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The matrix metalloproteinase MMP9 influences cellular morphology and function, and plays important roles in organogenesis and disease. It exerts both protective and deleterious effects in renal pathology, depending upon its specific substrates. To explore new functions for MMP9 in kidney cysts formation and disease progression, we generated a mouse model by breeding juvenile cystic kidney (jck) mice with MMP9 deficient mice. Specifically, we provide evidence that MMP9 is overexpressed in cystic tissue where its enzymatic activity is increased 7-fold. MMP9 deficiency in cystic kidney worsen cystic kidney diseases by decreasing renal function, favoring cyst expansion and fibrosis. In addition, we find that periostin is a new critical substrate for MMP9 and in its absence periostin accumulates in cystic lining cells. As periostin promotes renal cyst growth and interstitial fibrosis in polycystic kidney diseases, we propose that the control of periostin by MMP9 and its associated intracellular signaling pathways including integrins, integrin-linked kinase and focal adhesion kinase confers to MMP9 a protective effect on the severity of the disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Djaziri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

The ankyrin repeat and sterile α motif (SAM) domain-containing six gene (Anks6) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by deposition of IgA in the glomerular mesangium. The diagnosis of IgAN still requires a kidney biopsy that cannot easily be repeated in the same patient during follow-up. Therefore, identification of noninvasive urinary biomarkers would be very useful for monitoring patients with IgAN. We first used bidimensional electrophoresis (2DE) coupled to MALDI-TOF-TOF and Western blot to identify some urinary biomarkers associated with IgAN. Urine of IgAN patients showed an increase of albumin fragments, α-1-antitrypsin and α-1-β-glycoprotein, along with a decrease of a single spot that was identified as the laminin G-like 3 (LG3) fragment of endorepellin. The urinary proteomes of 43 IgAN patients were compared to those of 30 healthy individuals by ELISA. Quantification of LG3 confirmed a significant decrease in the urine of IgAN patients compared to healthy controls, except in ten patients in whom LG3 was increased. These ten patients had a more severe disease with lower glomerular filtration rate values. We found a significant inverse correlation between LG3 levels and glomerular filtration rate in the 43 patients with IgAN, which was not observed in 65 patients with other glomerular diseases including membranous nephropathy (23), lupus nephropathy (13), focal segmental glomerulosclerosis (15), diabetic nephropathy (14), and six patients with nonglomerular diseases. Therefore, we suggest that the LG3 fragment of endorepellin could be associated with IgAN severity and might be related to pathogenesis of IgAN., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Sustained neuroinflammation strongly contributes to the pathogenesis of pain. The clinical challenge of chronic pain relief led to the identification of molecules such as cytokines, chemokines and more recently matrix metalloproteinases (MMPs) as putative therapeutic targets. Evidence points to a founder member of the matricial CCN family, NOV/CCN3, as a modulator of these inflammatory mediators. We thus investigated the possible involvement of NOV in a preclinical model of persistent inflammatory pain., Methods: We used the complete Freund's adjuvant (CFA)-induced model of persistent inflammatory pain and cultured primary sensory neurons for in vitro experiments. The mRNA expression of NOV and pro-inflammatory factors were measured with real-time quantitative PCR, CCL2 protein expression was assessed using ELISA, MMP-2 and -9 activities using zymography. The effect of drugs on tactile allodynia was evaluated by the von Frey test., Results: NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1β- and TNF-α-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-α-induced MMP-9 expression through β1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia., Conclusions: This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.

Early events in kidney organogenesis involve reciprocal interactions between the ureteric bud and the metanephric mesenchyme, which lead to remodeling of the extracellular matrix. This remodeling involves matrix metalloproteases (MMPs), but the specific roles of individual MMPs in kidney development are not completely understood. Here, we analyzed MMP9-deficient mice at the first step of kidney development and found that MMP9 deficiency delayed embryonic kidney maturation and increased apoptosis ex vivo by 2.5-fold. These early defects resulted in a 30% decrease in nephron number, a 20% decrease in adult kidney weight, and altered kidney function and morphology at 12 mo. The membrane form of stem cell factor (SCF) increased, whereas the activated form of the SCF receptor, c-kit, decreased in MMP9-deficient embryonic kidneys. In organotypic culture, MMP9-deficient kidneys failed to secrete SCF, and addition of recombinant SCF partially rescued both apoptosis and the branching defect. In conclusion, these data show that MMP9 protects mesenchymal cells from apoptosis during kidney development and stimulates ureteric bud branching morphogenesis, most likely by releasing the soluble form of SCF, suggesting that normal renal development requires MMP9.

