Hypoxia-induced changes in protein s-nitrosylation in female mouse brainstem.

Exposure to hypoxia elicits an increase in minute ventilation that diminishes during continued exposure (roll-off). Brainstem N-methyl-D-aspartate receptors (NMDARs) and neuronal nitric oxide synthase (nNOS) contribute to the initial hypoxia-induced increases in minute ventilation. Roll-off is regulated by platelet-derived growth factor receptor-ß (PDGFR-ß) and S-nitrosoglutathione (GSNO) reductase (GSNOR). S-nitrosylation inhibits activities of NMDAR and nNOS, but enhances GSNOR activity. The importance of S-nitrosylation in the hypoxic ventilatory response is unknown. This study confirms that ventilatory roll-off is virtually absent in female GSNOR(+/-) and GSNO(-/-) mice, and evaluated the location of GSNOR in female mouse brainstem, and temporal changes in GSNOR activity, protein expression, and S-nitrosylation status of GSNOR, NMDAR (1, 2A, 2B), nNOS, and PDGFR-ß during hypoxic challenge. GSNOR-positive neurons were present throughout the brainstem, including the nucleus tractus solitarius. Protein abundances for GSNOR, nNOS, all NMDAR subunits and PDGFR-ß were not altered by hypoxia. GSNOR activity and S-nitrosylation status temporally increased with hypoxia. In addition, nNOS S-nitrosylation increased with 3 and 15 minutes of hypoxia. Changes in NMDAR S-nitrosylation were detected in NMDAR 2B at 15 minutes of hypoxia. No hypoxia-induced changes in PDGFR-ß S-nitrosylation were detected. However, PDGFR-ß phosphorylation increased in the brainstems of wild-type mice during hypoxic exposure (consistent with roll-off), whereas it did not rise in GSNOR(+/-) mice (consistent with lack of roll-off). These data suggest that: (1) S-nitrosylation events regulate hypoxic ventilatory response; (2) increases in S-nitrosylation of NMDAR 2B, nNOS, and GSNOR may contribute to ventilatory roll-off; and (3) GSNOR regulates PDGFR-ß phosphorylation.

The swan-neck lesion: proximal tubular adaptation to oxidative stress in nephropathic cystinosis.

Cystinosis is an inherited disorder resulting from a mutation in the CTNS gene, causing progressive proximal tubular cell flattening, the so-called swan-neck lesion (SNL), and eventual renal failure. To determine the role of oxidative stress in cystinosis, histologic sections of kidneys from C57BL/6 Ctns(-/-) and wild-type mice were examined by immunohistochemistry and morphometry from 1 wk to 20 mo of age. Additional mice were treated from 1 to 6 mo with vehicle or mitoquinone (MitoQ), an antioxidant targeted to mitochondria. The leading edge of the SNL lost mitochondria and superoxide production, and became surrounded by a thickened tubular basement membrane. Progression of the SNL as determined by staining with lectin from Lotus tetragonolobus accelerated after 3 mo, but was delayed by treatment with MitoQ (38 ± 4% vs. 28 ± 1%, P < 0.01). Through 9 mo, glomeruli had retained renin staining and intact macula densa, whereas SNL expressed transgelin, an actin-binding protein, but neither kidney injury molecule-1 (KIM-1) nor cell death was observed. After 9 mo, clusters of proximal tubules exhibited localized oxidative stress (4-hydroxynonenal binding), expressed KIM-1, and underwent apoptosis, leading to the formation of atubular glomeruli and accumulation of interstitial collagen. We conclude that nephron integrity is initially maintained in the Ctns(-/-) mouse by adaptive flattening of cells of the SNL through loss of mitochondria, upregulation of transgelin, and thickened basement membrane. This adaptation ultimately fails in adulthood, with proximal tubular disruption, formation of atubular glomeruli, and renal failure. Antioxidant treatment targeted to mitochondria delays initiation of the SNL, and may provide therapeutic benefit in children with cystinosis.

Tubular obstruction leads to progressive proximal tubular injury and atubular glomeruli in polycystic kidney disease.

