A new mini-invasive technique in treating pediatric diaphyseal forearm fractures by bioabsorbable elastic stable intramedullary nailing: a preliminary technical report.

BACKGROUND AND AIM: Operative treatment is often indicated in unstable pediatric diaphyseal forearm fractures. Recently minimally invasive reduction and elastic stable intramedullary nailing have been of increasing interest, instead of open reduction and internal fixation with plates. There are several disadvantages of metallic intramedullary implants, such as soft-tissue irritation and a risk of disturbing later imaging. Thus, they are generally removed in later operations. We aimed to develop a new technique to stabilize pediatric forearm fractures by the bioabsorbable intramedullary nailing. MATERIAL AND METHODS: We developed a new, two-stage mini-invasive surgical technique to stabilize the unstable diaphyseal fractures in children. The procedure is bioabsorbable elastic stable intramedullary nailing. Ultra-high-strength bioabsorbable intramedullary nails of poly(lactide-co-glycolide) were manufactured for our purpose. The material has been widely proven to be biocompatible and stable enough for fracture treatment as screws and pins. We have used the new technique in the unstable both-bone diaphyseal forearm fractures in children between the ages of 5 and 15 years. We report the technique and our clinical experience in the series of those three cases that have been followed up for at least 12 months. The present series has been randomized for the procedure instead for titanium elastic stable intramedullary nailing, and the series represents a part of ongoing randomized trial. RESULTS: The reported cases operated by the new technique referred good union in the fractured bones and acceptable alignment in the follow-up. Removal of the implants was not required. No troubles with the procedure or implant per se were noticed, indicating good feasibility. One high-energy refracture occurred half year after the primary trauma. Traditional titanium implants were used to control the refracture. CONCLUSIONS: We report our preliminary experience of a new surgical mini-invasive procedure to stabilize the unstable pediatric forearm shaft fractures by bioabsorbable elastic stable intramedullary nailing. Our clinical experience suggests that the procedure combined with long-arm casting is feasible in treating the pediatric forearm fractures. The technique may bring benefits to handling these challenging fractures. The disadvantages of metallic implants may be avoided. In addition, removal of the implant will not be required. There was one refracture in the series, but it was due to new high-energy trauma. According to our understanding, it was not related to the type of former osteosynthesis. However, ignoring the good preliminary experience, still we do not have results of the superiority of the procedure over traditional elastic stable intramedullary nailing. Our ongoing randomized multicenter study is aimed to determine its long-term outcome against the present golden standard. Nevertheless, due to encouraging preliminary results, we see it necessary to report the technique.

Association between sequence variations in genes encoding human zona pellucida glycoproteins and fertilization failure in IVF.

BACKGROUND: The zona pellucida (ZP) has multiple roles in reproductive processes, including oocyte maturation, fertilization and implantation. We used, for the first time, a genetic approach to study whether human ZP genes possess structural alterations in women with unsuccessful IVF trials. In theory, this may result in gradual reduction of sperm-zona interaction and eventually in total fertilization failure (TFF). METHODS: Eighteen infertile women (TFFs) whose IVF did not result in any fertilized oocytes, whereas fertilization by ICSI was successful, were screened for mutations in ZP genes by means of conformation-sensitive gel electrophoresis. Twenty-three fertilizers in IVF (FIVFs) and 68 women with proven fertility (WPFs) constituted the two control groups. RESULTS: Altogether, 20 sequence variations were found in the ZP genes. Two variations in ZP3, one in the regulatory region (c. 1-87 T --> G) and one in exon 6 [c. 894 G --> A (p. K298)] existed more frequently in TFFs than in FIVF and WPF groups (P-values 0.027 and 0.008, respectively). CONCLUSIONS: Our study on ZP genes of infertile women revealed a high degree of sequence variations. This may reflect gradual reduction of fertility among TFFs, but the putative roles and influences of single variations can only be hypothesized.

Lipid peroxidation in human proteinuric disease.

BACKGROUND: While metabolically generated oxidants are produced locally in experimental glomerular diseases, little is still known of their significance and the respective scavenger systems in human glomerular diseases. METHODS: Here we studied kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), a human model disease of isolated proteinuria. Expression of specific mRNAs for a major antioxidant system against lipoperoxidation [phospholipid hydroperoxide glutathione peroxidase (PHGPx)] and for mitochondrial proteins were studied in Northern blotting together with analysis of PHGPx in semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The respective proteins and lipoperoxide (LPO) adducts malonyldialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were analyzed in immunohistochemistry. RESULTS: PHGPx and the mitochondrially encoded subunits of cytochrome-c-oxidase were distinctly down-regulated within the glomeruli of CNF kidneys. These changes were confirmed in semiquantitative RT-PCR. Increases of lipoperoxidation products MDA and 4-HNE were constantly found in the glomeruli of CNF. In agreement with findings in CNF, similar results were obtained in biopsies from other human glomerular diseases. CONCLUSIONS: These findings suggest that local mitochondrial damage initiates LPO, which then causes deposition of the cytotoxic LPO products in glomeruli, as seen especially in CNF kidneys. Together with down-regulation of the local antioxidant protection, these may be important pathophysiologic mechanisms in human glomerular disease.