Nephronophthisis belongs to a family of recessive cystic kidney diseases and may arise from mutations in multiple genes. In this report we have used a spontaneous mouse mutant of type 3 nephronophthisis to examine whether the doxycycline-inducible synthesis of Timp-2, a natural inhibitor of matrix metalloproteinases, can influence renal cyst growth in transgenic mice. Metalloproteinases may exert either a negative or a positive effect on the progression of cystic kidney disease, and we reasoned that this may be most effectively examined by using a natural inhibitor. Surprisingly, already the application of doxycycline, which also inhibits matrix metalloproteinases, accelerated renal cyst growth and led to increased renal fibrosis, an additional effect of Timp-2 was not detected. The positive effect of doxycycline on kidney size was not due to a non-specific "anabolic effect" but was specific for cystic kidneys because it was not observed in non-cystic kidneys. When looking for potential metabolic changes we noticed that the urine of control animals led to an increase in the calcium response of LLC-PK(1) cells, whereas the urine of doxycycline-treated mice showed the opposite effect and even antagonized the urine of control animals. Further experiments demonstrated that the urine of control animals contained a heat-labile, proteinase K-resistant substance which appears to be responsible for the induction of a calcium response in LLC-PK(1) cells. We conclude that doxycycline accelerates cyst growth possibly by the induction of a substance which lowers the intracellular calcium concentration. Our data also add a note of caution when interpreting phenotypes of animal models based upon the tet system.

Apoptosis of tubular epithelial cells is a hallmark of acute kidney injury (AKI), but the cellular events preceding apoptosis in this setting are incompletely understood. Because matrix metalloproteinase 9 (MMP9) degrades matrix components involved in cell survival, we studied the role of MMP9 in AKI. In the mouse model of folic acid-induced AKI, we observed a marked increase of MMP9 activity in the S3 segment of the proximal tubule (S3PT), correlating with the apoptotic phase. MMP9 deficiency increased apoptosis and the severity of renal lesions and substantially delayed recovery of renal function. MMP9-/- mice exhibited significant apoptosis in the S3PT and the intercalated cells of the collecting duct (I-CD), whereas wild-type mice exhibited none in these segments. Stem cell factor (SCF), an MMP9 substrate, was identified in the S3PT, and its receptor, c-Kit, was expressed in both the S3PT and I-CD. MMP9 released the soluble form of SCF (sSCF) from kidney cells in vivo and in vitro. In addition, SCF inhibited apoptosis of tubular cells in vitro, rescued MMP9-/- S3PT and I-CD from apoptosis in vivo, and improved renal function. An ischemia-reperfusion model of AKI produced similar results. In patients with AKI, urinary sSCF increased with acute tubular necrosis but not with prerenal azotemia. In conclusion, these data show that MMP9 protects the S3 segment of the proximal tubule and the I-CD from apoptosis in AKI, most likely by releasing sSCF.

Remodeling of extracellular matrix (ECM) is an important physiological feature of normal growth and development. Recent studies have emphasized the role of matrix metalloproteinases (MMP-2 and MMP-9) in normal mouse nephrogenesis. We have demonstrated previously in the rat that in utero exposure to maternal diabetes impairs renal development leading to a 30% reduction in the nephron number. Transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) are known to mediate high glucose effects on matrix degradation. The aim of the present study was to address the expression of type IV collagenase and TGF-beta1/CTGF systems in rat kidney during normal development and after in utero exposure to maternal diabetes. Both MMP-2 and MMP-9 mRNA metanephric expressions and activities were dramatically downregulated in kidneys issued from diabetic fetuses and in metanephros cultured in the presence of high glucose concentration. TGF-beta1 and CTGF expressions were significantly enhanced in diabetic fetal kidneys and in high glucose cultured metanephroi. Conditioned media obtained from metanephroi grown with high glucose concentration upregulated functional TGF-beta activity in transfected ATDC5 cells. In conclusion, in impaired nephrogenesis resulting from in utero exposure to maternal diabetes, alteration of both type IV collagenase and TGF-beta1/CTGF systems may lead to abnormal remodeling of ECM, which may, in turn, induce defects in ureteral bud branching leading to the observed reduction in the nephron number with consequences later in life: progression of chronic renal disease and hypertension.

