffinized sections were rehydrated, washed in a phosphate-buffered saline buffer for 5 minutes, treated 5 minutes by 3% H2O2 in methanol to block any endogenous-peroxidase activity, and washed in a buffer for 2 minutes. Then they were incubated sequentially for 10 minutes with the protein-blocking agent and for 60 minutes with the primary antibody (see next section). After washing in a phospate-buffered saline, the sections were incubated for 30 minutes with the polyvalent secondary biotinylated antibody. Washed sections were incubated for 45 minutes with the streptavidin-peroxidase reagent. After washing, they were incubated for 10 to 20 minutes with the freshly prepared chromogen solution (H2O2 + the chromogen 3-amino-9 ethylcarbazole). The sections were counterstained with hematoxylin and mounted in aqueous media. The control sections were processed as above, but the primary antibodies were omitted.
Rabbit polyclonal anti-renin antibodies were raised against human renin
purified from a juxtaglomerular cell tumor.15
Their
specificity was previously established.16
The antiserum
with the highest antirenin titer, a gift from P. Corvol (Institut
National de la Santé et de la Recherche Médicale
Unité 36, Paris), was selected for the study. We used a
monoclonal anti-
-smooth muscle actin (SMA; Sigma Chemical, St.
Louis, MO), an Iopath monoclonal antibody anti-epithelial membrane
antigen (EMA), which is a marker for distal and collecting tubules
(Immunotech, Marseille, France), and a monoclonal antibody anti-CD15,
which recognizes an early myeloid differentiation antigen and is a
marker for proximal tubules (PTs; Immunotech). Antibodies were
used at the following concentrations: anti-EMA, pure (prediluted by the
manufacturer); anti-CD15, 1/100; anti-SMA, 1/500; and anti-renin,
1/500.
A semiquantitative evaluation of renin content was performed on the strictly corticomedullary sections taken through the hilus. Developing nephrons were not considered. We recorded the number of positive juxtaglomerular apparatus (JGA) for the 100 glomeruli examined and the number of renin-positive cells per section of JGA. We also evaluated the presence of renin-positive cells in the wall of afferent arterioles at a distance from glomeruli and in the interlobular arteries. Normal kidneys from single or multiple pregnancies served as controls.
In Situ Hybridization
The renin cDNA clone pGRh1417 was a gift from J. M. Gasc.18 Recombinant plasmids were linearized with the restriction enzymes HindIII or Sac. The in vitro transcription was carried out in the presence of digoxigenin (DIG)-uridine 5'-triphosphate, using the RNA polymerases T7 or SP6 probes to produce antisense or sense probes, by the manufacturer’s instructions (Boehringer Mannheim, Mannheim, Germany). These probes were DNase I-treated, purified, and stored at -80°C.
In situ hybridization was performed in seven twin pairs. Sections (6 µm thick) mounted on silanated slides were deparaffinized, postfixed in 4% paraformaldehyde before digestion for 20 minutes with proteinase K (Sigma), and again postfixed in paraformaldehyde. After dehydration and air-drying, the sections were prehybridized, drained, and hybridized overnight in a humid chamber at 52°C with DIG-labeled RNA sense or antisense probes (5–10 ng/ml) in the prehybridization buffer. Slides were washed at room temperature in solutions of various degrees of stringency (from 5x standard saline citrate with 50% formamide at 55°C to 0.1x standard saline citrate). DIG-labeled probes were detected by immunohistochemical methods with the Nonradioactive Nucleic Acid Detection Kit (Boehringer Mannheim). Briefly, slides were washed for 5 minutes at room temperature (RT) in a washing buffer and incubated for 30 minutes with a 0.5% Boehringer blocking reagent in the same buffer. They were then incubated for 1 hour at RT with alkaline phosphatase-conjugated sheep anti-DIG antibody at 1/500 dilution. After washing, the color was developed with the chromogenic agents nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate, and 2 mmol/L levamisole in the dark at RT. After the color was fully developed, all of the sections were washed at the same time with the buffer (100 mmol/L Tris-HCl, 1 mmol/L ethylenediaminetetraacetic acid, pH 8) to allow a comparison of the hybridization signal and then mounted in an aqueous medium.