In polycystic kidney disease (PKD), renal parenchyma is destroyed by cysts, hypothesized to obstruct nephrons. A signature of unilateral ureteral obstruction, proximal tubular atrophy leads to formation of atubular glomeruli. To determine whether this process occurs in PKD, kidneys from pcy mice (moderately progressive PKD), kidneys from cpk mice (rapidly progressive PKD), and human autosomal dominant PKD were examined in early and late stages. Integrity of the glomerulotubular junction and proximal tubular mass were determined in sections stained with Lotus tetragonolobus lectin. Development of proximal tubular atrophy and atubular glomeruli was determined in serial sections of individual glomeruli. In pcy mice, most glomerulotubular junctions were normal at 20 weeks, but by 30 weeks, 56% were atrophic and 25% of glomeruli were atubular; glomerulotubular junction integrity decreased with increasing cyst area (r = 0.83, P < 0.05). In cpk mice, all glomerulotubular junctions were normal at 10 days, but by 19 days, 26% had become abnormal. In early-stage autosomal dominant PKD kidneys, 50% of glomeruli were atubular or attached to atrophic tubules; in advanced disease, 100% were abnormal. Thus, proximal tubular injury in cystic kidneys closely parallels that observed with ureteral obstruction. These findings support the hypothesis that, in renal cystic disorders, cyst-dependent obstruction of medullary and cortical tubules initiates a process culminating in widespread destruction of proximal convoluted tubules at the glomerulotubular junction.

The Fate of Nephrons in Congenital Obstructive Nephropathy: Adult Recovery is Limited by Nephron Number Despite Early Release of Obstruction.

PURPOSE: Urinary tract obstruction and reduced nephron number often occur together as a result of maldevelopment of the kidneys and the urinary tract. We determined the role of nephron number on adaptation of the remaining nephrons of mice subjected to neonatal partial unilateral ureteral obstruction followed through adulthood. MATERIALS AND METHODS: Wild-type and Os/+ mice (the latter with 50% fewer nephrons) underwent sham operation or partial unilateral ureteral obstruction in the first 2 days of life. Additional mice underwent release of unilateral ureteral obstruction at 7 days. All kidneys were harvested at 3 weeks (weaning) or 6 weeks (adulthood). Glomerular number and area, glomerulotubular junction integrity, proximal tubular volume fraction and interstitial fibrosis were measured by histomorphometry. RESULTS: In the obstructed kidney unilateral ureteral obstruction caused additional nephron loss in Os/+ but not in wild-type mice. Glomerular growth from 3 to 6 weeks was impaired by ipsilateral obstruction and not preserved by release in wild-type or Os/+ mice. Proximal tubular growth was impaired and interstitial collagen was increased by ipsilateral obstruction in all mice. These conditions were attenuated by release of unilateral ureteral obstruction in wild-type mice but were not restored in Os/+ mice. Unilateral ureteral obstruction increased interstitial collagen in the contralateral kidney while release of obstruction enhanced tubular growth and reduced interstitial collagen. CONCLUSIONS: Unilateral ureteral obstruction in early postnatal development impairs adaptation to reduced nephron number and induces additional nephron loss despite release of obstruction. Premature and low birth weight infants with congenital obstructive nephropathy are likely at increased risk for progression of chronic kidney disease.

MRI-based glomerular morphology and pathology in whole human kidneys.

Nephron number (N(glom)) and size (V(glom)) are correlated with risk for chronic cardiovascular and kidney disease and may be predictive of renal allograft viability. Unfortunately, there are no techniques to assess N(glom) and V(glom) in intact kidneys. This work demonstrates the use of cationized ferritin (CF) as a magnetic resonance imaging (MRI) contrast agent to measure N(glom) and V(glom) in viable human kidneys donated to science. The kidneys were obtained from patients with varying levels of cardiovascular and renal disease. CF was intravenously injected into three viable human kidneys. A fourth control kidney was perfused with saline. After fixation, immunofluorescence and electron microscopy confirmed binding of CF to the glomerulus. The intact kidneys were imaged with three-dimensional MRI and CF-labeled glomeruli appeared as punctate spots. Custom software identified, counted, and measured the apparent volumes of CF-labeled glomeruli, with an ~6% false positive rate. These measurements were comparable to stereological estimates. The MRI-based technique yielded a novel whole kidney distribution of glomerular volumes. Histopathology demonstrated that the distribution of CF-labeled glomeruli may be predictive of glomerular and vascular disease. Variations in CF distribution were quantified using image texture analyses, which be a useful marker of glomerular sclerosis. This is the first report of direct measurement of glomerular number and volume in intact human kidneys.

