Kidneys Of Knockout Mice Research Articles

Na,K-ATPase from Mice Lacking the γ Subunit (FXYD2) Exhibits Altered Na+ Affinity and Decreased Thermal Stability

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype

The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by approximately 50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.

Smad‐3 as a mediator of the fibrotic response

Introduction Smad‐3, a key cytoplasmic mediator of transforming growth factor‐β (TGF‐β) signalling, mediates many of its inflammatory and fibrotic effects in vivo (Roberts et al. 2001). Smad‐3 null mice are protected against cutaneous injury induced by ionizing irradiation (Flanders et al. 2002). Here, we report on our continuing studies on radioprotection as well as protection against tubulointerstitial fibrosis following unilateral ureteral obstruction (UUO) in Smad‐3 null mice.Methods For radioprotection studies, the flank skin of Smad‐3+/+ wild‐type (WT) and Smad‐3–/– knockout (KO) mice was exposed to 30 Gy of localized γ‐irradiation and analysed for histology and gene expression at various times post irradiation. In the UUO model, the right proximal ureter of WT and KO mice was ligated, and 1–2 weeks later kidneys were analysed for inflammation, fibrosis and gene expression.Results Six weeks after exposure to irradiation, skin from KO mice shows less epidermal acanthosis and influx of mast cells, macrophages and neutrophils than skin of WT mice. Paradoxically, at 6–8 h post irradiation, KO skin shows a significantly greater number of neutrophils. Irradiated KO skin also exhibits less immunoreactive TGF‐β, fewer myofibroblasts and less scarring than does WT. Smad‐3 null dermal fibroblasts do not respond to the chemotactic effects of TGF‐β and show less induction of fibrogenic cytokines when treated with irradiation plus TGF‐β compared to WT cells. Following UUO, normal kidney architecture is preserved in KO mice, while kidneys from WT mice are enlarged with an influx of mononuclear cells and increased expression of collagen and TGF‐β1. Additionally, renal tubules in obstructed kidneys of KO mice remain positive for E‐cadherin without expression of α‐smooth muscle actin, while the opposite expression pattern is seen in obstructed kidneys of WT mice. TGF‐β treatment of primary cultures of WT renal tubular epithelial cells results in a phenotypic change from a cobblestone pattern to a spindle‐shaped fibroblastic appearance, while KO cells treated with TGF‐β maintain their original appearance.Conclusion Smad‐3 plays an important role in mediating pathogenic inflammation and fibrosis in several model systems and is also essential for TGF‐β1‐induced epithelial–mesenchymal transition in renal tubular epithelial cells. Inhibitors of the Smad‐3 pathway may have clinical applications in the treatment of a number of fibrotic conditions.

Expression profiling confirms the role of endocytic receptor megalin in renal vitamin D3 metabolism

The endocytic receptor megalin constitutes the major pathway for clearance of low-molecular weight plasma proteins from the glomerular filtrate into the renal proximal tubules. Furthermore, the receptor has been implicated in a number of other functions in the kidney including uptake and activation of 25-(OH) vitamin D3, calcium and sodium reabsorption as well as signal transduction. We used genome-wide expression profiling by microarray technology to detect changes in the gene expression pattern in megalin knockout mouse kidneys and to uncover some of the renal pathways affected by megalin deficiency. Alterations were identified in several (patho)physiologic processes in megalin-deficient kidneys including the renal vitamin D metabolism, transforming growth factor (TGF)-beta1 signal transduction, lipid transport and heavy metal detoxification. Most importantly, changes were detected in the mRNA levels of 25-(OH) vitamin D-24-hydroxylase and 25-(OH) vitamin D-1alpha-hydroxylase as well as strong up-regulation of TGF-beta1 target genes. Both findings indicate plasma vitamin D deficiency and lack of vitamin D signaling in renal tissues. Expression profiling confirms a crucial role for megalin in renal vitamin D metabolism.

Megalin and Cubilin are Endocytic Receptors Involved in Renal Clearance of Hemoglobin

The kidney is the main site of hemoglobin clearance and degradation in conditions of severe hemolysis. Herein it is reported that megalin and cubilin, two epithelial endocytic receptors, mediate the uptake of hemoglobin in renal proximal tubules. Both receptors were purified by use of hemoglobin-Sepharose affinity chromatography of solubilized renal brush-border membranes. Apparent dissociation constants of 1.7 microM for megalin and 4.1 microM for cubilin were determined by surface plasmon resonance analysis. The binding was calcium dependent in both cases. Uptake of fluorescence-labeled hemoglobin by BN-16 cells was inhibited by anti-megalin and anti-cubilin antibodies as well as by receptor-associated protein, a chaperone for LDL-receptor family proteins. Partial inhibition by myoglobin was observed, whereas bovine serum albumin, intrinsic factor-cobalamin complexes, and beta2-microglobulin did not affect the uptake. By use of immunohistochemistry, it was demonstrated that uptake of hemoglobin in proximal tubules of rat, mouse, and dog kidneys occurs under physiologic conditions. Studies on normal and megalin knockout mouse kidney sections showed that megalin is responsible for physiologic clearance of hemoglobin. Labeling intensities in kidneys from normal and cubilin-malexpressing dogs were similar, which suggests that, in the normal state, the role of cubilin in uptake of hemoglobin is rather limited. However, cubilin is likely to assist hemoglobin endocytosis in settings of hemoglobinuria. In conclusion, the study provides a molecular explanation for long-standing observations of hemoglobin uptake in renal proximal tubules that involve the endocytic receptors megalin and cubilin. The findings may prove to be essential for further research on the pathophysiology of hemoglobinuric acute renal failure and proteinuria-associated tubulointerstitial nephritis.

Increased susceptibility of decay-accelerating factor deficient mice to anti-glomerular basement membrane glomerulonephritis.

To prevent complement-mediated autologous tissue damage, host cells express a number of membrane-bound complement inhibitors. Decay-accelerating factor (DAF, CD55) is a GPI-linked membrane complement regulator that is widely expressed in mammalian tissues including the kidney. DAF inhibits the C3 convertase of both the classical and alternative pathways. Although DAF deficiency contributes to the human hematological syndrome paroxysmal nocturnal hemoglobinuria, the relevance of DAF in autoimmune tissue damage such as immune glomerulonephritis remains to be determined. In this study, we have investigated the susceptibility of knockout mice that are deficient in GPI-anchored DAF to nephrotoxic serum nephritis. Injection of a subnephritogenic dose of rabbit anti-mouse glomerular basement membrane serum induced glomerular disease in DAF knockout mice but not in wild-type controls. When examined at 8 days after anti-glomerular basement membrane treatment, DAF knockout mice had a much higher percentage of diseased glomeruli than wild-type mice (68.8 +/- 25.0 vs 10.0 +/- 3.5%; p < 0.01). Morphologically, DAF knockout mice displayed increased glomerular volume (516 +/- 68 vs 325 +/- 18 x 10(3) microm(3) per glomerulus; p < 0.0001) and cellularity (47.1 +/- 8.9 vs 32.0 +/- 3.1 cells per glomerulus; p < 0.01). Although the blood urea nitrogen level showed no difference between the two groups, proteinuria was observed in the knockout mice but not in the wild-type mice (1.4 +/- 0.7 vs 0.02 +/- 0.01 mg/24 h albumin excretion). The morphological and functional abnormalities in the knockout mouse kidney were associated with evidence of increased complement activation in the glomeruli. These results support the conclusion that membrane C3 convertase inhibitors like DAF play a protective role in complement-mediated immune glomerular damage in vivo.