Pharmacological inhibition of Vanin-1 is not protective in models of acute and chronic kidney disease.

Oxidative stress is a key concept in basic, translational, and clinical research to understand the pathophysiology of various disorders, including cardiovascular and renal diseases. Although attempts to directly reduce oxidative stress with redox-active substances have until now largely failed to prove clinical benefit, indirect approaches to combat oxidative stress enzymatically have gained further attention as potential therapeutic strategies. The pantetheinase Vanin-1 is expressed on kidney proximal tubular cells, and its reaction product cysteamine is described to negatively affect redox homeostasis by inhibiting the replenishment of cellular antioxidative glutathione stores. Vanin-1-deficient mice were shown to be protected against oxidative stress damage. The aim of this study was to elucidate whether pharmacological inhibition of Vanin-1 protects mice from oxidative stress-related acute or chronic kidney injury as well. By studying renal ischemia-reperfusion injury in Col4α3-/- (Alport syndrome) mice and in vitro hypoxia-reoxygenation in human proximal tubular cells we found that treatment with a selective and potent Vanin-1 inhibitor resulted in ample inhibition of enzymatic activity in vitro and in vivo. However, surrogate parameters of metabolic and redox homeostasis were only partially and insufficiently affected. Consequently, apoptosis and reactive oxygen species level in tubular cells as well as overall kidney function and fibrotic processes were not improved by Vanin-1 inhibition. We thus conclude that Vanin-1 functionality in the context of cardiovascular diseases needs further investigation and the biological relevance of pharmacological Vanin-1 inhibition for the treatment of kidney diseases remains to be proven.

Laminin α2-mediated focal adhesion kinase activation triggers Alport glomerular pathogenesis.

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.

Assessment of 24,25(OH)2D levels does not support FGF23-mediated catabolism of vitamin D metabolites.

Progressive elevations of fibroblastic growth factor 23 (FGF23) in chronic kidney disease may reduce serum 25-hydroxyvitamin D (25(OH)) and 1,25-dihydroxyvitamin D (1,25(OH)(2)D) levels, via stimulation of 24-hydroxylase (Cyp24a1)-mediated catabolism of these vitamin D metabolites. To test this possibility, we measured serum concentrations of 24,25-dihydroxyvitamin D (24,25(OH)(2)D), a product of Cyp24a1 hydroxylation of 25(OH)D, in the Col4a3 knockout mouse, a model of Alport syndrome-derived chronic kidney disease, and in patients with chronic kidney disease of variable severity. There was an inverse correlation between serum FGF23 and both 25(OH)D and 1,25(OH)(2)D in the mouse model, but no significant relationship was observed in the cross-sectional patient cohort. The FGF23-dependent increase in Cyp24a1 mRNA expression in the mouse kidneys was consistent with the possibility that FGF23 induces vitamin D catabolism. There was, however, a reduction in serum 24,25(OH)(2)D levels, rather than the expected elevation, in both the mice and patients with chronic kidney disease. Low 25(OH)D and elevated FGF23 and parathyroid hormone levels were correlated with the reduced serum 24,25(OH)(2)D concentrations of these patients. Thus, we failed to find support for FGF23-mediated catabolism of vitamin D metabolites in chronic kidney disease assessed by 24,25(OH)(2)D levels.

Trypsin digestion on paraffin sections is a useful tool for diagnosis of alport syndrome.

Trypsin digestion for 90 min revealed the alpha(5) chain of type IV collagen along the glomerular basement membrane and Bowman's capsule in paraffin-embedded renal sections of controls. In the 9 patients with the ultrastructures suggestive of Alport syndrome (AS), 8 patients were classified as X-linked dominant type due to the lack or mosaic pattern of alpha(5) chain in paraffin sections of renal biopsies by trypsin digestion, and 1 patient was classified as autosomal recessive type due to the lack of alpha(5) chain in the glomerular basement membrane only. Trypsin digestion is useful for the diagnosis of AS in paraffin-embedded renal tissue.

Integrin alpha1beta1 regulates matrix metalloproteinases via P38 mitogen-activated protein kinase in mesangial cells: implications for Alport syndrome.

