Matrix metalloproteinase-10 deficiency has protective effects against peritoneal inflammation and fibrosis via transcription factor NFκΒ pathway inhibition.

One of the most common causes of discontinued peritoneal dialysis is impaired peritoneal function. However, its molecular mechanisms remain unclear. Previously, by microarray analysis of mouse peritoneum, we showed that MMP (matrix metalloproteinase)-10 expression is significantly increased in mice with peritoneal fibrosis, but its function remains unknown. Chlorhexidine gluconate (CG) was intraperitoneally injected to wild-type and MMP-10 knockout mice to induce fibrosis to elucidate the role of MMP-10 on peritoneal injury. We also examined function of peritoneal macrophages and mesothelial cells obtained from wild-type and MMP-10 knockout mice, MMP-10-overexpressing macrophage-like RAW 264.7 cells and MeT-5A mesothelial cells, investigated MMP-10 expression on peritoneal biopsy specimens, and the association between serum proMMP-10 and peritoneal solute transfer rates determined by peritoneal equilibration test on patients. MMP-10 was expressed in cells positive for WT1, a mesothelial marker, and for MAC-2, a macrophage marker, in the thickened peritoneum of both mice and patients. Serum proMMP-10 levels were well correlated with peritoneal solute transfer rates. Peritoneal fibrosis, inflammation, and high peritoneal solute transfer rates induced by CG were all ameliorated by MMP-10 deletion, with reduction of CD31-positive vessels and VEGF-A-positive cells. Expression of inflammatory mediators and phosphorylation of NFκΒ subunit p65 at S536 were suppressed in both MMP-10 knockout macrophages and mesothelial cells in response to lipopolysaccharide stimulation. Overexpression of MMP-10 in RAW 264.7 and MeT-5A cells upregulated pro-inflammatory cytokines with phosphorylation of NFκΒ subunit p65. Thus, our results suggest that inflammatory responses induced by MMP-10 are mediated through the NFκΒ pathway, and that systemic deletion of MMP-10 ameliorates peritoneal inflammation and fibrosis caused by NFκΒ activation of peritoneal macrophages and mesothelial cells.

Dual deletion of guanylyl cyclase-A and p38 mitogen-activated protein kinase in podocytes with aldosterone administration causes glomerular intra-capillary thrombi.

Natriuretic peptides exert not only blood-lowering but also kidney-protective effects through guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. Signaling through GC-A has been shown to protect podocytes from aldosterone-induced glomerular injury, and a p38 mitogen-activated protein kinase (MAPK) inhibitor reduced glomerular injury in aldosterone-infused podocyte-specific GC-A knockout mice. To explore the role of p38 MAPK in podocytes, we constructed podocyte-specific p38 MAPK and GC-A double knockout mice (pod-double knockout mice). Unexpectedly, aldosterone-infused and high salt-fed (B-ALDO)-treated pod-double knockout mice resulted in elevated serum creatinine, massive albuminuria, macrophage infiltration, foot process effacement, nephrin and podocin reduction, and additionally, intra-capillary fibrin thrombi, indicating endothelial injury. Microarray analysis showed increased plasminogen activator inhibitor-1 (PAI-1) in glomeruli of B-ALDO-treated pod-double knockout mice. In B-ALDO-treated pod-double knockout mice, PAI-1 increased in podocytes, and treatment with PAI-1 neutralizing antibody ameliorated intra-capillary thrombus formation. In vitro, deletion of p38 MAPK by the CRISPR/Cas9 system and knockdown of GC-A in human cultured podocytes upregulated PAI-1 and transforming growth factor- ß1 (TGF-ß1). When p38 MAPK knockout podocytes, transfected with a small interfering RNA to suppress GC-A, were co-cultured with glomerular endothelial cells in a transwell system, the expression of TGF-ß1 was increased in glomerular endothelial cells. PAI-1 inhibition ameliorated both podocyte and endothelial injury in the transwell system signifying elevated PAI-1 in podocytes is a factor disrupting normal podocyte-endothelial crosstalk. Thus, our results indicate that genetic dual deletion of p38 MAPK and GC-A in podocytes accelerates both podocyte and endothelial injuries, suggesting these two molecules play indispensable roles in podocyte function.