A semiquantitative evaluation of renin expression was performed by the same method as described for the assessment of renin protein. Four normal fetal kidneys served as controls.
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Results |
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Normal Controls (18–30 Weeks)
In the 39 control fetuses, the various steps of nephron
differentiation were observed from the outer to the deep cortex.
Undifferentiated mesenchyme and immature structures were located
beneath the capsula, and the most mature nephrons were seen at the
juxtamedullary junction. Even at the early development stages, the
different tubular segments could be identified by immunostaining. PTs
were labeled with CD15 antibodies from the glomerular capillary stage
onwards, whereas distal tubules and collecting ducts were stained with
anti-EMA antibodies. The vascular smooth muscle cells strongly
expressed -SMA. No expression was detected in the interstitial or
blastema cells, whereas occasional expression could be seen in a few
glomerular mesangial cells.
Donor Twins (18–30 Weeks)
In 10 of the 21 fetuses (20–30 weeks), renal tubular dysgenesis
(RTD) was observed (Figure 1a)
. The
cortical tubules were lined by small cuboidal or columnar epithelial
cells, none showing an apical brush border characteristic of the
proximal tubules. With the CD15 antibody, a marker of PT, there was no
or only very occasional labeling of the small sections of poorly
differentiated tubules of the deep cortex. Conversely, strong and
uniform tubular apical labeling was seen with the anti-EMA antibody,
which normally recognizes distal tubules and collecting ducts (Figure 1b)
. Glomeruli were crowded together because of the poor
differentiation of tubules. They appeared ischemic, with small
glomerular tufts within relatively large Bowman’s capsules, and the
capillary loops lined by podocytes were closely distributed at the
periphery of the tuft. The increase in glomerular size from the
superficial to the deep cortex normally associated with maturation was
not observed. The large arteries were normal, but interlobular and
preglomerular arteries showed muscular-wall thickening also evidenced
by -SMA labeling (Figure 2a)
.
Occasionally, mesangial cells of developing glomeruli were
-SMA-positive. In the medulla, the interstitial mesenchyme was
strikingly increased, resulting in an abrupt corticomedullary
delimitation (Figure 1a)
.
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Recipient Twins (21 Fetuses, 18–30 Weeks)
In all cases, the recipient’s kidneys were enlarged (Table 2)
and
congested. Hemorrhagic infarction was present in 19/21 cases. It was
extensive in four fetuses, involved diffusely the corticomedullary
junctions in four, and was focal in 11. Glomeruli were strikingly
enlarged due to the diffuse mesangial matrix expansion and mesangial
hypercellularity (Figure 2b)
. Hypertrophy always predominated in the
most mature glomeruli located in the deep cortex. Focal (three cases)
or diffuse (seven cases) glomerular lesions were superimposed on
glomerulomegaly and congestion. They consisted in thickening and the
double-contour appearance of the capillary wall due to mesangial
interposition and to the presence of clear subendothelial deposits.
Focal mesangiolysis was observed in three of the seven cases with
diffuse glomerular lesions. With anti--SMA antibodies, all mesangial
cells and their subendothelial expansion were strongly labeled
(Figure 2c)
. Thickening of preglomerular and interlobular arteries was
observed in 11 fetuses, due to smooth muscle cell hypertrophy and focal
vacuolation (Figure 2d)
. Tubular differentiation was normal with
positive CD15 labeling of proximal segments and EMA labeling of distal
and collecting tubules.