Matrix metalloproteinases (MMPs) have pleiotropic enzymatic actions that go far beyond degradation of extracellular matrix. Both the multiplicity of their targets and the complexity of their regulation account for a variety of biological effects. In renal diseases, MMP effects may be different and/or opposite during the different phases of the pathology evolution. The major challenge with future therapeutic interventions using MMP inhibitors remains how to accomplish temporal and spatial control of their activity without flipping the coin.

We have analyzed the expression and regulation of plasminogen activators (PA) in principal cells of the renal collecting duct. We used a rabbit principal cell line (RC.SVtsA58) infected with the temperature-sensitive SV40 strain tsA58. Transformed cells cultured at permissive temperature (33 degrees C) produced only tissue-type plasminogen activator (t-PA). Shifting the cells to nonpermissive temperature (39.5 degrees C) induced their differentiation and a marked increase in total fibrinolytic activity due to the induction of urokinase-type plasminogen activator (u-PA) synthesis and secretion. The effect on u-PA was post-transcriptional and it could be attributed to large-T inactivation at 39.5 degrees C since it was abolished by re-infecting the cells with wild-type SV40. Run-on assay and real-time RT-PCR of u-PA transcripts indicated that large-T altered post-transcriptional regulation. u-PA was also produced by primary cultures of collecting duct cells and was present in the rabbit urine. In the kidney, u-PA and its receptor (u-PAR) were almost exclusively expressed at the apex of collecting duct cells. We then analyzed the regulation of u-PA by arginine vasopressin (AVP) and epidermal growth factor (EGF), two key regulators of principal cell functions. We found that AVP and EGF, which have opposite hydro-osmotic effects in the collecting duct, also exhibited contrasted effects on u-PA synthesis in differentiated RC.SVtsA58 cells. EGF increased but AVP suppressed u-PA activity and protein, and these regulations occurred at post-transcriptional level. These results point to a physiological role of u-PA in principal cells of the renal collecting duct., (Copyright (c) 2005 Wiley-Liss, Inc.)

Type IV collagenases matrix metalloproteinase-2 (MMP2) and MMP9 and their related proteins, MT1-MMP, tissue inhibitor of metalloproteinases 1 (TIMP1), TIMP2, and TIMP3, are expressed during kidney morphogenesis and nephrogenesis, but the renal ontogeny of these proteins is only partially known, and their persistence in the adult remains controversial. Their expression was analyzed from early metanephric stages to adulthood by Western blot semiquantitative analysis; laser confocal microscopy of whole-mount kidneys; and a two-step immunoperoxidase labeling procedure using specific markers of proximal tubule (megalin), ascending limb of Henle's loop (Tamm Horsfall protein), and collecting duct (Dolichos biflorus agglutinin lectin). By Western blot, all antigens were detected at day 11.5, peaked at day 16.5, and persisted in the adult at lower levels, although MMP2 was less modulated. All antigens were expressed in metanephric mesenchyme at embryonic day 11.5 and became concentrated in neural cell adhesion molecule-positive-induced mesenchymal cells at day 12.5. Only MT1-MMP and to a lesser extent MMP2 were detected in the ureter bud. At day 16.5, all antigens predominated in the cytoplasm of the proximal tubule, except TIMP1, which was mostly expressed in the ascending limb of Henle's loop and distal tubule. During tubule segmentation, components of the type IV collagenase system showed both spatial and temporal regulation. The distribution of gelatinases was not strictly superimposable to that of their natural inhibitors TIMP, especially for MMP9 and TIMP1. All components persisted in specific segments of the adult renal tubule, where MMP9, MMP2, and MT1-MMP showed an apical expression, suggesting that substrates for these enzymes should be in the tubule lumen or in the apical cell domain and not in the extracellular matrix. These results suggest that a regulated balance of gelatinase activity is required during kidney organogenesis and that gelatinases continue to play a role in adult renal tubule physiology.

