X-linked Hyper IgM Syndrome Presenting as Pulmonary Alveolar Proteinosis.

PURPOSE: X-linked hyper IgM syndrome (XHIGM) is a combined immunodeficiency caused by mutations in the CD40 ligand (CD40L) gene that typically results in decreased or absent CD40L expression on activated T cells, leading to defective class switching and somatic hypermutation. We describe an infant who presented with respiratory failure due to pulmonary alveolar proteinosis (PAP) with a novel damaging missense mutation in the CD40L gene. METHODS: Whole exome sequencing (WES) was used to identify a mutation in the CD40L gene. CD40L expression and function were determined by flow cytometry. RESULTS: A 5-month-old previously-healthy male presented with respiratory failure and diffuse pulmonary ground glass opacities on CT scan of the chest. Laboratory evaluation revealed an undetectable IgG, normal IgA, and elevated IgM. A bronchoalveolar lavage demonstrated pulmonary alveolar proteinosis. WES demonstrated a c.608G > C mutation in the CD40L gene resulting in p.R203T. Flow cytometry demonstrated normal CD40L expression on activated T cells but absent binding of CD40-Ig to CD40L on activated patient T cells. CONCLUSIONS: The clinical manifestations of XHIGM in our patient had several unique features, including the presentation with PAP, normal serum IgA, and expression of non-functional CD40L on activated T cells. To our knowledge, this is the first published case of PAP in a patient with XHIGM.

Successful hematopoietic cell transplantation in a patient with X-linked agammaglobulinemia and acute myeloid leukemia.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency characterized by marked reduction in all classes of serum immunoglobulins and the near absence of mature CD19(+) B-cells. Although malignancy has been observed in patients with XLA, we present the first reported case of acute myeloid leukemia (AML) in a patient with XLA. We also demonstrate the complete correction of the XLA phenotype following allogeneic hematopoietic cell transplantation for treatment of the patient's leukemia.

Cysteamine modulates oxidative stress and blocks myofibroblast activity in CKD.

Therapy to slow the relentless expansion of interstitial extracellular matrix that leads to renal functional decline in patients with CKD is currently lacking. Because chronic kidney injury increases tissue oxidative stress, we evaluated the antifibrotic efficacy of cysteamine bitartrate, an antioxidant therapy for patients with nephropathic cystinosis, in a mouse model of unilateral ureteral obstruction. Fresh cysteamine (600 mg/kg) was added to drinking water daily beginning on the day of surgery, and outcomes were assessed on days 7, 14, and 21 after surgery. Plasma cysteamine levels showed diurnal variation, with peak levels similar to those observed in patients with cystinosis. In cysteamine-treated mice, fibrosis severity decreased significantly at 14 and 21 days after unilateral ureteral obstruction, and renal oxidized protein levels decreased at each time point, suggesting reduced oxidative stress. Consistent with these results, treatment of cultured macrophages with cysteamine reduced cellular generation of reactive oxygen species. Furthermore, treatment with cysteamine reduced α-smooth muscle actin-positive interstitial myofibroblast proliferation and mRNA levels of extracellular matrix proteins in mice and attenuated myofibroblast differentiation and proliferation in vitro, but did not augment TGF-ß signaling. In a study of renal ischemia reperfusion, cysteamine therapy initiated 10 days after injury and continued for 14 days decreased renal fibrosis by 40%. Taken together, these data suggest previously unrecognized antifibrotic actions of cysteamine via TGF-ß-independent mechanisms that include oxidative stress reduction and attenuation of the myofibroblast response to kidney injury and support further investigation into the potential benefit of cysteamine therapy in the treatment of CKD.

Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection.