Renin Expression
Immunohistochemistry
Normal fetal kidneys. In 18 of the 21 singleton
fetuses, a few cells (1–4) in 5 to 10% of the JGA were
renin-positive, whereas renin labeling was observed in 10 to 20% of
JGA in three fetuses (Table 3)
. In twin
pregnancies, renin-positive cells were observed in 10 to 25% of JGA in
the fetal kidneys of eight twin pairs, with usually less than four
renin-positive cells by positive JGA (Figure 3a)
. No mesangial cell was
renin-positive. In each fetus of one twin pair, 30 to 40% of JGA were
labeled with a variable number (1 to 8) of positive cells in each JGA.
These 22-week-old fetuses with dichorionic, diamniotic placenta were
female and 590 and 500 g, respectively, at birth, with
morphologically normal kidneys and no sign of TTS.
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. The number of renin-positive cells by JGA was
less than four in three donors and reached six cells in some JGA
sections in the other two. In 14 twin donors, 40 to 90% of glomeruli
had renin-positive JGA, with more than four renin-positive cells by
positive JGA (Figure 3d)
. The labeling was always prominent in mature
nephrons. In these patients, the renin-positive cells were also
frequently observed in the muscular layer of the afferent arterioles at
a distance from the glomeruli and in the interlobular arteries in three
patients. In addition, positive mesangial cells were observed in eight
of these patients and were very abundant in the five patients with 80
to 90% of renin-positive JGA (Figure 3e)
.
Recipient twins (20 fetuses, 18–30 weeks). Immunolabeling
with anti-renin antibodies detected no renin-positive cells in 19
recipients (Figure 3b
; Table 3
) . Conversely, in one fetus with severe
arteriolar thickening, renin-positive cells were present in the JGA of
about 20% of the glomeruli. It is interesting that only the developing
glomeruli had positive JGA, which were associated with the presence of
renin-positive cells in numerous sections of arterioles located in the
outer cortex. JGA of the enlarged glomeruli of the deep cortex were
renin-negative.
In Situ Hybridization
Normal fetal kidneys (four kidneys). Renin expression was observed in the JGA at the vascular poles of 10 to 30% of the mature glomeruli. In these four fetuses, renin protein was seen in 10 to 20% of JGA. Renin transcripts were also present in the dispersed cells of preglomerular arteries.
Donor twins (seven kidneys). A strong expression of renin
was observed in the JGA of 30 to 90% of the glomeruli, with more than
six cells labeled in most JGA. The extensive expression of renin
messenger RNA (mRNA) was also observed in afferent arteriole segments
at a distance from the glomeruli. Correlations between protein and mRNA
expression were as follows: 30% of the glomeruli had RNA-positive JGA
in one fetus expressing the protein in 40% of JGA; 50 to 60% of JGA
were mRNA positive in the four fetuses expressing the renin protein in
20, 30, 40, and 50% of JGA, respectively; and 90% of JGA were
positive in the two patients showing the renin protein in 80 and
90% of JGA, respectively (Figure 3f)
. In these two patients, the renin
transcript, as well as the protein, were also observed focally in the
mesangial cells and in the smooth muscle cells of interlobular
arteries.
Recipient twins (seven kidneys). In 6/7 fetuses tested,
there was either no renin expression (2 cases) or minor renin
expression restricted to about 10% of JGA (four cases; Figure 3c
).
However, in the kidney of the one fetus expressing renin protein, renin
transcripts were also detected in JGA of 30% of glomeruli, mostly in
the developing nephrons located in the intermediate and outer cortex.
They were also observed in the smooth muscle cells of arterioles of the
superficial cortex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The renin-angiotensin system is highly active during fetal life,26 and is regulated by the same mechanisms as in postnatal life. One major mechanism stimulating renin synthesis and release is volume depletion and decreased renal perfusion pressure.27,28 The endpoint of the stimulation of the renin-angiotensin cascade is the generation of angiotensin II, a potent vasoconstrictor peptide whose expected beneficial effects are to restore extracellular volume through stimulation of aldosterone release and to maintain peripheral blood pressure by acting directly on the vascular smooth muscle cells. However, these adaptive mechanisms may fail to compensate volume depletion in the donor. In this case, angiotensin II-induced intrarenal vasoconstriction may act as an additional deleterious factor by further reducing renal blood flow and fetal diuresis, which in turn aggravates oligohydramnios.
