Abstract: Basement membrane of glomerular mesangium (BMG) is one of important components which play a key role to support of the capillary loops in a renal glomerulus and completeness of BMG due to interaction of ureteric bud and metanephric mesenchyme during glomerulogenesis. As laminin contribute in extra cellular matrix and especially in basement membrane, the aim of the present study was to demonstrate the distribution of this molecule so, in this investigation specific antibody against laminin have been used in light microscopy to study development of BMG of fetal and postnatal mouse glomerular mesangium. Female inbred Balb/c mice were selected and were kept under normal condition and finding vaginal plug was assumed as day zero of pregnancy. Two pregnant mice were sacrificed by cervical dislocation in one of gestational days 13-18, respectively and their fetuses were fixed, serially sectioned and by using antibody against laminin in BMG were carried out. The same process was used for kidneys preparation at 15 postnatal days. Present data revealed that laminin showed weak reaction on day 14 of gestation. The amount of laminin increased continuously until next days of fetal life and primary of 10 days postnatal in BMG. After this period, laminin reaction did not show significant change in newborns. These data indicate that laminin appears just during the glomerulogenesis and because of continuity with vasculature which is required for Extra Cellular Matrix (ECM) and glomerular endothelial cell differentiation, laminin, is the one of major structural proteins in BMG.
INTRODUCTION
Basement membrane is a specialized component of extra cellular matrix which is consists of different compositions such as glycoproteins (Xue et al., 1998; Berkholtz et al., 2006). In previous investigation, we showed that one of the major basement membrane proteins is type IV collagen (Nikravesh et al., 2009; Jalali et al., 2009). In addition, there are some other several important protein such as laminin, fibronectin, sulfated and nonsulfated glycosaminoglycans (Berkholtz et al., 2006; Mates et al., 2004).
Laminin, an extra cellular element is a large, flexible protein composed of three very long polypeptide chains arranged in the shape an asymmetric cross and held together by disulfide bonds. Previous studies showed that laminin plays important roles during development by special regard in extracellular matrix, in addition other studies revealed that this molecule capable to selectively stimulating axonal extension and nurite outgrowth and in Glomerular and Mesenchymal Basement Membrane During Fetal and Postnatal Period of Balb/c Mice (Jalali, 2005). Based on some other findings the BMG not only changes during embryonic period, but also alters at later stages of life and its alterations considers as an index of tissue changes in pathologic studies (Poschl et al., 2004; Cosgrove et al., 2008) and ECM components turn over continually in developmental organs (Carnegie and Cabac, 1993; Chai et al., 2003). Studies have been shown the expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature (Durbeej et al., 1996). Other investigation also demonstrated that an interaction between basement membrane proteins such as collagen type IV and laminins are required for early kidney morphogenesis in vivo (Willem et al., 2002). Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in ureteric bud branching during nephrogenesis (Zent et al., 2001; Ekblom et al., 1990; Klein et al., 1988).
In other words, molecules and matrix components are required in cell differentiation and among them, laminin play complex roles in cell behavior such as development, migration, proliferation, morphogenesis and metabolism (Durbeej, 2010). One of the most prominent role of ECM is migration and cell adhesion that laminin molecule serves them (Durbeej, 2010; Urbano et al., 2009). Therefore, considering laminin roles in vital organs changes, is necessary to be investigated.
MATERIALS AND METHODS
Twenty four female Balbc/c mice were obtained from animal house of Mashhad University of Medical Sciences in 2009 and selected randomly and finding vaginal plug were assumed as day zero of pregnancy. Two mice per each group were anesthetized by chloroform and were sacrificed by cervical dislocation in one of gestational days 13-18. Their fetuses were collected and were processed for histological studies. The similar processes were carried out for newborns on 1, 5, 10 and 15 of postnatal days. Kidneys were prepared from newborns of 3 mothers for each day. Finally, all samples of fetuses and new borns were placed in paraffin blocks and sectioned serially at a thickness of 7 μm. After deparaffination and rehydration, sections of kidneys were washed twice for 5 min with Tris buffer (containing 1.5% sodium chloride at pH = 7). None-specific antibodies were blocked with 3% Triton X-100 and goat serum for 3 h. For blocking endogenous peroxides activity, sections were treated with 3% H2O2- methanol for 1 h and were incubated with the antibody collagen IV (conjugated with Horse radish peroxidase) at a dilution 1:50 overnight. Then sections again were placed in Tris buffer solution containing 3% Triton and 2% goat serum and were washed three times for 10 min with Tris buffer. After this stage, sections were placed for 15 min in Di-aminobenzidine containing 0.03% H2O2 and after washing samples, were counterstained with hematoxylin. The sections were mounted with glycerol gel. In this method, collagen was showed positive reaction according to amount of appearance and the coloring reaction was from light to dark brown. The coloring reaction of laminin was a proper index for determination of BMG. The images of glomerular regions of kidneys were obtained by a camera microscope and were saved as a file. The intensity of staining laminin was graded by two separate individual according to firth method (Firth and Reade, 1996).
Statistical analysis: The data were analyzed by using SPSS software and Kruskal Wallis and Mann-Withney tests. p-values<0.05 was considered as significant.