Staphylococcus aureus are gram-positive bacteria that cause clinically significant infections in humans. Severe S. aureus infections are particularly problematic in hospitalized patients and reoccur despite therapeutic measures. The absence of natural protective immune responses and the lack of high-throughput approaches to identify S. aureus antigens have imposed constraints in the development of effective vaccines. Here, we showed that vaccination with the genetically attenuated S. aureus mutant, inactivated using UV irradiation rather than heat, significantly increased survival and diminished bacterial burden and kidney abscesses when mice were challenged with virulent methicillin-sensitive or methicillin-resistant S. aureus. Protection conferred by immunization could be transferred to the naive host and was not observed in B-cell-deficient mice. Using a novel S. aureus whole-proteome microarray, we show that immunoglobulin G antibody responses to 83 proteins were observed in the immunized mice. These results suggest that protection against S. aureus infections requires antibody responses to the wide repertoire of antigens/virulence factors. Vaccination using UV-irradiated genetically attenuated S. aureus induces humoral immunity and provides a vaccine strategy for pathogens that fail to induce protective immunity. We also describe a novel, high-throughput technology to easily identify S. aureus antigens for vaccine development.

Airway epithelial cells from asthmatic children differentially express proremodeling factors.

BACKGROUND: The airway epithelium can express factors that drive subepithelial airway remodeling. TGF-ß2, vascular epithelial growth factor (VEGF), a disintegrin and metalloprotease 33 (ADAM33), and periostin are hypothesized to be involved in subepithelial remodeling and are overexpressed in adult asthmatic airways. Epidemiologic data suggest that lung function deficits in asthmatic patients are acquired in childhood. OBJECTIVES: We sought to determine whether airway epithelial cells (AECs) from asthmatic children differentially express TGF-ß2, VEGF, ADAM33, or periostin compared with cells from atopic nonasthmatic and healthy children intrinsically or in response to IL-4/IL-13 stimulation. METHODS: Bronchial and nasal epithelial cells were obtained from brushings from well-characterized asthmatic (n = 16), atopic nonasthmatic (n = 9), and healthy (n = 15) children after achievement of anesthesia for elective procedures. After differentiation at an air-liquid interface (ALI) for 3 weeks, conditioned media were sampled and RNA was extracted from unstimulated and IL-4/IL-13-stimulated cultures. TGF-ß2 and VEGF levels were measured with ELISA. ADAM33 and periostin expression was assessed by using real-time PCR. RESULTS: TGF-ß2 and VEGF production was significantly greater in bronchial and nasal ALI cultures from asthmatic children than in cultures from atopic nonasthmatic and healthy children. TGF-ß2 levels increased significantly in asthmatic cultures after IL-4/IL-13 stimulation. Within-subject correlation between nasal and bronchial ALI production of TGF-ß2 (r = 0.64, P = .001) and VEGF (r = 0.73, P < .001) was good. Periostin expression was 3.7-fold higher in bronchial cells (P < .001) and 3.9-fold higher in nasal cells (P < .004) from asthmatic children than in cells from atopic nonasthmatic or healthy children. ADAM33 was not differentially expressed by AECs from asthmatic patients compared with that from cells from atopic nonasthmatic or healthy children. CONCLUSION: AECs from asthmatic children differentially express TGF-ß2, VEGF, and periostin compared with cells from atopic nonasthmatic and healthy children. Nasal epithelial cells might be a suitable surrogate for bronchial cells that could facilitate investigation of the airway epithelium in future longitudinal pediatric studies.

Mannose receptor 2 attenuates renal fibrosis.

Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3(-/-) mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3(-/-) mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway.

Investigating mechanisms of chronic kidney disease in mouse models.

Animal models of chronic kidney disease (CKD) are important experimental tools that are used to investigate novel mechanistic pathways and to validate potential new therapeutic interventions prior to pre-clinical testing in humans. Over the past several years, mouse CKD models have been extensively used for these purposes. Despite significant limitations, the model of unilateral ureteral obstruction (UUO) has essentially become the high-throughput in vivo model, as it recapitulates the fundamental pathogenetic mechanisms that typify all forms of CKD in a relatively short time span. In addition, several alternative mouse models are available that can be used to validate new mechanistic paradigms and/or novel therapies. Here, we review several models-both genetic and experimentally induced-that provide investigators with an opportunity to include renal functional study end-points together with quantitative measures of fibrosis severity, something that is not possible with the UUO model.