Responses of proximal tubular cells to injury in congenital renal disease: fight or flight.

Most chronic kidney disease in children results from congenital or inherited disorders, which can be studied in mouse models. Following 2 weeks of unilateral ureteral obstruction (UUO) in the adult mouse, nephron loss is due to proximal tubular mitochondrial injury and cell death. In neonatal mice, proximal tubular cell death is delayed beyond 2 weeks of complete UUO, and release of partial UUO allows remodeling of remaining nephrons. Progressive cyst expansion develops in polycystic kidney disease (PKD), a common inherited renal disorder. The polycystic kidney and fibrosis (pcy)-mutant mouse (which develops late-onset PKD) develops thinning of the glomerulotubular junction in parallel with growth of cysts in adulthood. Renal insufficiency in nephropathic cystinosis, a rare inherited renal disorder, results from progressive tubular cystine accumulation. In the Ctns knockout mouse (a model of cystinosis), proximal tubular cells become flattened, with loss of mitochondria and thickening of tubular basement membrane. In each model, persistent obstructive or metabolic stress leads ultimately to the formation of atubular glomeruli. The initial "fight" response (proximal tubular survival) switches to a "flight" response (proximal tubular cell death) with ongoing oxidative injury and mitochondrial damage. Therapies should be directed at reducing proximal tubular mitochondrial oxidative injury to enhance repair and regeneration.

Transforming growth factor-ß1 receptor inhibition preserves glomerulotubular integrity during ureteral obstruction in adults but worsens injury in neonatal mice.

Unilateral ureteral obstruction (UUO), a widely used model of chronic kidney disease and congenital obstructive uropathy, causes proximal tubular injury and formation of atubular glomeruli. Because transforming growth factor-ß1 (TGF-ß1) is a central regulator of renal injury, neonatal and adult mice were subjected to complete UUO while under general anesthesia and treated with vehicle or ALK5 TGF-ß1 receptor inhibitor (IN-1130, 30 mg·kg(-1)·day(-1)). After 14 days, glomerulotubular integrity and proximal tubular mass were determined by morphometry of Lotus tetragonolobus lectin distribution, and the fraction of atubular glomeruli was determined by serial section analysis of randomly selected individual glomeruli. Glomerular area, macrophage infiltration, fibronectin distribution, and interstitial collagen were measured by morphometry. Compared with placebo, inhibition of TGF-ß1 by IN-1130 decreased apoptosis and formation of atubular glomeruli, prevented parenchymal loss, increased glomerular area and glomerulotubular integrity, and increased proximal tubule fraction of the adult obstructed kidney parenchyma from 17 to 30% (P < 0.05, respectively). IN-1130 decreased macrophage infiltration and fibronectin and collagen deposition in the adult obstructed kidney by ∼50% (P < 0.05, respectively). In contrast to these salutary effects in the adult, IN-1130 caused widespread necrosis in obstructed neonatal kidneys. We conclude that whereas IN-1130 reduces obstructive injury in adult kidneys through preservation of glomerulotubular integrity and proximal tubular mass, TGF-ß1 inhibition aggravates obstructive injury in neonates. These results indicate that while caution is necessary in treating congenital uropathies, ALK5 inhibitors may prevent nephron loss due to adult kidney disease.

Chronic unilateral ureteral obstruction in the neonatal mouse delays maturation of both kidneys and leads to late formation of atubular glomeruli.

Unilateral ureteral obstruction (UUO) in the adult mouse is the most widely used model of progressive renal disease: the proximal tubule is the nephron segment most severely affected and atubular glomeruli are formed after only 7 days of UUO. To determine the proximal nephron response to UUO in the maturing kidney, neonatal mice were examined 7 to 28 days following complete UUO under general anesthesia. Proximal tubular mass and maturation were determined by staining with Lotus tetragolonobus lectin. Superoxide was localized by nitroblue tetrazolium and collagen by Sirius red. Cell proliferation, cell death, PAX-2, megalin, α-smooth muscle actin (α-SMA), renin, and fibronectin were identified by immunohistochemistry. During the first 14 days of ipsilateral UUO, despite oxidative stress (4-hydroxynonenal staining), glomerulotubular continuity was maintained and mitochondrial superoxide production persisted. However, from 14 to 28 days, papillary growth was impaired and proximal tubules collapsed with increased apoptosis, autophagy, mitochondrial loss, and formation of atubular glomeruli. Fibronectin, α-SMA, and collagen increased in the obstructed kidney. Oxidative stress was present also in the contralateral kidney: renin was decreased, glomerulotubular maturation and papillary growth were delayed, followed by increased cortical and medullary growth. We conclude that neonatal UUO initially delays renal maturation and results in oxidative stress in both kidneys. In contrast to the adult, proximal tubular injury in the neonatal obstructed kidney is delayed at 14 days, followed only later by the formation of atubular glomeruli. Antioxidant therapies directed at proximal tubular mitochondria during early renal maturation may slow progression of congenital obstructive nephropathy.