Matrix metalloproteinase (MMP)9/gelatinase B is increased in various nephropathies. To investigate its role, we used a genetic approach. Adult MMP9-deficient (MMP9(-/)-) mice showed normal renal histology and function at 3 mo. We investigated the susceptibility of 3-mo-old mice to the accelerated model of anti-glomerular basement membrane nephritis, in which fibrin is an important mediator of glomerular injury and renal impairment. Unexpectedly, nephritis was more severe in MMP9(-/)- than in control mice, as attested by levels of serum creatinine and albuminuria, and the extent of crescents and fibrin deposits. Circulating or deposited immunoglobulin G, interleukin (IL)-1beta, or IL-10 were the same in MMP9(-/-) and MMP9(+/+) mice. However, we found that fibrin is a critical substrate for MMP9, and in its absence fibrin accumulated in the glomeruli. These data indicate that MMP9 is required for a novel protective effect on the development of fibrin-induced glomerular lesions.

To investigate the role of angiotensin II (ANG II) in nephrogenesis, a developmental study of renal AT1 and AT2 receptor mRNA expression was performed in parallel with the quantitative and qualitative analysis of metanephros development in fetal lamb from 60 to 140 days of gestation. Both ANG II receptor subtypes were expressed early during nephrogenesis but displayed specific spatial and temporal distribution during gestation. High-AT2 mRNA expression took place in the outermost nephrogenic area and in the undifferentiated mesenchymal cells surrounding the ampulla; level of AT2 expression in this localization followed closely glomeruli proliferation rate and disappeared after nephrogenesis completion (>120 days). AT2 mRNA was also detected in the differentiated epithelial cells of macula densa of maturing glomeruli. Although most of AT1 mRNA labeling was found in the mesangial cells of maturing glomeruli, where it persisted after nephrogenesis completion, additional labeling was found in undifferentiated cells, in cells invading the inferior cleft of S-shaped bodies (80 days), and in medullar cells between tubules (120 days). Our results suggest that each receptor subtype has a specific role in renal morphogenesis, i.e., AT2 in mesenchymal proliferation or apoptosis and AT1 in vascular smooth muscle cells differentiation.

The first step of renal development is characterized by the invasion of a mesenchymal tissue, the metanephric blastema, by an epithelial structure, the ureter bud. Reciprocal inductive interactions between these two tissues, involving the sequential activation of genes that begin to be identified, are required for the successive stages of kidney morphogenesis. Recent technological advances such as gene targeting and improvement of organotypic culture have revealed the role of these genes and of several essential factors in kidney development. This review will focus on the genes which govern renal organogenesis, with special emphasis on those coding transcription and diffusible factors.

We analyzed matrix metalloproteinase (MMP) production by 11-d embryonic mouse kidneys and the effects of these enzymes on subsequent renal organogenesis. In vivo, immunolocalization of metalloproteinases by laser scanning confocal microscopy and zymograms of kidney lysates showed that the mesenchyme of embryonic kidneys synthesized both MMP9 and MMP2 enzymes. In vitro, embryonic kidneys also secreted both enzymes when cultured in a medium devoid of hormone, growth factor, and serum for 24 h during which T-shaped branching of the ureter bud appeared. We then evaluated the role of MMP2 and MMP9 in kidney morphogenesis by adding anti-MMP2 or anti-MMP9 IgGs to the culture medium of 11-d kidneys for 24 or 72 h. Although it inhibited activity of the mouse enzyme, anti-MMP2 IgGs had no effect on kidney morphogenesis. In contrast, anti-MMP9 IgGs with enzyme-blocking activity impaired renal morphogenesis, in a concentration-dependent manner, by inhibiting T-shaped branching and further divisions of the ureter bud. This effect was irreversible, still observed after inductive events and reproduced by exogenous tissue inhibitor of metalloproteinase 1 (TIMP1), the natural inhibitor of MMP9. These data provide the first demonstration of MMP9 and MMP2 production in vivo by 11-d embryonic kidneys and further show that MMP9 is required in vitro for branching morphogenesis of the ureter bud.