Chimeric maternal cells in offspring do not respond to renal injury, inflammatory or repair signals.

Maternal microchimerism (MMc) can persist for years in a child, and has been implicated in the pathogenesis of chronic inflammatory autoimmune diseases. Chimeric cells may either contribute to disease by acting as immune targets or expand in response to signals of injury, inflammation or repair. We investigated the role of maternal cells in tissue injury in the absence of autoimmunity by quantifying MMc by quantitative PCR in acute and chronic models of renal injury: (1) reversible acute renal injury, inflammation and regeneration induced by rhabdomyolysis and (2) chronic injury leading to fibrosis after unilateral ureteral obstruction. We found that MMc is common in the mouse kidney. In mice congenic with their mothers neither acute nor chronic renal injury with fibrosis influenced the levels or prevalence of MMc. Maternal cells expressing MHC antigens not shared by offspring (H2(b/d)) were detected at lower levels in all groups of homozygous H2(b/b) or H2(d/d) offspring, with or without renal injury, suggesting that partial tolerance to low levels of alloantigens may regulate the homeostatic levels of maternal cells within tissues. Maternal cells homozygous for H2(b) were lost in H2(b/d) offspring only after acute renal failure, suggesting that an inflammatory stimulus led to loss of tolerance to homozygous maternal cells. The study suggests that elevated MMc previously found in association with human autoimmune diseases may not be a response to non-specific injury or inflammatory signals, but rather a primary event integral to the pathogenesis of autoimmunity.

Vitronectin accumulates in the interstitium but minimally impacts fibrogenesis in experimental chronic kidney disease.

Vitronectin (Vtn) is a glycoprotein found in normal serum and pathological extracellular matrix. Given its known interactions with plasminogen activator inhibitor-1 (PAI-1) and Vtn cellular receptors, especially αvß3 integrin and the urokinase receptor (uPAR), this study was designed to investigate its role in renal fibrogenesis in the mouse model of unilateral ureteral obstruction (UUO). Kidney Vtn mRNA levels were increased ×1.8-5.1 and Vtn protein levels ×1.9-3 on days 7, 14, and 21 after UUO compared with sham kidney levels. Groups of age-matched C57BL/6 wild-type (Vtn+/+) and Vtn-/- mice (n = 10-11/group) were killed 7, 14, or 21 days after UUO. Absence of Vtn resulted in the following significant differences, but only on day 14: fewer αSMA+ interstitial myofibroblasts (×0.53), lower procollagen III mRNA levels (×0.41), lower PAI-1 protein (×0.23), higher uPA activity (×1.1), and lower αv protein (×0.32). The number of CD68+ macrophages did not differ between the genotypes. Despite these transient differences on day 14, the absence of Vtn had no effect on fibrosis severity based on both picrosirius red-positive interstitial area and total kidney collagen measured by the hydroxyproline assay. These findings suggest that despite significant interstitial Vtn deposition in the UUO model of chronic kidney disease, its fibrogenic role is either nonessential or redundant. These data are remarkable given Vtn's strong affinity for the potent fibrogenic molecule PAI-1.

Galectin-3 preserves renal tubules and modulates extracellular matrix remodeling in progressive fibrosis.

