Expression of collagen type IV in human kidney during prenatal development

Background / Aim. Type IV collagen belongs to the group of non-fibrillar
 collagens and is an important component of the basement membranes where it
 accounts for approximately 50% of its structural elements. The aim of the
 paper was to describe the expression and distribution of collagen type IV in
 embryonic and fetal metanephric kidney, and to determine the volume density
 of collagen type IV in kidney tissue in each trimester of development.
 Methods. The material consisted of 19 human embryos/fetuses, in the
 gestational age from 8th to 37th week. Kidney tissue specimens were
 routinely processed to paraffin molds and stained with hematoxylin and eosin
 and immunohistochemically using polyclonal anti-collagen IV antibody.
 Stained slides were examined using light microscope and images of the
 selected areas, under different lens magnification were captured with
 digital camera. Volume density of collagen type IV was determined by using
 ImageJ 1.48v and a plugin of the software which inserted a grid system with
 336 points. For the data comparison One-Way Analysis of Variance was used.
 Results. Strong collagen IV immunopositivity was seen in all specimens, with
 a distribution in the basement membranes of urinary bud, parietal leaf of
 Bowman?s capsule, glomerular basement membrane, basement membrane of
 interstitial blood vessels, and basement membranes of nephron tubules and
 collecting ducts. No statistically significant difference in the volume
 density of type IV collagen was found between the different trimesters of
 development. Conclusion. The synthesis and secretion of collagen type IV
 simultaneously follows the development of nephron structures, collecting
 system and blood vessels. The volume density of collagen type IV remains
 constant throughout all the trimesters of metanephric kidney development,
 indicating that it plays a crucial role in normal development of nephron and
 collecting system structures, as well as in maintaining the normal kidney
 function.


Introduction
The human kidney is a complex organ consisting of functional unitsnephrons, that are connected to a highly branched collecting duct system. Nephrogenesis, the process of kidney formation, ends around the 36 th week of development and comprises a plethora of intertwined processes such as epithelial-mesenchymal interactions, epithelial branching, cell migration, differentiation and cell division, as well as cell-extracellular matrix interactions 1,2,3 . The unique feature of kidney development is the mesenchymal-epithelial transition that occurs during the formation of the nephron, and the differentiation of highly specialized structures, such as the glomerulus 1,3 .
The definitive mammalian kidney development is a complex process that occurs trough the formation of three excretory structures from intermediate mesoderm: pronephros, mesonephros and metanephros, of which the first two are temporary and involute, while the metanephros will give rise to the definitive kidney 4 . The pronephros and mesonephros formation are necessary for the development of metanephric kidney, and the interruption in development of these two precursor excretory structures will lead to renal agenesis 5 . The metanephric kidney occurs during the 5 th week of development and is result of interaction of nephric duct and metanephric mesenchyme, both of which originate from the intermediate mesoderm 6 . The intermediate mesoderm is a narrow strip of mesoderm located between the somite and lateral plate mesoderm 7 . Its ventral part will give rise to nephric duct, while the posterior part of intermediary mesoderm, that is referred to as the nephrogenic cord, will become condensed near the hindlimb buds thus giving rise to metanephric mesenchyme 8 . Nephric duct is a tubular structure, covered with simple cuboidal epithelium, that is directed toward cloaca of the embryo with whom it connects. It is shown that glial cell-line-derived neurotrophic factor (GDNF), a protein secreted by the metanephric mesenchyme cells, binds to the Ret receptors on the epithelial cells of the distal part of the nephric duct and initiates the formation of the ureteric bud 9 . The ureteric bud plays a crucial role in the formation of metanephros and its branching in the metanephric mesenchyme will give rise to collecting system of the kidney 10 . The metanephric mesenchyme contains multipotent self-renewing Six2+ progenitors that will give rise to the main body of the nephron, as well as self-renewing Foxd-1 progenitor cells that will give rise to the stroma of the interstitium, mesangium and pericytes in kidney 11 .
Type IV collagen belongs to the group of non-fibrillar collagens and is an important component of the basement membranes (BM) where it accounts for approximately 50% of its structural elements 12,13 . In BM, collagen type IV forms a polygonal network that, along with other molecular components of BM, has a supporting and barrier function 12 . Also, it has a role in supporting tissue integrity, cell survival, cell signaling, morphogenesis, and tissue regeneration 14 . The type IV collagen molecule is a heterotrimer (protomer) consisting of three alpha chains that have a similar primary structure 12,15 . To date, six genes encoding alpha chains, denoted COL4A1-COL4A6 were identified 16 . Alpha chains are interconnected into a three-helix structure of type IV collagen molecules. Three molecular isoforms of type IV collagen are described: α1(2)α2, α3α4α5 and α5(2)α6 17 . The 6 α1(2)α2 collagen IV isoform is found in all basement membranes, the α3 (IV) α4 (IV) α5 (IV) isoform is found in the kidney, lung, and at the neuromuscular junction, while the α5 (IV) 2 α6 (IV) isoform is present in smooth muscle and at the neuromuscular junction 17,18 .
The aim of the paper was to describe the expression and distribution of collagen type IV in embryonic and fetal metanephric kidney, and to determine the volume density of collagen type IV in kidney tissue in each trimester of development.