RESULTS
The results of present study showed that mesenchyme cells are enclosed uretric bud and glomerular primordium and rudimentary tubules observed on day 13 of gestation (Fig. 1). But it was not observed laminin reaction until this period of time.
| Fig. 1: | Transverse section through kidney parenchyme on day 13 of
gestation, laminin indicated no reaction in glomerular primordium (arrows)
as well as rudimentary tubules (arrowheads) |
| Fig. 2: | |
Glomerular development completed on embryonic day 14 and laminin just showed weak reaction in Parenchyma Vessels (PV), (Fig. 2). Laminin showed first reaction on day 15 of gestation in cortical glomerulus (Fig. 3). The intensity of staining increased continually in next days and detected on day 18 of gestation in other parts of glomerulus and tubules of kidney (Fig. 4). The results of this stage also showed that sever reaction in tufts of capillaries. The observations of postnatal days indicated that laminin reaction was more intensive on 10 days of postnatal in glomerular and tubular basement membrane (Fig. 5, 6) but did not showed significant changes on after this period (Table 1).
| Fig. 3: | Transverse section of glomerulus on day 15 of gestation, The
first reaction was observed in parenchyma of cortical regions (arrows) |
| Fig. 4: | Sections of glomerulus on 18 day of gestation. In this aspect
it have been showed labeling in both cortical glomerulus (arrows) and primary
tubules (arrowheads), urethric bud sections (stars), a macro vessel with
blood cells (bc) and (X 200) |
| Table 1: | Laminin reaction during kidney glomerular morphogenesis |
This gradation was scored ranging from negative (zero) to
4 positive in conformity with the severe of reaction from negative, weak,
moderate, strong and high strong. Values represent Means±SE of the
mean (SEM), compared to embryonic with postnatal days: *p≤0.002, **p≤0.005
and ***p≤0.05 |
|
| Fig. 5: | Transverse sections through glomerulus on 5th postnatal day,
arrows refer to strong reaction in glomerular mesangium, tubules (arrowheads)
and micro vessels (V) |
| Fig. 6: | Glomerulus on 10th postnatal day, that laminin glomerulus
reaction in (arrows), tubules (arrowheads) and parenchyma vessels (V) labeling
indicate more intensive reaction than previous days |
DISCUSSION
The kidney is one of the main parts of urinary system that its mesenchyme forms tubular epithelium and renal glomerular during different stages of development (Abrass et al., 2006; Barasch et al., 1999). The embryologic studies show that rudimentary glomerulus appear on 12.5-13 days of gestation in mouse (Ekblom et al., 1980). In connection with kidney development, there are 2 groups of fibrillar proteins in extra cellular matrix: structural proteins such as collagen and elastin and proteins which play adhesion role such as laminin and fibronectin (Olson et al., 1991). It has been suggested that the vessel endothelial formation in glomerulus is required to basement membrane support that its proteins especially laminin plays a crucial role (Urbano et al., 2009). The present data show that, laminin indicated weak reaction on 14 day of gestation in glomerular endothelial formations. The first evidence of laminin was appeared around the inductor tissue in the undifferentiated mesenchyme. These results also showed that laminin not only increased during final stage of embryonic but also followed on 1-10 of postnatal days. Of course, it does not mean that its density does not change by factors such as long-time activity of kidney and increasing age (Karttunen et al., 1986). These data are apparently confirmed by other investigation that suggest laminin was regularly found distributed as spots around the tips of the ureter at places known to aggregate first (Ekblom et al., 1980). The studies have been shown that in pathological condition, such as diabetes and renal failure may influence on thickness of basement membrane and change of Laminin (Pugliese et al., 1997). This protein effects on glomerular formation and renal filtration at early stages of kidney development (Abrahamson, 2009). It also contributes in an immune response by signal transduction to adjacent tissues and extra cellular matrix that increase thickness of basement membrane (Abrass et al., 1995). Of course, this autoimmune response supports glomerular endothelium against chemical factors, which results in decrease of glomerular filtration and renal failure in acute cases (Yasuda et al., 1996). It have been also believed that compositions of basement membrane play a structural role in epithelial cells, while have been distinguished basement membrane plays complexity roles in cell behavior such as development, proliferation, morphogenesis, metabolism and pathologic changes (Gullberg and Ekblom, 1995). Glomerular extra cellular matrix contributes in different cellular activities such as adhesion, migration that fibrillar proteins such as laminins, serve them (Hamill et al., 2009). Interestingly, it has verified that fibrillar proteins such as laminin play unique and synergistic role with other proteins during development (Peterson and Henry, 2010). Also the Laminins are key components of basement membranes and are thought to be essential for initiation of basement membrane assembly (Anderson et al., 2009). At this rate, laminin is one of the most remarkable component of glomerular basement membrane synthesizes under inductions mechanisms and it reserved in glomerular primordium and increasing of this molecule represents that glomerular development is dependent to laminin that appeared during mesangeal formations. In the other hand, laminn detected on 14 day of gestation in glomerular basement membrane and increased in next days that suggests gelumerular development is dependent to basement membrane formation. After birth, when glomerulus developed laminin density did not show change in newborns. This data indicate that the expression of laminin in the developing kidney is a necessary for shaping of basement membranes and glomerulogenesis.
ACKNOWLEDGMENT
The authors wish to thank Mrs. Motejadded for her excellent technical assistance.