Previous work has shown that integrin alpha1-null Alport mice exhibit attenuated glomerular disease with decreased matrix accumulation and live much longer than strain-matched Alport mice. However, the mechanism underlying this observation is unknown. Here we show that glomerular gelatinase expression, specifically matrix metalloproteinase-2 (MMP-2), MMP-9, and MMP-14, was significantly elevated in both integrin alpha1-null mice and integrin alpha1-null Alport mice relative to wild-type mice; however, only MMP-9 was elevated in glomeruli of Alport mice that express integrin alpha1. Similarly, cultured mesangial cells from alpha1-null mice showed elevated expression levels of all three MMPs, whereas mesangial cells from Alport mice show elevated expression levels of only MMP-9. In both glomeruli and cultured mesangial cells isolated from integrin alpha1-null mice, activation of the p38 and ERK branches of the mitogen-activated protein kinase pathway was also observed. The use of small molecule inhibitors demonstrated that the activation of the p38, but not ERK, pathway was linked to elevated MMP-2, -9, and -14 expression levels in mesangial cells from integrin alpha1-null mice. In contrast, elevated MMP-9 levels in mesangial cells from Alport mice were linked to ERK pathway activation. Blockade of gelatinase activity using a small molecule inhibitor (BAY-12-9566) ameliorated progression of proteinuria and restored the architecture of the glomerular basement membrane in alpha1 integrin-null Alport mice, suggesting that elevated gelatinase activity exacerbates glomerular disease progression in these mice.

Role for macrophage metalloelastase in glomerular basement membrane damage associated with alport syndrome.

Alport syndrome is a glomerular basement membrane (GBM) disease caused by mutations in type IV collagen genes. A unique irregular thickening and thinning of the GBM characterizes the progressive glomerular pathology. The metabolic imbalances responsible for these GBM irregularities are not known. Here we show that macrophage metalloelastase (MMP-12) expression is >40-fold induced in glomeruli from Alport mice and is markedly induced in glomeruli of both humans and dogs with Alport syndrome. Treatment of Alport mice with MMI270 (CGS27023A), a broad spectrum MMP inhibitor that blocks MMP-12 activity, results in largely restored GBM ultrastructure and function. Treatment with BAY-129566, a broad spectrum MMP inhibitor that does not inhibit MMP-12, had no effect. We show that inhibition of CC chemokine receptor 2 (CCR2) receptor signaling with propagermanium blocks induction of MMP-12 mRNA and prevents GBM damage. CCR2 receptor is expressed in glomerular podocytes of Alport mice, suggesting MCP-1 activation of CCR2 on podocytes may underlie induction of MMP-12. These data indicate that the irregular GBM that characterizes Alport syndrome may be mediated, in part, by focal degradation of the GBM due to MMP dysregulation, in particular, MMP-12. Thus, MMP-12/CCR2 inhibitors may provide a novel and effective therapeutic stra-tegy for Alport glomerular disease.

Matrix metalloproteinase dysregulation in the stria vascularis of mice with Alport syndrome: implications for capillary basement membrane pathology.

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.

Gelatinase B (MMP-9) is not essential in the normal kidney and does not influence progression of renal disease in a mouse model of Alport syndrome.

Matrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB-/- and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB-/- embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred with Col4a3-/- mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome.

FACL4, a new gene encoding long-chain acyl-CoA synthetase 4, is deleted in a family with Alport syndrome, elliptocytosis, and mental retardation.

We observed a family in which two boys were diagnosed with Alport syndrome, elliptocytosis, and mental retardation and carried a large deletion of the Xq22.3-q23 region, encompassing the COL4A5 gene. This suggests the possibility of a new contiguous gene syndrome. In an attempt to characterize the genes contributing to this complex phenotype, we have isolated a gene encoding a new long-chain acyl-CoA synthetase (FACL4 or LACS4) from the region deleted in these patients. Among several ESTs identified by searching the human gene map database maintained at the National Center for Biotechnology Information, using the map position as a query, only one was deleted in the patients. RACE products containing the entire ORF were subsequently generated. Northern blot analysis showed a 5-kb mRNA expressed in several tissues except for liver and lung. Brain shows a longer transcript, possibly reflecting the use of a brain-specific upstream ATG start codon. FACL4 encodes a predicted protein product of 670 amino acids (711 in brain), with a remarkable level of conservation compared to the rat acyl-CoA synthetases ACS4 and brain-specific ACS3 protein sequences. We are investigating the possibility that the absence of this enzyme may play a role in the development of mental retardation or other signs associated with Alport syndrome in the family.