Fight-or-flight: murine unilateral ureteral obstruction causes extensive proximal tubular degeneration, collecting duct dilatation, and minimal fibrosis.

Unilateral ureteral obstruction (UUO) is the most widely used animal model of progressive renal disease. Although renal interstitial fibrosis is commonly used as an end point, recent studies reveal that obstructive injury to the glomerulotubular junction leads to the formation of atubular glomeruli. To quantitate the effects of UUO on the remainder of the nephron, renal tubular and interstitial responses were characterized in mice 7 and 14 days after UUO or sham operation under anesthesia. Fractional proximal tubular mass, cell proliferation, and cell death were measured by morphometry. Superoxide formation was identified by nitro blue tetrazolium, and oxidant injury was localized by 4-hydroxynonenol and 8-hydroxydeoxyguanosine. Fractional areas of renal vasculature, interstitial collagen, α-smooth muscle actin, and fibronectin were also measured. After 14 days of UUO, the obstructed kidney loses 19% of parenchymal mass, with a 65% reduction in proximal tubular mass. Superoxide formation is localized to proximal tubules, which undergo oxidant injury, apoptosis, necrosis, and autophagy, with widespread mitochondrial loss, resulting in tubular collapse. In contrast, mitosis and apoptosis increase in dilated collecting ducts, which remain patent through epithelial cell remodeling. Relative vascular volume fraction does not change, and interstitial matrix components do not exceed 15% of total volume fraction of the obstructed kidney. These unique proximal and distal nephron cellular responses reflect differential "fight-or-flight" responses to obstructive injury and provide earlier indexes of renal injury than do interstitial compartment responses. Therapies to prevent or retard progression of renal disease should include targeting proximal tubule injury as well as interstitial fibrosis.

Proximal tubular injury and rapid formation of atubular glomeruli in mice with unilateral ureteral obstruction: a new look at an old model.

Unilateral ureteral obstruction (UUO), employed extensively as a model of progressive renal interstitial fibrosis, results in rapid parenchymal deterioration. Atubular glomeruli are formed in many renal disorders, but their identification has been limited by labor-intensive available techniques. The formation of atubular glomeruli was therefore investigated in adult male mice subjected to complete UUO under general anesthesia. In this species, the urinary pole of Bowman's capsule is normally lined by tall parietal epithelial cells similar to those of the proximal tubule, and both avidly bind Lotus tetragonolobus lectin. Following UUO, these cells became flattened, lost their affinity for Lotus lectin, and no longer generated superoxide (revealed by nitroblue tetrazolium infusion). Based on Lotus lectin staining, stereological measurements, and serial section analysis, over 80% of glomeruli underwent marked transformation after 14 days of UUO. The glomerulotubular junction became stenotic and atrophic due to cell death by apoptosis and autophagy, with concomitant remodeling of Bowman's capsule to form atubular glomeruli. In this degenerative process, transformed epithelial cells sealing the urinary pole expressed α-smooth muscle actin, vimentin, and nestin. Although atubular glomeruli remained perfused, renin immunostaining was markedly increased along afferent arterioles, and associated maculae densae disappeared. Numerous progressive kidney disorders, including diabetic nephropathy, are characterized by the formation of atubular glomeruli. The rapidity with which glomerulotubular junctions degenerate, coupled with Lotus lectin as a marker of glomerular integrity, points to new investigative uses for the model of murine UUO focusing on mechanisms of epithelial cell injury and remodeling in addition to fibrogenesis.

Formation of atubular glomeruli in the developing kidney following chronic urinary tract obstruction.