Background: Several halogenated anesthetics induce a urinary concentrating defect, partly related to fluoride ion toxicity in collecting duct cells. The aim of this study was to investigate the effects of fluoride ion in human kidney cells., Methods: Immortalized human collecting duct cells were used. In a first set of experiments, the toxicity threshold concentration was determined by exposing cell cultures for 24 h to increasing concentrations of fluoride ion in the medium: 0, 1, 5, and 10 mM. The second set of experiments was a time- effect study in which cells were exposed to 5 mM fluoride for 2, 6, and 24 h. Assessment of toxicity was based on several endpoints: cell number, protein content, (3)H-leucine incorporation in newly synthesized proteins, extracellularly released lactate dehydrogenase, Na-K-ATPase pump activity, and electron microscope studies., Results: After 24 h of exposure, fluoride ion decreased cell number (-23%, P<0.05), total protein content (-30%, P<0.05) and increased lactate dehydrogenase release (+236%, P<0.05) at a threshold concentration of 5mM. Fluoride ion also inhibited Na-K- ATPase activity at 5 mM (-58%, P<0.05). Major morphologic alterations of mitochondria, including crystal formation, were detected from 1 mM fluoride concentration. Time-effect studies showed that, after only 6 h of exposure at 5 mM, fluoride decreased cell number (-13%, P<0.05), (3)H-leucine incorporation (-48%, P<0.05), and Na-K-ATPase activity (- 20%, P<0.05) and increased lactate dehydrogenase release (+145%, P<0.05). Crystal deposits in mitochondria again were a more sensitive marker of cell injury, detectable after only 2 h of exposure., Conclusions: these results suggest that the mitochondrion is a target of fluoride toxicity in human collecting duct cells, and its alteration is partly responsible for the sodium and water disturbances observed in patients.

Kidney tubule dysfunction and lesions are frequent complications of myeloma, related to unknown properties of the monoclonal light chain. We have analyzed protease sensitivity and binding properties of urinary light chains from four patients with Fanconi's syndrome, 12 with cast nephropathy, and four control patients without myeloma-associated tubulopathy. All light chains were normal-sized, monomeric and/or dimeric, and none was N-glycosylated. Kinetic studies of light chain digestion by pepsin and the lysosomal enzyme cathepsin B showed the generation of a protease-resistant 12 kDa fragment, corresponding to the V domain of the kappa chain in the four Fanconi's syndrome patients; in two out of four the V domain was also completely resistant to trypsin. Western and dot blots revealed similar patterns of reactivity of light chains from patients with the Fanconi's syndrome towards other light chains. Properties of cast-nephropathy light chains were more heterogeneous but clearly differed from those of Fanconi's syndrome: (i) 9 out of 12 were of the lambda-type; (ii) only four yielded a transient 12 kDa fragment after cathepsin B digestion, but all showed some resistance to proteolysis of the entire molecule or a fragment thereof to at least one protease, at variance with control light chains; (iii) they displayed various patterns of reactivity with other light chains; (iv) 7 out of 12 reacted specifically with Tamm-Horsfall protein (THP) by ELISA, in contrast with those of Fanconi's syndrome. In one patient who presented with cast nephropathy and the Fanconi's syndrome, the light chain exhibited both partial resistance of the V kappa domain to cathepsin B and the highest reactivity with THP.(ABSTRACT TRUNCATED AT 250 WORDS)