Deletion of connective tissue growth factor ameliorates peritoneal fibrosis by inhibiting angiogenesis and inflammation.

Background: Connective tissue growth factor (CTGF/CCN2) regulates the signalling of other growth factors and promotes fibrosis. CTGF is increased in mice and humans with peritoneal fibrosis. Inhibition of CTGF has not been examined as a potential therapeutic target for peritoneal fibrosis because systemic CTGF knockout mice die at the perinatal stage. Methods: To study the role of CTGF in peritoneal fibrosis of adult mice, we generated CTGF conditional knockout (cKO) mice by crossing CTGF floxed mice with RosaCreERT2 mice. We administered tamoxifen to Rosa-CTGF cKO mice to delete the CTGF gene throughout the body. We induced peritoneal fibrosis by intraperitoneal injection of chlorhexidine gluconate (CG) in wild-type and Rosa-CTGF cKO mice. Results: Induction of peritoneal fibrosis in wild-type mice increased CTGF expression and produced severe thickening of the peritoneum. In contrast, CG-treated Rosa-CTGF cKO mice exhibited reduced thickening of the peritoneum. Peritoneal equilibration test revealed that the excessive peritoneal small-solute transport in CG-treated wild-type mice was normalized by CTGF deletion. CG-treated Rosa-CTGF cKO mice exhibited a reduced number of αSMA-, Ki67-, CD31- and MAC-2-positive cells in the peritoneum. Analyses of peritoneal mRNA showed that CG-treated Rosa-CTGF cKO mice exhibited reduced expression of Cd68, Acta2 (αSMA), Pecam1 (CD31) and Vegfa. Conclusions: These results indicate that a deficiency of CTGF can reduce peritoneal thickening and help to maintain peritoneal function by reducing angiogenesis and inflammation in peritoneal fibrosis. These results suggest that CTGF plays an important role in the progression of peritoneal fibrosis.

Natriuretic peptide receptor guanylyl cyclase-A pathway counteracts glomerular injury evoked by aldosterone through p38 mitogen-activated protein kinase inhibition.

Guanylyl cyclase-A (GC-A) signaling, a natriuretic peptide receptor, exerts renoprotective effects by stimulating natriuresis and reducing blood pressure. Previously we demonstrated massive albuminuria with hypertension in uninephrectomized, aldosterone-infused, and high salt-fed (ALDO) systemic GC-A KO mice with enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) in podocytes. In the present study, we examined the interaction between p38 MAPK and GC-A signaling. The administration of FR167653, p38 MAPK inhibitor, reduced systolic blood pressure (SBP), urinary albumin excretion, segmental sclerosis, podocyte injury, and apoptosis. To further investigate the local action of natriuretic peptide and p38 MAPK in podocytes, we generated podocyte-specific (pod) GC-A conditional KO (cKO) mice. ALDO pod GC-A cKO mice demonstrated increased urinary albumin excretion with marked mesangial expansion, podocyte injury and apoptosis, but without blood pressure elevation. FR167653 also suppressed urinary albumin excretion without reducing SBP. Finally, we revealed that atrial natriuretic peptide increased phosphorylation of MAPK phosphatase-1 (MKP-1) concomitant with inhibited phosphorylation of p38 MAPK in response to MAPK kinase 3 activation, thereby resulting in decreased mRNA expression of the apoptosis-related gene, Bax, and Bax/Bcl2 ratio in cultured podocytes. These results indicate that natriuretic peptide exerts a renoprotective effect via inhibiting phosphorylation of p38 MAPK in podocytes.

Crucial Role of Mesangial Cell-derived Connective Tissue Growth Factor in a Mouse Model of Anti-Glomerular Basement Membrane Glomerulonephritis.

Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-ß1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.