Congenital urinary tract obstruction is a major cause of progressive renal disease in children. We developed a model of partial unilateral ureteral obstruction (UUO) in the neonatal mouse, in which nephrogenesis at birth is similar to that of the midtrimester human fetus. The proximal tubule responds to UUO by undergoing apoptosis and necrosis, likely due to mitochondrial sensitivity to hypoxia and reactive oxygen species in the face of reduced endogenous antiapoptotic factors such as eNOS. Damage to the glomerulotubular junction is followed by scission and formation of atubular glomeruli and aglomerular tubules. This is an orchestrated process, with atubular glomeruli surrounded by a continuous layer of regenerated parietal epithelial cells. Relief of UUO at 7 days of age results in remodeling of the renal parenchyma by adulthood. In contrast to proximal tubular destruction, collecting ducts remain dilated and patent, with remodeling due to apoptosis and proliferation (a process associated with recruitment of intercalated cells as progenitor cells following UUO in the fetal monkey). Formation of atubular glomeruli occurs in other renal disorders (congenital nephrotic syndrome and cystinosis), and may represent a maladaptive response to proximal tubular injury reflecting an evolutionary adaptation by an ancestor we share with aglomerular marine fish.

Inducible nitric oxide synthase modulates hydronephrosis following partial or complete unilateral ureteral obstruction in the neonatal mouse.

To investigate the role of endogenous inducible nitric oxide synthase (iNOS) in the response of the developing kidney to unilateral ureteral obstruction (UUO), neonatal iNOS null mutant (-/-) and wild-type (WT) mice were subjected to partial or complete UUO. At 7 and 21 days of age, apoptosis, renin, vascular endothelial growth factor (VEGF), fibroblasts (anti-fibroblast-specific peptide 1), myofibroblasts (alpha-smooth muscle actin), macrophages (F4/80), and collagen were measured in kidney tissue. Compared with WT, renal parenchymal thickness was increased, with preservation of the papilla, in -/- mice with partial UUO, but decreased in -/- mice with complete UUO. Ureteral peristalsis increased with severity of pelvic dilatation in WT, and increased further in -/- mice with partial UUO. Apoptosis, fibroblasts, and macrophages were increased in -/- mice with complete UUO, but there was no effect of iNOS on other histological parameters following complete UUO. Renin was decreased in -/- mice with partial UUO. There was no effect of iNOS genotype on renal collagen accumulation at either 7 or 21 days of age. These results are consistent with an injurious role for endogenous iNOS following partial UUO by inhibiting ureteral peristalsis and increasing renal renin although renal fibrosis is not affected. In contrast, in mice with complete UUO, iNOS attenuates apoptosis and enhances renal parenchymal thickness. Alterations in the severity of ureteral obstruction may therefore influence the effect of iNOS on long-term renal injury.

S-nitrosothiols signal hypoxia-mimetic vascular pathology.

NO transfer reactions between protein and peptide cysteines have been proposed to represent regulated signaling processes. We used the pharmaceutical antioxidant N-acetylcysteine (NAC) as a bait reactant to measure NO transfer reactions in blood and to study the vascular effects of these reactions in vivo. NAC was converted to S-nitroso-N-acetylcysteine (SNOAC), decreasing erythrocytic S-nitrosothiol content, both during whole-blood deoxygenation ex vivo and during a 3-week protocol in which mice received high-dose NAC in vivo. Strikingly, the NAC-treated mice developed pulmonary arterial hypertension (PAH) that mimicked the effects of chronic hypoxia. Moreover, systemic SNOAC administration recapitulated effects of both NAC and hypoxia. eNOS-deficient mice were protected from the effects of NAC but not SNOAC, suggesting that conversion of NAC to SNOAC was necessary for the development of PAH. These data reveal an unanticipated adverse effect of chronic NAC administration and introduce a new animal model of PAH. Moreover, evidence that conversion of NAC to SNOAC during blood deoxygenation is necessary for the development of PAH in this model challenges conventional views of oxygen sensing and of NO signaling.

Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy.