Renal tubular cell apoptosis is a critical detrimental event that leads to chronic kidney injury in association with renal fibrosis. The present study was designed to investigate the role of galectin-3 (Gal-3), an important regulator of multiple apoptotic pathways, in chronic kidney disease induced by unilateral ureteral obstruction (UUO). After UUO, Gal-3 expression significantly increased compared with basal levels reaching a peak increase of 95-fold by day 7. Upregulated Gal-3 is predominantly tubular at early time points after UUO but shifts to interstitial cells as the injury progresses. On day 14, there was a significant increase in TdT-mediated dUTP nick end labeling-positive cells (129%) and cytochrome c release (29%), and a decrease in BrdU-positive cells (62%) in Gal-3-deficient compared with wild-type mice. The degree of renal damage was more extensive in Gal-3-deficient mice at days 14 and 21, 35 and 21% increase in total collagen, respectively. Despite more severe fibrosis, myofibroblasts were significantly decreased by 58% on day 14 in the Gal-3-deficient compared with wild-type mice. There was also a corresponding 80% decrease in extracellular matrix synthesis in Gal-3-deficient compared with wild-type mice. Endo180 is a recently recognized receptor for intracellular collagen degradation that is expressed by interstitial cells during renal fibrogenesis. Endo180 expression was significantly decreased by greater than 50% in Gal-3-deficient compared with wild-type mice. Taken together, these results suggested that Gal-3 not only protects renal tubules from chronic injury by limiting apoptosis but that it may lead to enhanced matrix remodeling and fibrosis attenuation.

CD36 regulates oxidative stress and inflammation in hypercholesterolemic CKD.

Scavenger receptors play a central role in atherosclerosis by processing oxidized lipoproteins and mediating their cellular effects. Recent studies suggested that the atherogenic state correlates with progression of chronic kidney disease (CKD); therefore, scavenger receptors are candidate mediators of renal fibrogenesis. Here, we investigated the role of CD36, a class B scavenger receptor, in a hypercholesterolemic model of CKD. We placed CD36-deficient mice and wild-type male mice on a high-fat Western diet for 7 to 8 wk and then performed either sham or unilateral ureteral obstruction surgery. CD36-deficient mice developed significantly less fibrosis compared with wild-type mice at days 3, 7, and 14 after obstruction. Compared with wild-type mice, CD36-deficient mice had significantly more interstitial macrophages at 7 d but not at 14 d. CD36-deficient mice exhibited reduced levels of activated NF-kappaB and oxidative stress (assessed by measuring fatty acid-derived hydroxyoctadecadienoic acid and protein carbonyl content) and decreased accumulation of interstitial myofibroblasts compared with wild-type mice. These data suggest that CD36 is a key modulator of proinflammatory and oxidative pathways that promote fibrogenesis in CKD.

Atherogenic scavenger receptor modulation in the tubulointerstitium in response to chronic renal injury.

Oxidized low-density lipoproteins (oxLDL) and their scavenger receptor (SR) binding partners play a central role in atherosclerosis and by analogy may play a role in chronic kidney disease pathogenesis. The present study was designed to investigate in C57BL/6 mice the effects of hypercholesterolemia on renal injury severity and oxLDL generation after unilateral ureteral obstruction (UUO). The expression profiles of CD36, SR class AI/II (SR-A), lectin-like receptor for oxidized low-density lipoprotein-1 (Lox-1), and SR that binds phosphatidylserine and oxLDL (SR-PSOX/CXCL16) were examined. Four experimental groups were studied: sham and UUO male mice on either a high-fat Western diet or a control diet. Significantly more oxLDL accumulated in the tubulointerstitium of hypercholesterolemic mice compared with normocholesterolemic mice after 14 days of UUO (P < 0.01). Total kidney collagen was significantly higher in the obstructed kidneys of hypercholesterolemic mice compared with normocholesterolemic mice on day 14 (P < 0.01). After 14 days of obstruction, the number of interstitial F4/80+ macrophages and NF-kappaB activation increased in hypercholesterolemic mice compared with normocholesterolemic mice (P < 0.01). In normal kidneys, CD36, SR-A, Lox-1, and CXCL16 were primarily localized to renal tubular epithelia. After ureteral obstruction, CD36 increased at day 7; SR-A and Lox-1 progressively decreased in a time-dependent manner; and CXCL16 increased significantly with the onset of obstruction (P < 0.01). Strong tubular expression suggests that in addition to inflammatory interstitial cells, renal tubular scavenger receptors may help to orchestrate the inflammatory and fibrogenic pathways that are activated by oxLDL.