Nephrotic range proteinuria as a strong risk factor for rapid renal function decline during pre-dialysis phase in type 2 diabetic patients with severely impaired renal function.

BACKGROUND: Proteinuria is an established risk factor for progression of renal disease, including diabetic nephropathy. The predictive power of proteinuria, especially nephrotic range proteinuria, for progressive renal deterioration has been well demonstrated in diabetic patients with normal to relatively preserved renal function. However, little is known about the relationship between severity of proteinuria and renal outcome in pre-dialysis diabetic patients with severely impaired renal function. METHODS: 125 incident dialysis patients with type 2 diabetes were identified. This study was aimed at retrospectively evaluating the impact of nephrotic range proteinuria (urinary protein-creatinine ratio above 3.5 g/gCr) on renal function decline during the 3 months just prior to dialysis initiation. RESULTS: In total, 103 patients (82.4 %) had nephrotic range proteinuria. The median rate of decline in estimated glomerular filtration rate (eGFR) in this study population was 0.98 (interquartile range 0.51-1.46) ml/min/1.73 m(2) per month. Compared to patients without nephrotic range proteinuria, patients with nephrotic range proteinuria showed significantly faster renal function decline (0.46 [0.24-1.25] versus 1.07 [0.64-1.54] ml/min/1.73 m(2) per month; p = 0.007). After adjusting for gender, age, systolic blood pressure, serum albumin, calcium-phosphorus product, hemoglobin A1c, and use of an angiotensin-converting enzyme inhibitor or an angiotensin II receptor blocker, patients with nephrotic range proteinuria showed a 3.89-fold (95 % CI 1.08-14.5) increased risk for rapid renal function decline defined as a decline in eGFR ≥0.5 ml/min/1.73 m(2) per month. CONCLUSION: Nephrotic range proteinuria is the predominant renal risk factor in type 2 diabetic patients with severely impaired renal function receiving pre-dialysis care.

Systemic mycobacteriosis in an aborted thoroughbred fetus in Japan.

A male Thoroughbred fetus was aborted on day 251 of pregnancy. Gross and histological examinations detected systemic granulomatous lesions in many superficial and visceral lymph nodes and organs including the liver, tonsils, lungs, thymus, spleen, right thyroid gland and gastrointestinal tract, and suppurative placentitis, pyogranulomatous amnionitis and intralesional acid-fast bacilli were also detected. An examination of the DNA base sequence of the ß subunit of RNA polymerase demonstrated that Mycobacterium avium strain 104 had infected several organs. To the best of our knowledge, this is the first report of equine fetal mycobacterial infection in Japan.

Distal renal tubular acidosis that became exacerbated by proton pump inhibitor use.

Acid-base imbalances and electrolyte disorders induced by proton pump inhibitors (PPIs) are extremely rare. However, under certain conditions, PPIs may cause metabolic acidosis or hypokalemia, probably due to an inhibitory action on the proton pump that contributes to H(+) and K(+) homeostasis in the kidney. We herein present a case of marked hypokalemia accompanied by distal renal tubular acidosis in which a PPI appeared to contribute to the pathophysiology of metabolic acidosis.

A case of neurofibromatosis type 1 coinciding with bilateral pheochromocytomas, multiple gastrointestinal stromal tumors, and malignant peripheral nerve sheath tumor.

Neurofibromatosis type 1 (NF1) is associated with benign and malignant neoplasms, but the coincidence of abdominal neoplasms is rare. A 65-year-old woman with NF1 had episodes of nausea, tachycardia, hypertension, and loss of consciousness. Bilateral adrenal tumors were detected by abdominal computed tomography, and plasma and urinary catecholamine levels were elevated. Open bilateral adrenalectomy and histological findings revealed bilateral pheochromocytomas (PCCs). Furthermore, malignant peripheral nerve sheath tumor (MPNST) and multiple gastrointestinal stromal tumors (GISTs) were incidentally found in the abdominal cavity. Early diagnosis of abdominal neoplasms in NF1 patients is important because of the risk of malignancy, organic complications and hemorrhagic-obstructive complications.