The recipient kidneys were large and congested, showing more or less
extensive hemorrhagic infarction. The proximal tubule differentiation
was normal, whereas prominent changes involved the glomeruli and, to a
lesser extent, arteries. Glomeruli were strikingly enlarged due to
mesangial matrix expansion. Mesangial hypercellularity and hypertrophy
were outlined by an increased expression of -SMA. -SMA is a
marker of mesangial cell proliferation and activation in human and
experimental glomerulonephritis.29,30
In the human fetal
kidney, it is normally expressed by mesangial cells in the developing
glomeruli as soon as the mesangium and capillary loops become
identifiable.31
Its expression decreases substantially in
mature human fetal glomeruli.31
The strong mesangial
expression in the recipient fetuses may result from hypertension as
up-regulation of -SMA by mesangial cells is observed in
hypertension-injured glomeruli.29,32
Half of the recipient twins had additional glomerular lesions consisting of the presence of clear subendothelial deposits associated with focal or diffuse peripheral mesangial interposition between the glomerular basement membrane and endothelial cells, causing a double-contour appearance of the capillary wall. These lesions are reminiscent of those observed in polycythemia or in hypertension-induced thrombotic microangiopathy in children.24,33 The occurrence of focal mesangiolysis in some fetuses and of severe arterial and arteriolar changes strongly suggests that not only polycythemia but also hypertension participates in the development of renal lesions. Hypertension has been documented recently in a recipient fetus by ultrasonography.14 Hypertension could be a direct consequence of hypervolemia, but other pathogenic factors may be involved and particularly the inappropriate production of angiotensin II.
Down-regulation of renin synthesis is expected in hypervolemic recipients, resulting from the inhibiting action of high-perfusion pressure on renal baroreceptors. Actually, no renin protein was detected in 20/21 recipient fetuses, whereas none or a reduced number of JGA expressed the renin transcripts, suggesting a double transcriptional and post-transcriptional regulation of renin synthesis. In the fetus with severe hypertensive-glomerular and arteriolar lesions, renin expression, observed in the distal part of the renal vasculature (JGA of the developing nephrons and arterioles of the outer cortex), was probably secondary to arteriolar changes. Plasma renin concentration or activity has not been measured in this series of patients. However, we may speculate that circulating renin from the donor is transferred to the recipient through placental vascular anastomoses. Indeed, renin activity was previously detected in both twins in three cases of TTS.13 This inappropriate presence of renin in hypervolemic fetuses, with the eventual increase of angiotensin II generation and aldosterone synthesis, represents a potential factor in aggravating hypervolemia and its renal and extrarenal consequences for the recipient, including systemic hypertension.
In conclusion, in TTS the renal lesions observed in donors and recipients are partly due to hemodynamic changes in both fetuses: hypovolemia in the donor and hypervolemia/polycythemia in the recipient. Hypovolemia-induced increased renin synthesis in the donor may negatively affect both fetuses by aggravating hypoperfusion in the donor and increasing blood pressure and volemia in the recipient. Further prospective studies are needed to evaluate the precise role of vasoactive hormones in the cascade of events leading from twin-to-twin transfusion to severe renal dysfunction and extrarenal complications in both fetuses.
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Footnotes |
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Supported by the Institut National de la Santé et de la Recherche Médicale, the Association Claude-Bernard, and the Association pour l’Utilisation du Rein Artificiel.
Accepted for publication October 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin expression in human glomerulogenesis. Kidney Int 1992, 42:390-399[Medline]
-smooth muscle actin and non-muscle myosin. Kidney Int 1996, 55(Suppl):S169-S172
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