Endogenous urokinase lacks antifibrotic activity during progressive renal injury.

Interstitial fibrosis is a universal feature of progressive kidney disease. Urokinase-type plasminogen activator (uPA) is thought to participate for several reasons: 1) uPA is produced predominantly in kidney, 2) its inhibitor plasminogen activator inhibitor-1 (PAI-1) is a strong promoter of interstitial fibrosis, whereas its receptor (uPAR) attenuates renal fibrosis, 3) uPA reduces fibrosis in liver and lung, and 4) uPA can activate hepatocyte growth factor (HGF), a potent antifibrotic growth factor. The present study tested the hypothesis that endogenous uPA reduces fibrosis severity by investigating the unilateral ureteral obstruction (UUO) model in wild-type (WT) and uPA-/- mice. Several outcomes were measured: renal collagen 3-21 days after UUO, macrophage accumulation (F4/80 Western blotting), interstitial myofibroblast density (alpha-smooth muscle actin immunostaining), and tubular injury (E-cadherin and Ksp-cadherin Western blotting). None of these measures differed significantly between WT and uPA-/- mice. uPA genetic deficiency was not associated with compensatory changes in renal uPAR mRNA levels, PAI-1 protein levels, or tissue plasminogen activator activity levels after UUO. Despite the known ability of uPA to activate latent HGF, immunoblotting failed to detect significant differences in levels of the active HGF alpha-chain and phosphorylated cMET (the activated HGF receptor) between the WT and uPA-/- groups. These findings suggest that the profibrotic actions of PAI-1 are uPA independent and that an alternative pathway must activate HGF in kidney. Finally, these results highlight a significant organ-specific difference in basic fibrogenic pathways, as enhanced uPA activity has been reported to attenuate pulmonary and hepatic fibrosis.

Plasmin(ogen) promotes renal interstitial fibrosis by promoting epithelial-to-mesenchymal transition: role of plasmin-activated signals.

Plasminogen (Plg) activator inhibitor-1 (PAI-1) is an important fibrosis-promoting molecule. Whether this effect can be attributed to PAI-1's activity as an inhibitor of plasmin generation is debated. This study was designed to investigate the role of Plg in renal fibrosis using in vivo and in vitro approaches. Plg-deficient (Plg-/-) and wild-type (Plg+/+) C57BL/6 mice were subjected to unilateral ureteral obstruction or sham surgery (n = 8/group; sham, days 3, 7, 14, and 21). Plg deficiency was confirmed by the absence of Plg mRNA, protein, and plasmin activity. After 21 d of unilateral ureteral obstruction, total kidney collagen was significantly reduced by 35% in the Plg-/- mice. Epithelial-to-mesenchymal transition (EMT), as typified by tubular loss of E-cadherin and acquisition of alpha-smooth muscle actin, was also significantly reduced in Plg-/- mice, 76% and 50%, respectively. Attenuation of EMT and fibrosis severity in the Plg-/- mice was associated with significantly lower levels of phosphorylated extracellular signal-regulated kinase (ERK) and active TGF-beta. In vitro, addition of plasmin (20 microg/ml) to cultures of murine tubular epithelial cells initiated ERK phosphorylation within minutes, followed by phenotypic transition to fibroblast-specific protein-1+, alpha-smooth muscle actin+, fibronectin-producing fibroblast-like cells. Both plasmin-induced ERK activation and EMT were significantly blocked in vitro by the protease-activated receptor-1 (PAR-1) silencing RNA; by pepducin, a specific anti-PAR-1 signaling peptide; and by the ERK kinase inhibitor UO126. Plasmin-induced ERK phosphorylation was enhanced in PAR-1-overexpressing tubular cells. These findings support important profibrotic roles for plasmin that include PAR-1-dependent ERK signaling and EMT induction.