Congenital obstructive nephropathy accounts for the greatest fraction of chronic kidney disease in children. Genetic and nongenetic factors responsible for the lesions are largely unidentified, and attention has been focused on minimizing obstructive renal injury and optimizing long-term outcomes. The cellular and molecular events responsible for obstructive injury to the developing kidney have been elucidated from animal models. These have revealed nephron loss through cellular phenotypic transition and cell death, leading to the formation of atubular glomeruli and tubular atrophy. Altered renal expression of growth factors and cytokines, including angiotensin, transforming growth factor-beta, and adhesion molecules, modulate cell death by apoptosis or phenotypic transition of glomerular, tubular, and vascular cells. Mediators of cellular injury include hypoxia, ischemia, and reactive oxygen species, while fibroblasts undergo myofibroblast transformation with increased deposition of extracellular matrix. Progression of the lesions involves interstitial inflammation and interstitial fibrosis, both of which impair growth of the obstructed kidney and result in compensatory growth of the contralateral kidney. The long-term outcome depends on timing and severity of the obstruction and its relief, minimizing ongoing injury, and enhancing remodeling. Advances will depend on new biomarkers to evaluate the severity of obstruction, to determine therapy, and to follow the evolution of lesions.

Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy.

Renal fibrosis is the hallmark of progressive renal disease of virtually any etiology. The model of unilateral ureteral obstruction (UUO) in the rodent generates progressive renal fibrosis. Surgically created UUO can be experimentally manipulated with respect to timing, severity, and duration, while reversal of the obstruction permits the study of recovery. The use of genetically engineered mice has greatly expanded the utility of the model in studying molecular mechanisms underlying the renal response to UUO. Ureteral obstruction results in marked renal hemodynamic and metabolic changes, followed by tubular injury and cell death by apoptosis or necrosis, with interstitial macrophage infiltration. Proliferation of interstitial fibroblasts with myofibroblast transformation leads to excess deposition of the extracellular matrix and renal fibrosis. Phenotypic transition of resident renal tubular cells, endothelial cells, and pericytes has also been implicated in this process. Technical aspects of the UUO model are discussed in this review, including the importance of rodent species or strain, the age of the animal, surgical procedures, and histological methods. The UUO model is likely to reveal useful biomarkers of progression of renal disease, as well as new therapies, which are desperately needed to allow intervention before the establishment of irreversible renal injury.

Generation and evolution of atubular glomeruli in the progression of renal disorders.

Functional nephrons can be lost through a process of glomerulotubular disconnection. Progressive development of atubular glomeruli seems to play a major role in a number of renal disorders, including glomerular diseases, ascribed to injury to the glomerulotubular junction as a result of proteinuria; however, formation of atubular glomeruli is even more common in tubulointerstitial disorders, such as obstructive nephropathy. Toxic nephropathy is also associated with the formation of atubular glomeruli, suggesting susceptibility of the glomerulotubular junction to toxic injury. Narrowing or other abnormalities of the glomerulotubular junction are described as precursors of glomerulotubular disconnection. Cystinosis represents a dramatic example of progressive injury to the glomerulotubular junction, with formation of the "swan-neck deformity" following degenerative tubular cell changes attributable to apoptosis. Significant numbers of atubular glomeruli have been reported in chronic pyelonephritis and renal allograft rejection; this suggests interstitial inflammation as a stimulus for the formation of atubular glomeruli. Because of difficulties in morphologic recognition, it is likely that glomerulotubular disconnection is an underappreciated mechanism in the progression of renal disease. A better understanding of the vulnerability of the glomerulotubular junction and its protection from injury should lead to better strategies for preserving renal function in many nephropathies.

A2A adenosine receptor agonist and PDE4 inhibition delays inflammation but fails to reduce injury in experimental obstructive nephropathy.

BACKGROUND: Renal interstitial inflammation is a consequence of unilateral ureteral obstruction (UUO). Following ischemia/reperfusion, adenosine reduces renal inflammation and injury, effects which are potentiated by type 4 phosphodiesterase inhibitors. We therefore studied the effects of A2A adenosine receptor agonist (ATL146e), and PDE4 inhibitor (rolipram) in mice subjected to UUO. METHODS: Mice were subjected to UUO or sham operation, and received either vehicle or ATL146e + rolipram by osmotic minipump for 1 or 7 days. At 1, 3, 7, or 14 days after operation, renal macrophage infiltration, apoptosis, proliferation, tubular atrophy, and interstitial fibrosis were quantitated, and expressions of IL-6 and TGF-beta mRNA were determined. RESULTS: ATL146e + rolipram reduced macrophage infiltration by 40% after 3 days UUO (p < 0.05). Tubular apoptosis, tubular atrophy, and interstitial fibrosis were increased by 7 or 14 days UUO, but were unaffected by ATL146e + rolipram. However, cellular proliferation was increased by ATL146e + rolipram in the obstructed kidney. ATL146e + rolipram had no effect on the renal expression of IL-6 and TGF-beta mRNA. CONCLUSIONS: A2A receptor activation and PDE4 inhibition transiently reduce renal macrophage infiltration, but do not ameliorate the renal response to UUO. We speculate that the persistent stimulus for inflammation triggered by UUO cannot be reversed by agents that suppress inflammatory cell activation alone.