Altered gene expression and functions of mitochondria in human nephrotic syndrome.

The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.

mRNA differential display analysis of nephrotic kidney glomeruli.

BACKGROUND: Differential display RT-PCR (DDRT-PCR) is a new powerful technique for identification and characterization of altered gene expression in eukaryotic cells and tissues. We studied here changes in kidney glomerular gene expression in patients with congenital nephrotic syndrome of the Finnish type (CNF), an inherited kidney disease with heavy proteinuria already in utero. METHODS: Using the DDRT-PCR approach and isolated glomeruli from removed human kidneys, we compared the gene expression patterns of normal human and CNF glomeruli. Differential expression of candidate genes was verified by Northern blotting, and the corresponding PCR fragments were sequenced and compared to known sequences in databanks. RESULTS: We found several genes and sequence tags with altered expression in nephrotic glomeruli including fragments with close homologies to cytochrome c oxidase subunit I, integrin-linked kinase, insulin-like growth factor II receptor and eotaxin, and also clones resembling anchyrin and cadherin-like consensus sequences. CONCLUSION: All the sequences identified are of interest in respect to pathogenesis of proteinuria. Furthermore, this study reveals potentially new members to known gene families with tissue and cell type-specific expression.

Morphologic changes suggesting abnormal renal differentiation in congenital nephrotic syndrome.

Retrograde differentiation (or dedifferentiation) has recently been proposed as a pathogenetic mechanism involved also in various renal diseases. Here we studied whether evidence of these mechanisms can be found in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF). These patients show isolated massive proteinuria but no primary symptoms from any other organ systems. For the analysis we used antibody markers of early (fibronectin, stem cell factor, Wilms' tumor gene product, cytokeratin) and later (laminin, midgestation and kidney, heparin binding growth-associated molecule) stages of nephron differentiation as well as for apoptosis (acridine orange staining), rescue from apoptosis (anti-Bcl-2 antibodies) and cell proliferation (antibodies to proliferating cell nuclear antigen). In the peritubular spaces atypically organized areas were found which appeared positive with markers of low stages of differentiation, but neither abnormal cell proliferation nor activation of the apoptotic pathway could be detected. As morphologic signs of abnormal tissue organization, we found clusters of tightly compacted and large glomeruli corresponding to the size of two to three normal glomeruli. However, all individual glomerular cell compartments (mesangial, endothelial, visceral epithelial cells) appeared balanced in relative cell numbers. Together these results may indicate abnormal early mesenchymoepithelial tissue interaction leading to excessive and poorly organized formation of glomeruli. This could be causally related also to the serious functional immaturity of CNF kidneys presented as isolated proteinuria.

Expression of nine developmental stage-specific genes in human kidney and cultured renal cells.

BACKGROUND: Several genes transiently expressed during the maturation of the metanephrogenic mesenchyme have been reported in recent years while there is accumulating evidence of a reverted developmental pathway during tissue damage and loss of function. METHODS: Here we studied the expression of nine genes associated with kidney maturation from samples of normal human fetal, juvenile and adult kidneys and cultured glomerular cells using Northern blotting analysis. Subsequently, kidneys from patients with congenital nephrotic syndrome of the Finnish type (CNF), presenting with heavy proteinuria, and Wilms' tumor tissue were studied for the corresponding expression pattern for evidence of dedifferentiation/persistence of a fetal expression pattern. RESULTS: All the genes studied had their distinct expression pattern within the tissues and cells tested. No conclusive evidence of dedifferentiation could be obtained in the samples from CNF kidneys, whereas, as expected, the gene expression pattern of Wilms' tumor tissue was highly similar to that of fetal kidney tissue. CONCLUSION: Some genes thought to be only transiently expressed during kidney maturation were, however, constantly found in the samples from fetal to newborn and adult kidneys.

Mechanisms of proteinuria: vascular permeability factor in congenital nephrotic syndrome of the Finnish type.