Plasminogen activator inhibitor-1 deficiency has renal benefits but some adverse systemic consequences in diabetic mice.

BACKGROUND: Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are observed in patients with obesity, hypertension and diabetes, and several observations suggest that PAI-1 mediates diabetic vascular complications. Although increased intrarenal expression of PAI-1 is also a feature of diabetic nephropathy, evidence that PAI-1 plays a primary pathogenetic role in the renal pathology is lacking. METHODS: This study was designed to investigate the renal effects of genetic PAI-1 deficiency in db/db mice with obesity, hyperinsulinemia and hyperglycemia. For comparison the effects of PAI-1 deficiency were also examined in a cohort of mice with insulin-deficient streptozotocin (STZ)-induced diabetes. The findings are reported for 4 study groups at 8 months of age: PAI-1+/+ controls, PAI-1+/+ diabetics, PAI-1-/- controls and PAI-1-/- diabetics. RESULTS: PAI-1 deficiency had an unexpected negative impact on the db/db mice. Overall 33% of the diabetic mice died prematurely, and 63% of the db/db PAI-1-/- males had an obese body habitus but were runts. The final analyses were limited to the female db/db mice. Several nephropathy parameters were improved in the db/db PAI-1-/- group compared to the db/db PAI-1+/+ group including: albumin-to-creatinine ratios (57 +/- 45 vs. 145 +/- 71 microg/mg x10), change in glomerular extracellular matrix (ECM) area (decrease of 10% compared to controls vs. an increase of 31%) and increased total kidney collagen (47% increased vs. 96% in the PAI-1+/+ diabetics). The serum glucose levels were 15-25% lower in the PAI-1-/- nondiabetic control groups and remained lower in the db/dbPAI-1-/- mice. The STZ study was performed in males. None of the mice developed a runted phenotype or died prematurely. After diabetes of 6 months' duration changes in glomerular ECM area (-15 vs. +64%) and total kidney collagen (+8 vs. +40%) were lower in the PAI-1-/- mice compared to the PAI-1+/+ mice. The serum cholesterol levels were significantly lower in the PAI-1-/- mice, both controls (47 +/- 3 vs. 53 +/- 10 mg/dl) and diabetics (48 +/- 3 vs. 74 +/- 9 mg/dl). CONCLUSION: These data suggest a direct role for PAI-1 in renal matrix expansion and metabolic control in diabetes, but they also highlight important adverse outcomes that include male runting and premature death in mice with diabetes due to an inactive leptin receptor.

Thyroid hormone receptor isoforms localize to cardiac mitochondrial matrix with potential for binding to receptor elements on mtDNA.

Thyroid hormone (T(3)) rapidly promotes both nuclear and mitochondrial DNA transcription in cardiomyocytes, suggesting that T3 directly binds and activates mitochondrial genes. We showed for the first time mitochondrial localization for multiple TRalpha isoforms in heart, including truncated versions. Additionally, we demonstrated novel mitochondrial localization for versions of TRalpha(2), the dominant negative isoform lacking a functional ligand-binding domain. We also confirmed by electromobility shift assays, that TRalpha(2) in mitochondrial extracts binds to thyroid receptor response elements present in the 12S rRNA (DRO) and D-loop region (DR2) of mitochondrial DNA. Thus, TRalpha isoforms may directly regulate T(3) responses at mtDNA in the heart.

Thyroid hormone interacts with PPARalpha and PGC-1 during mitochondrial maturation in sheep heart.