The apical domain of epithelial cells lining the proximal tubule and the yolk sac is characterized by the development of extensive microvilli which limit intermicrovillar spaces backed on their cytoplasmic aspect by a coat of clathrin. These membrane areas which give rise to endocytic vesicles are characterized by the expression on their outer aspect of two high molecular weight glycoproteins: gp330 and gp280. In this study we report on an epithelial cell line, BN/MSV, derived from a yolk sac carcinoma which expresses these two glycoproteins. By indirect immunofluorescence, gp330 and gp280 were detectable on the cell surface and after permeabilization in intracytoplasmic vesicles. At the ultrastructural level they were concentrated in clathrin-coated membrane areas and although gp280 could also be detected in non-coated areas. The two proteins were synthesized independently in the form of high molecular weight polymers by biosynthetically labeled BN/MSV cells. Both were released in the supernatant, but, in spite of previously reported similarities by peptide mapping, only gp330 coprecipitated with a 45 kDa protein comigrating with the alpha 2-macroglobulin receptor-associated protein (MRAP). Culture of the cells in the presence of antibodies to gp280 and to a lesser extent of antibodies to gp330 inhibited the internalization of [14C]sucrose and peroxidase. When followed intracellularly at the ultrastructural level, the compartments containing peroxidase in the presence of anti-gp280 or gp330 antibodies were morphologically distinct from those observed under control conditions: vesicles were of smaller size and irregular shape and accumulation in lysosomes was delayed.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephritogenic potential of antibodies directed against one of the glomerular basement membrane (GBM) components, i.e., heparan sulfate-proteoglycan (HS-PG), was investigated in different strain of rats, i.e., Brown Norway, Lewis, Long Evans, and Sprague-Dawley. The rats were given two intravenous injections of anti-HS-PG antibody on days 1 and 3, and killed 2 to 8 weeks later. Before killing, blood and urine were collected for determination of anti-rabbit IgG levels and excretion of proteins, respectively. In addition, the right kidney was perfused with 125I-anti-rat IgG to quantitate the amount of immune-complexes present within the GBM. The tissues were processed for morphologic, autoradiographic, and immunofluorescent studies. The anti-HS-PG antibody was seen uniformly bound to GBM equally in all strains of rats. However, the protein-uric response was as follows: Brown Norway much much greater than Lewis much greater than Long Evans greater than Sprague Dawley. Also, the glomerular cells, monocytes in the glomerular capillaries, immunoreactivity of rat IgG and C3 frequency of subepithelial immune deposits, serum levels of anti-rabbit IgG, and the amount of 125I-anti-rat IgG bound to the GBM were proportionately increased among different strains of rats. The data suggest that the sustained presence of anti-HS-PG antibodies in the subepithelial aspect of the GBM with differential humoral response in the production of the antibody by the host most likely attributed to the variable glomerular damage in different strains of rats. Thus, it seems that the genetic makeup of a given strain of rat heavily influences the nephritogenic potential of an antibody and consequentially the outcome of the immune complex-mediated glomerular injury.

Biology of glomerular visceral epithelial cells ("podocytes") and their role in inflammatory process remain obscure, partly because of the lack of well-differentiated podocyte cultures. We have established a human cell line by transfecting with a replication-defective SV40 plasmid (pSVHB1), a primary culture of podocytes derived from an enriched preparation of unencapsulated glomeruli free of tubule and Bowman's capsule contaminants. Podocyte specificity of the primary culture was assessed by a dual immunomorphological and functional approach. The resulting cell line (HGVEC.SV1) was cloned and the clonal cells were adapted to hormonally defined medium supplemented with only 2% newborn bovine serum. Clone A4 has been exhibiting over 35 passages, a combination of markers unique to podocytes, including expression of vimentin, podocalyxin, ectoenzymes (CALLA antigen and mRNA), heparan-sulfate proteoglycans (molecular mass of core protein = 75 kDa), and production of type IV collagen (alpha 1 and alpha 5 chains) established by immunoprecipitation and Northern blot analysis. Cytokeratin was detected in rare cellular foci and the search of Von Willebrand factor was negative. This clonal cell line has been used to demonstrate: (1) that human podocytes are highly sensitive to atrial natriuretic peptide (ANP) which induced a dose-dependent increase in cGMP production (x20 at 0.5 microM ANP), and (2) that secretion of ANP-stimulated cGMP is dramatically polarized as 93% of extracellular cGMP were released in the apical medium when filter-grown HGVEC. SV1A4 cells were stimulated at their basal pole.