Vascular permeability factor (VPF) is the most potent known mediator of vessel wall permeability. In the kidney, it is expressed preferentially in the glomerular visceral epithelial cells. The present study was designed to clarify the proposed role of VPF in diseases with increased glomerular permeability as here exemplified by the congenital nephrotic syndrome of the Finnish type (CNF). For this, we studied the expression levels and the sites of synthesis of VPF and its kinase-insert domain receptor (KDR) in kidneys of patients with CNF using Northern and in situ hybridization techniques and immunohistologic staining with anti-VPF antibody. In addition, we extended the study to include analysis of fetal kidney tissue and cultured glomerular cells of normal and CNF kidneys. In CNF and in normal kidneys VPF was localized in the visceral epithelial aspect of the glomeruli and in the collecting ducts, as also earlier described. A new finding was its localization also in the juxtaglomerular area. The VPF receptor KDR was found in glomeruli in the endothelial cells, but it was not detected in the peritubular capillaries. no consistent differences in the levels of VPF or KDR mRNAs or in their sites of production were seen in CNF and control samples. Also the distribution of VPF antigen in the CNF kidneys and normal kidneys was similar. Thus, we propose that VPF and KDR are not directly involved in the pathogenesis of the proteinuria in CNF.

Sphingolipid activator proteins in a human hereditary renal disease with deposition of disialogangliosides.

Congenital nephrotic syndrome of the Finnish type is a recessively inherited renal disease with glomerular deposits of the disialoganglioside O-acetyl-GD3. Sphingolipid activator proteins (saposins) stimulate the degradation of glycosphingolipids by lysosomal enzymes, and defects in saposins cause accumulation of substrate lipids in the affected tissues in lysosomal storage disease. Here we report a study of the role of saposins in the accumulation of O-acetyl-GD3 in kidneys of congenital nephrotic syndrome patients. At the mRNA level, the expression of saposin precursor in diseased kidneys appeared normal, and the nucleotide sequence analysis of cDNA clones did not reveal abnormalities in the prosaposin gene. Immunohistologically, saposins were localized mainly to the epithelial cells of the distal renal tubules or to the parietal epithelial cells of glomeruli. In the nephrotic syndrome kidneys, the staining pattern was highly granular and appeared mostly in the apical part of the epithelial lining, unlike the control kidneys. These results show that a major site of ganglioside metabolism is located in the distal nephron. Furthermore, these results suggest that saposins are not directly involved in the metabolism of the terminal sialic acids of disialogangliosides in the nephrotic syndrome kidneys.

Decrease of glomerular disialogangliosides in puromycin nephrosis of the rat.

Puromycin aminonucleoside nephrosis (PAN) is a model for human minimal change nephropathy induced in rats by injection of puromycin. In PAN, defective sialylation of a major sialoprotein of podocytes, podocalyxin, has been demonstrated and the consequent decrease of anionic charge suggested as a causative factor for increased glomerular permeability and proteinuria. Whether defective sialylation is a general feature of PAN affecting also glomerular glycosphingolipids is not known. We have shown that rat glomeruli are rich in disialogangliosides GD3 and O-acetyl GD3, the functions of which are not known. Here, we made a sequential analysis of the glomerular gangliosides, especially of GD3 and its O-acetyl derivative in acute PAN using immunohistochemical and biochemical techniques and compared the results with another rat model of glomerular disease, Heymann nephritis. The prominent immunohistochemical finding was the almost total disappearance of glomerular O-acetyl GD3 and a substantial decrease of its precursor GD3 peaking at 10 days after injection of puromycin. Segmental areas lacking these gangliosides remained in glomeruli still at 30 days after injection. The response was dose dependent. Semiquantitative analysis by thin layer chromatograms showed that O-acetyl GD3 was decreased by 41% already at 3 days and by 60% at 10 days after injection of puromycin. Also GD3, the immediate precursor of O-acetyl GD3, was decreased by 20 and 19%, respectively, at 3 and 10 days after injection. At 3 days after injection, overt proteinuria had not started. At these times, no other changes were observed in the glomerular gangliosides. The decrease of glomerular GD3 and O-acetyl GD3 indicates a decrease of GD3 synthase activity and perhaps of O-acetyltransferase activity in PAN nephrosis. As these changes preceded the overt proteinuria, they may have a causal relationship to it. In the glomeruli of Heymann nephritic rats, no similar changes were seen, suggesting that the sialylation defect is not due to proteinuria but is a consequence of targeted puromycin action on cells.

Noncollagenous matrix components of glomeruli in congenital nephrotic syndrome of the Finnish type: evidence of abnormal splitting of nidogen?