Thyroid hormone (TH) promotes cardiac mitochondrial maturation and substrate metabolism after birth. This regulation involves ligand-dependent binding of nuclear TH receptors to target gene elements. TH also putatively controls genes indirectly by modulating transcription and/or translation of other nuclear steroid receptors and coactivators, such as peroxisome proliferator-activated receptor-alpha (PPARalpha) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1). We tested the hypothesis that TH influences PPARalpha and PGC-1 regulation of metabolic genes during postnatal maturation in sheep heart in vivo. We measured their mRNAs and/or protein levels and downstream targets in left ventricle from lambs: fetal (F), 30-day-old after postnatal thyroidectomy (THY), and 30-day-old euthyroid (Con). Both PPARalpha and PGC-1 mRNA expression decreased from F to Con, while PGC-1 protein increased substantially and PPARalpha did not change. THY limited this mRNA response and attenuated the paradoxical postnatal PGC-1 protein elevation but did not alter mRNA levels for PPARalpha, nuclear respiratory factor-1 and hypoxia-inducible factor-1alpha. THY promotion in PPARalpha mRNA did not change PPARalpha protein or mRNA for PPARalpha target genes, pyruvate-dehydrogenase kinase 4 (PDK4) and muscle type carnitine palmitoyltransferase I (mCPTI). THY reduction in PGC-1 protein occurred, while reducing cytochrome c oxidase and cytochrome c content and decreasing cardiac maximal inherent respiratory capacity. These data imply that TH modulates mitochondrial maturation partly through posttranscriptional control of PGC-1, while any important regulation of PDK4 and mCPTI by change in PPARalpha protein expression remains doubtful. Also, the paradoxical expression pattern between mRNA and protein, particularly for PGC-1, suggests a feedback control mechanism.

Multifunctionality of PAI-1 in fibrogenesis: evidence from obstructive nephropathy in PAI-1-overexpressing mice.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of chronic kidney disease based on its up-regulated expression and on the beneficial effects of PAI-1 inhibition or depletion in experimental models. PAI-1 is a multifunctional protein and the mechanisms that account for its profibrotic effects have not been fully elucidated. METHODS: The present study was designed to investigate PAI-1-dependent fibrogenic pathways by comparing the unilateral ureteral obstruction model (UUO) (days 3, 7, and 14) in PAI-1-overexpressing mice (PAI-1 tg) to wild-type mice, both on a C57BL6 background. RESULTS: Following UUO, total kidney PAI-1 mRNA and/or protein levels were significantly higher in the PAI-1 tg mice (N= 6 to 8/group) and fibrosis severity was significantly worse (days 3, 7, and 14), measured both as Sirius red-positive interstitial area (e.g., 10 +/- 3.2% vs. 4.5 +/- 1.0%) (day 14) and total kidney collagen (e.g., 11.1 +/- 1.7 vs. 6.2 +/- 1.3 microg/mg) (day 14). By day 14, the expression of two normal tubular proteins, E-cadherin and Ksp-cadherin, were significantly lower in the PAI-1 tg mice (3.2 +/- 0.5% vs. 11.7 +/- 5.9% and 2.6 +/- 1.6) vs. 6.2 +/- 0.8%, respectively), implying more extensive tubular damage. At least four fibrogenic pathways were differentially expressed in the PAI-1 tg mice. First, interstitial macrophage recruitment was more intense (P < 0.05 days 3 and 14). Second, interstitial myofibroblast density was greater (P < 0.05 days 3 and 7) despite similar numbers of proliferating tubulointerstitial cells. Third, transforming growth factor-beta1 (TGF-beta1) and collagen I mRNA were significantly higher. Finally, urokinase activity was significantly lower (P < 0.05 days 7 and 14) despite similar mRNA levels. Gene microarray studies documented that that the deletion of this single profibrotic gene had far-reaching consequences on renal cellular responses to chronic injury. CONCLUSION: These data provide further evidence that PAI-1 is directly involved in interstitial fibrosis and tubular damage via two primary overlapping mechanisms: early effects on interstitial cell recruitment and late effects associated with decreased urokinase activity.

Exogenous bone morphogenetic protein-7 fails to attenuate renal fibrosis in rats with overload proteinuria.