Methods
Material. The material consisted of 19 human embryos/fetuses, in the gestational age from 8 th to 37 th week, that were obtained following all legal and ethical guidelines. The material was obtained after spontaneous or artificial miscarriages and premature births due to prepartal deaths. There was not macroscopic damage, or any pathological/ autolytic changes of the specimens, and both sexes were represented in the sample. Gestation week was determined using medical history, as well as by measuring the crown-rump length. The study was performed at the Department of Histology and embryology, Faculty of Medicine, University of Nis. All examined samples were allocated into three groups, based on trimester of development (Table 1). Morphometric and statistical analysis. Volume density is relative variable, which shows how much overall space is occupied by the observed space in volume units (19). Volume density (Vv) of collagen type IV was determined by using ImageJ v. of interstitial blood vessels, tubules and collecting ducts (Fig.1a). and tubules of the nephron, collecting ducts, and blood vessels (Fig 1b, 1c. 1d).
The volume densities of type IV collagen in the kidney, presented by trimesters of development, are shown in Table 2. No statistically significant difference in the volume density of type IV collagen was found between the different trimesters of development.
Volume densities of type IV collagen in the elements of the renal cortex and medulla in the third trimester of development are shown in Fig. 2 and 3.  Although there is a number of papers dealing with the temporal and spatial expression of different isoforms of collagen type IV in developing kidney, there are virtually no data concerning the quantification of collagen type IV in prenatal human kidney. Jalali et al.
were using a semiquantitative approach to determine the amount of collagen type IV in murine model of kidney development. Their results indicate that the first traces of collagen type IV were observed during the E13, and that its amount was gradually increasing until the E18, to finally reach its maximum around day 5 postnatally 36 . The earliest specimen used in our research was at the 8 th week of gestation and strong collagen IV positivity was already seen around all the tubular structures in developing kidney. The quantified volume density of collagen type IV showed no significant statistical difference compared to the later stages of development. Also, the volume density is a relative variable and does not reflect the absolute amount of collagen type IV in developing kidney, but rather its volume presence expressed in percentages within the organ and compared to all the other structural kidney components.
The genetic disorders of collagen IV synthesis especially affect kidney, due to the dependence of its functions on the stability and normal morphology of the basement membranes. Glomerular filtration and tubular filtration are highly specialized kidney features, and the failure of kidney to perform these functions may lead to the end-stage renal disease with life threatening consequences. The two major syndromes occurring as a result of mutation of genes for collagen type IV are Alport's and Goodpasture's syndromes 37 . Alport's syndrome occurs as a result of mutations in any of the three genes encoding components of the collagen α3α4α5(IV) network (COL4A3, COL4A4, and COL4A5).
Most mutations prevent assembly and/or secretion of collagen α3α4α5(IV) heterotrimers such that all 3 proteins are absent from the GBM 38 . Clinically, it manifests with persistent hematuria, sensorineural hearing loss and ocular abnormalities 39 . Goodpasture syndrome is an autoimmune disease caused by the development of auto-antibodies against the glomerular basement membrane that leads to the kidney failure 40 .

Conclusion
The collagen type IV is an important part of basement membranes in kidney, whose synthesis and secretion simultaneously follows the development of nephron structures, collecting system and blood vessels. The volume density of collagen type IV remains constant throughout all the trimesters of metanephric kidney development, indicating that it plays a crucial role in normal development of nephron and collecting system structures, as well as in maintaining the normal kidney function.