The role of proteoglycans (PGs) in morphogenesis was investigated. Fetal kidneys were obtained from 13-day-old mouse embryos and maintained for 7 days in culture. The biosynthesis of PGs was perturbed by addition of p-nitrophenyl-beta-D-xylopyranoside in the culture medium. The kidneys were processed for morphological and biochemical studies. The morphological studies included staining of tissues with anti-basement membrane antibodies and ruthenium red. [35S]sulfate was used as the precursor product for biosynthetic and autoradiographic studies. The kidneys treated with xyloside had loose mesenchyme, inhibition of ureteric bud branching, diminution in the population of developing nephron elements, decreased immunofluorescence with anti-proteoglycan antibodies and staining with ruthenium red, and a reduced [35S]sulfate incorporation into poorly organized extracellular matrices. The biochemical studies included characterization of PGs/glycosaminoglycans (GAGs) by Sepharose CL-4B, -6B, and DEAE-Sephacel chromatographies and cellulose acetate electrophoresis. Under the influence of xyloside, the total radioactivities decreased 2 to 4-fold in tissues and increased 18 to 42-fold in media fractions. A reduction in the size of macromolecular form of PGs, i.e., from MW approximately 2.5 X 10(6) to approximately 2.5 X 10(4), was noted. The PGs/GAGs synthesized were mainly made up of heparan sulfate and small amounts of chondroitin sulfate. They eluted at a lower salt concentration as compared to the controls. A similar diminution in the size of media PGs, i.e., from MW approximately 1.8 X 10(5) to approximately 2.8 X 10(4), was observed. Additional studies with [3H]xyloside indicated that the chains initiated on xyloside residues were similar in size and composition to GAG-chains. These findings indicate that a perturbance in the biosynthesis of PGs/GAGs leads to abnormalities in renal organogenesis.

Status of glomerular proteoglycans (PGs) in puromycin aminonucleoside nephrosis was investigated. Rats were made nephrotic and sacrificed 0, 7, 14, and 21 days later. Maximal proteinuric response was observed between 7 and 14 days. Prior to sacrifice, they received injections of conjugated or unconjugated anti-heparan-sulfate-proteoglycan antibody, directed against its core protein (Mr = 18,000). Their kidneys were processed for direct and indirect immunofluorescence, immunoperoxidase, tannic-acid staining, and tissue autoradiography (ARG). By tannic-acid staining, antibody binding sites identical to the anionic sites described previously were discovered. No qualitative differences were observed by these immunohistochemical techniques. Quantitative tissue ARG did not reveal any statistical differences in the binding of antibody between the control and nephrotic groups. For de novo biosynthetic studies, rats were sacrificed on day 10. Their kidneys were utilized for labeling of basement membrane PGs by employing [35S]-sulfate as the precursor product. Tissue ARG, as well as biochemical studies, were performed on the radiolabeled glomeruli. PGs were extracted with 4 M GuCl and characterized by Sepharose CL-6B and DEAE-Sephacel chromatography. There was an overall increase in the total incorporated radioactivities in the glomerular and media fractions. No differences were observed in the macromolecular size characteristics of intact PGs and glycosaminoglycan chains of either glomerular or media fractions. However, an increase in the charge-density characteristics was observed in PGs of the nephrotic group. By tissue ARG, an increase in the grain densities over the basement membrane and mesangial matrices of the glomerulus was noted. These data indicate that the intact PGs, their chains and core protein do not undergo significant biochemical alterations; however, de novo synthesized PGs have higher charge-density characteristics which may be related to a higher degree of sulfation that occurs during the course of aminonucleoside nephrosis.

