Cleavage of periostin by MMP9 protects mice from kidney cystic disease.

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.

ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

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 SAM domain of ANKS6 has different interacting partners and mutations can induce different cystic phenotypes.

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.

LG3 fragment of endorepellin is a possible biomarker of severity in IgA nephropathy.

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-ß-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.

NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9.

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-1ß- and TNF-α-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-α-induced MMP-9 expression through ß1 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.

MMP9 limits apoptosis and stimulates branching morphogenesis during kidney development.

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.

Doxycycline accelerates renal cyst growth and fibrosis in the pcy/pcy mouse model of type 3 nephronophthisis, a form of recessive polycystic kidney disease.

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.

MMP9 and SCF protect from apoptosis in acute kidney injury.

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.

Matrix metalloproteinases in kidney disease progression and repair: a case of flipping the coin.

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.

Expression of matrix metalloproteinases MMP-2 and MMP-9 is altered during nephrogenesis in fetuses from diabetic rats.

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.

Urokinase (u-PA) is produced by collecting duct principal cells and is post-transcriptionally regulated by SV40 large-T, arginine vasopressin, and epidermal growth factor.

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.

Role of extracellular matrix in kidney development and repair.

Extracellular matrix (ECM) molecules and their receptors exert a dynamic role in cell-matrix interactions during kidney development and repair processes. They provide a physical substratum for the spatial organization of the cells, but also regulate cell growth and proliferation by interacting with growth factors. In addition, they can regulate signal transduction pathways by binding to integrins or by modulating the activity of signaling molecules such as Wnts. ECM and ECM-related molecules control multiple (if not all) steps of kidney development, including ureteric bud branching morphogenesis, mesenchymal condensation, nephron formation, terminal differentiation of renal tubules, and glomerular basement membrane assembly. Their role still needs to be better documented in renal repair. The emergence of conditionally mutated mice for basement membrane components will provide a useful tool to demonstrate further the involvement of ECM and ECM-related proteins in development and repair.

Role of matrix metalloproteinases in kidney development and glomerulopathy: lessons from transgenic mice.

Matrix metalloproteinase-9 (MMP9) is required for renal organogenesis in vitro and is increased in various nephropathies. We analysed the renal phenotype of MMP9-deficient mice and their susceptibility to a murine model of proliferative glomerulonephritis. MMP9 deficiency resulted in adult mice in a 12% nephronic reduction. Histological appearance and renal function of these mice was normal up to 12 months, at which time histological lesions appeared. In addition, glomerulonephritis was more severe in MMP9-deficient mice than in their control 3-month-old mates. In particular, the extent of crescent formation and fibrin deposition was greater, which led us to show that fibrin is a critical substrate for MMP9. These data provide the first demonstration in vivo that MMP9 is required for nephron mass formation and renal function in elderly mice, and further evidence of a novel protective effect of MMP9 on the development of fibrin-induced glomerular lesions.

Expression of the type IV collagenase system during mouse kidney development and tubule segmentation.

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.