BACKGROUND: Bone morphogenetic protein-7 (BMP-7) plays a critical role in renal development, accelerates recovery from acute renal injury, and more recently it has been shown to delay progressive renal disease. The present study was designed to investigate the effect of BMP-7 on interstitial fibrosis in the rat protein-overloaded model. METHODS: Renal disease was induced in 26 rats by daily intraperitoneal injections of bovine serum albumin (BSA); controls (n = 28) were injected with saline. Half of the rats in each group were treated with human recombinant BMP-7 (300 microg/kg i.p. 3 times weekly) and half with placebo. Animals were killed after 3 or 6 weeks. RESULTS: Compared to the saline control groups, the BSA groups had evidence of chronic renal disease: significantly increased urinary protein excretion rates; total kidney collagen content, and increased fibronectin and collagen III interstitial areas. By 6 weeks the BSA + BMP-7 group compared to the BSA + placebo group had a nonsignificant decrease in blood urea nitrogen (40 +/- 13 vs. 46 +/- 11 mg/dl), total kidney collagen (10.8 +/- 2.1 vs. 12.2 +/- 3.5 microg/kidney), fibronectin interstitial area (23 +/- 4 vs. 25 +/- 8%) and collagen III interstitial area (22 +/- 6 vs. 28 +/- 7%). Despite these results, renal gene expression profiles actually predicted worse fibrosis in the BSA + BMP-7 group with significantly higher total kidney mRNA levels for alpha(1)(III) procollagen (2.8 +/- 0.5 vs. 1.6 +/- 0.6, p < 0.05) and fibronectin at 6 weeks (1.9 +/- 0.3 vs. 1.2 +/- 0.5, p < 0.05). Renal BMP-7 mRNA levels at 6 weeks were significantly increased in the BSA + placebo group compared to the saline + placebo group with no difference between the BSA + BMP-7 and the BSA + placebo groups. Both cortical and medullary tubules expressed BMP-7 protein but BMP-7 was only detected in the tubular lumina and urine of proteinuric animals. CONCLUSIONS: In rats with protein-overload proteinuria, renal tubules continue to express BMP-7 but some of the endogenous protein is secreted into the urinary space. Administration of exogenous recombinant BMP-7 had no effect on proteinuria but was associated with a nonsignificant trend towards less interstitial fibrosis at 6 weeks despite significantly higher kidney extracellular matrix gene mRNA levels. These findings suggest that BMP-7 treatment may have anti-fibrotic effects through enhancement of matrix turnover, although overall these effects are modest in proteinuric states in the absence of significant tubular epithelial cell apoptosis and epithelial-mesenchymal transition.

Mitogenic signaling of urokinase receptor-deficient kidney fibroblasts: actions of an alternative urokinase receptor and LDL receptor-related protein.

The urokinase receptor (uPAR) attenuates myofibroblast recruitment and fibrosis in the kidney. This study examined the role of uPAR and its co-receptor LDL receptor-related protein (LRP) in the regulation of kidney fibroblast proliferation and extracellular signal-regulated kinase (ERK) signaling. Compared with uPAR+/+ cells, uPAR-/- kidney fibroblasts were hyperproliferative. UPAR-/- fibroblast proliferation was 60% inhibited by an ERK kinase inhibitor. LRP protein was reduced and extracellular accumulation of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) proteins were greater in uPAR-/- cultures. Addition of functional uPA protein or LRP antisense RNA significantly increased ERK signaling and cell mitosis in both genotypes. Enhanced uPAR-/- fibroblast proliferation was reversed by a recombinant nonfunctional uPA peptide. The density of cell-bound fluor-uPA was similar between uPAR-/- and uPAR+/+ fibroblasts (78 +/- 6 versus 92 +/- 16 units). These data suggest that uPAR-deficient kidney fibroblasts express lower levels of its scavenger co-receptor LRP, resulting in greater extracellular accumulation of uPA and PAI-1. Enhanced proliferation of uPAR-/- fibroblasts seems to be mediated by uPA-dependent ERK signaling via an alternative urokinase receptor.