Antibodies to glomerular basement membrane, heparan sulfate-proteoglycans are nephrotoxic but possess a weak nephritogenic potential. In order to enhance the nephritogenic potential, the antibodies were intravenously administered into rats presensitized with heterologous rabbit IgG. This resulted in the integration of heterologous and autologous phases, the two phases characteristic of the traditional model of nephrotoxic serum nephritis. The presensitization caused a dramatic shift in the binding characteristics of the heterologous antibodies between the kidney and lymphoid tissues. A proliferative form of immune complex glomerulonephritis associated with a remarkable proteinuric response was observed. In addition, a moderate degree of hematuria was noted as well. The proteinuria was largely complement-dependent and may possibly be cell-mediated as well. The proteinuria became severe with increasing production of host IgG antibodies and with their subsequent sequestration in the glomeruli. The predominant glomerular lesions were in the form of epimembranous/subepithelial immune deposits, which became more frequent with timely increasing titer of host autologous IgG antibodies. These findings indicate that antibodies to heparan sulfate-proteoglycan, an authentic component of the basement membrane, are capable of mediating a glomerular injury with acquisition of nephritogenic potential in an appropriate environment of the host. At present, it seems that this is the sole constituent of the basement membrane whose antibodies are capable of inducing an immune complex nephritis.

Administration of antibody, directed against glomerular basement membrane (GBM) heparan sulfate-proteoglycan, into a presensitized rat results in the induction of membranous nephropathy with subepithelial immune-complex deposits. In this investigation, we examined the mechanisms responsible for the formation of subepithelial immune-complex deposits in the anti-HS-PG model. In initial experiments, the intravenously administered radioiodinated antibody was seen exclusively localized in the regions of the glomerular capillary wall where the subepithelial deposits were observed. To determine their exclusive localization in the subepithelial space, kinetics of movement of the intravenously administered antibody was investigated. The antibody localized in the inner layers of the GBM within a few minutes after its administration. It equilibrated in the inner and outer layers of the GBM in a matter of a few hours. Then, after 24 hours, it gradually disappeared from the inner layers of the GBM and persisted in the outer layers only. The ready clearance of the antibody from the inner layers may be related to the differential in the kinetics of lateral intrinsic plasma fluid currents within the GBM. The persistence of heterologous antibody exclusively in the outer layers and the availability of host autologous antibodies probably resulted in the development of immune complex deposits in the subepithelial space. The glomeruli devoid of plasma water currents showed no change in the concentration of the antibody in the inner and outer layers of the GBM or mesangial matrix. Also, no antibody binding was observed with the plasmalemma of either the foot processes or visceral epithelia. The data suggest that the biochemical-biophysical properties of the glomerular capillary wall, in concert with its intraglomerular hemodynamics, most likely played a significant role in the development of subepithelial immune-complex deposits in this model.

Maturation of extracellular matrices in relation to heparan sulfate proteoglycans was investigated during glomerular development. Antibodies directed against the core-protein (Mr = 18,000) of the basement membrane heparan sulfate-proteoglycan were utilized. The IgG fraction of the antibodies was conjugated with either fluorescein or rhodamine or 125I-iodine. The fluoresceinated and radioiodinated antibodies were given intravenously to three-day-old rats; their kidneys were obtained 6, 48, and 120 hours later and processed for immunohistochemical and tissue autoradiographic studies. At six hours (day 0), matrices of all the stages were labeled. The labeling was lowest in the vesicle stage and highest in the capillary stage. The autoradiographic grain-densities of vesicle, S-shaped, precapillary, and capillary stages were 10.62 +/- 0.54, 7.44 +/- 0.65, 11.37 +/- 1.03, and 18.35 +/- 1.97, respectively. After 48 hours (day 5), the grain-density for S-shaped body decreased from 7.44 +/- 0.65 to 4.38 +/- 0.61, while no appreciable change in the precapillary and capillary stages was observed. At this time interval, the fluorescein-labeled glomeruli were seen in the deeper cortex only. After 120 hours (day 8), the grain-density for the capillary stage decreased from 18.35 +/- 1.97 to 2.82 +/- 0.92. The double-labeling experiments included a second injection of rhodaminated antibody at 48 hours (day 5). In these experiments, newly developed glomeruli of varying stages were seen in the superficial cortex. With the use of double filters, the immature glomeruli in the superficial cortex and mature ones in the deeper cortex were visualized.(ABSTRACT TRUNCATED AT 250 WORDS)