The congenital nephrotic syndrome of the Finnish type (CNF) is a rare autosomal recessive disease with proteinuria starting already in utero, prematurity and nephrotic syndrome developing within the first weeks of life. The basic defect of this disease is unknown but has been suggested to be restricted to the kidney glomeruli and especially to the glomerular basement membrane (GBM). The location of the major matrix components in the glomeruli of CNF patient kidneys has previously been reported. Using indirect immunofluorescence microscopy we here describe the more recently characterized components of the glomerular extracellular matrix, including nidogen, tenascin, vitronectin and chondroitin sulfate proteoglycan in CNF and control kidney glomeruli. The accumulation of tenascin and chondroitin sulfate in the renal interstitium as well as a more granular deposition pattern of vitronectin in the mesangium of CNF glomeruli as compared to the control kidneys were observed. These changes were considered secondary to the massive proteinuria, reflected also by the presence of glomerular sclerosis and interstitial fibrosis in the CNF kidney samples. Additionally, analysis of GBM components by immunoblotting revealed either increased or decreased proportionate amounts of fibronectin and laminin in the GBM of CNF kidneys, respectively. Interestingly, different proportionate amounts of proteolytic fragments of nidogen were found in CNF glomeruli as compared to controls. Equal levels of nidogen mRNA were found in the cortical tissue of CNF and control glomeruli. Since nidogen is crucial for the supramolecular organization of basement membranes, these results suggest that an unusual fragmentation of nidogen, due to abnormal assembly, degradation or reorganization of glomerular extracellular matrix, may be associated with the basic defect of CNF.

Glomerular antigens in severe hereditary nephrosis.

In search of the basic defect and cell type responsible for the massive treatment-resistant proteinuria of congenital nephrotic syndrome of the Finnish type (CNF), we examined tissue samples of CNF kidneys using established antibody and lectin markers of various glomerular cell types. Markers of vascular endothelium (antibodies to factor VIII and a human homologue of podocalyxin (anti-PHM5) and UEA I lectin) showed no qualitative changes in the endothelial cells of glomeruli or peritubular areas in CNF as compared with controls. Markers of glomerular mesangial cells (antibodies to desmin, smooth muscle actin, RCA I lectin) revealed a secondary increase in mesangial reactivity reflecting the sclerosis and expansion of the mesangial areas in CNF. Markers of visceral epithelial cells (antibodies to a human homologue of podocalyxin, C3b receptor, vimentin, common lymphocytic leukemia antigen, gp44, and the WGA, LFA and, after neuraminidase treatment, PNA lectin) failed to show appreciable qualitative changes in CNF kidney samples. Interestingly, the alpha 2 beta 1 integrins appeared greatly reduced in all CNF samples studied, possibly explaining the mechanisms of CNF-associated proteinuria.

Establishment and characterization of a rat glomerular endothelial cell line.

BACKGROUND: Primary cell cultures have been widely used for research purposes, but kidney glomerular and vascular endothelial cells and, particularly, stable endothelial cell lines from these have been difficult to obtain. EXPERIMENTAL DESIGN: We used electroporation and lipofectin transfection to introduce various transforming constructs, and direct infection with an oncogenic adeno 31 (Ad31) virus to immortalize cells from freshly isolated rat kidney glomeruli and characterized the cells obtained at their 20th, 40th, 60th, and 80th passage for markers of various glomerular cell types. RESULTS: Direct infection with an oncogenic adenovirus type 31 (Ad31) resulted in a stable cell line (over 120 passages) morphologically resembling epithelial/endothelial cells line (over 120 passages) morphologically resembling epithelial/endothelial cells. These cells were constantly positive for factor VIII-related antigen, podocalyxin and for OX-43 anti-rat endothelial cell antibodies, Bandeiraea simplicifolia (BSI-B4) lectin, and expressed receptors for acetylated low density lipoprotein. Anti-Thy 1.1 and anti-desmin antibodies recognizing glomerular mesangial cells and antibodies against O-acetyl GD3 and gp330 antigens of podocytes, as well as anti-thrombospondin and anti-cytokeratin antibodies identifying parietal epithelial cells failed to bind to our cells. These rat glomerular endothelial cells (RGE cells) showed decreased serum requirements for growth, changes in morphology, loss of contact inhibition, and loose adherence to growth support. The transforming E1A gene could not be found by Southern blotting or polymerase chain reaction amplification analysis. Neither did the RGE cells produce any adenoviral proteins tested by immunoprecipitation analysis. CONCLUSIONS: The RGE cells represent a stable cell line with various characteristics of endothelial cells. Furthermore, they remain responsive to basic FGF and grow on type IV collagen and fibronectin matrices forming capillary-like structures. Thus, we consider RGE cells a cell line of rat glomerular endothelial origin.