Aluminum-induced dendritic pathology revisited: cytochemical and electron microscopic studies of rabbit cortical pyramidal neurons.

Intracisternal administration of aluminum maltolate induces biochemical and histological changes in the rabbit brain. The primary histological response to this aluminum intoxication is the appearance within many neuronal somata and dendrites of intensely argyrophilic masses of fibrillar material. Ultrastructural examination of these bodies in both conventionally-prepared and silver-stained sections shows them to be composed of neurofilaments. For this reason, we have elected to call these argyrophilic masses "neurofilamentous arrays (NFAs)." At their zenith, NFAs in cortical pyramidal neurons comprise thousands of filaments interconnected by periodic crossbridges. NFAs begin to be formed within both somata and dendrites as isolated groups of neurofilaments, which apparently go on to assemble en masse within the cytoplasm. In symptomatic animals, many cortical neurons are rich in NFAs, yet lack classic cytological signs of degeneration, such as nuclear pyknosis. Though silver staining reveals extensive NFAs only in aluminum-exposed brains, there is a strong degree of immunostaining for phosphorylated neurofilamentous epitopes in both untreated and Al-injected animals. This suggests that protein subunits that are already present in the neurons under normal circumstances are recruited, in the presence of aluminum, to form NFAs through the directed assembly of masses of oriented filaments.

Renal vascular endothelial growth factor in neonatal obstructive nephropathy. I. Endogenous VEGF.

Obstructive nephropathy constitutes a major cause of renal impairment in children. Chronic unilateral ureteral obstruction (UUO) impairs maturation of the developing kidney and leads to tubular apoptosis and interstitial inflammation. Vascular endothelial growth factor (VEGF) is involved in recovery from various forms of renal injury. We questioned whether the renal expression of endogenous VEGF and its receptor (VEGFR2/Flk-1) is modified by UUO in early development. Neonatal rats were subjected to partial or complete UUO or sham operation. The distribution of immunoreactive VEGF in each kidney was examined after 7, 14, or 28 days. Adult rats were also subjected to sham operation or complete UUO. Tubular VEGF increased between 14 and 28 days in sham-operated rats and in some partially obstructed neonatal rats but decreased with complete UUO. Parallel changes were found by Western blotting, but not by RT-PCR. Immunoreactive VEGF colocalized with mitochondria in proximal and distal tubules and also appeared in type A intercalated cells, glomerular vascular endothelium, and podocytes. While neonatal microvascular renal VEGFR2 receptor staining was strongly positive regardless of UUO, staining was weak in sham-operated adults but increased following UUO. Parallel changes in VEGFR2 expression were verified by RT-PCR and Western blotting. We conclude that endogenous renal VEGF is developmentally regulated in the neonatal rat and is differentially regulated by partial and complete UUO. Following UUO in the adult, the VEGF receptor is upregulated. Endogenous VEGF may serve an adaptive role in responding to tubular injury caused by UUO and may modulate adaptation by the contralateral kidney.

Lack of endothelial nitric-oxide synthase leads to progressive focal renal injury.

Because endothelial nitric-oxide synthase (eNOS) is generally considered protective against renal injury, we examined eNOS knockout mice for kidney pathology. In 80% of the adults examined, the renal surface was marked by distinct indented scars containing crowded small glomeruli but lacking attached tubules. Although vasculature was intact in the scars, Bowman's space was dilated and glomerular tufts were degenerated. The atubular glomeruli were embedded in a dense interstitial matrix composed of cells positive for fibroblast (FSP-1) or macrophage (F4/80) markers, degenerated proximal tubules and collecting ducts, and diffuse fibrotic deposits. Surrounding regions of kidney contained mostly normal-appearing tubules, but enlarged or sclerotic glomeruli were also present. In neonatal animals, apoptosis and necrosis were concentrated in tubules within focal parenchymal zones, with narrowing of the glomerulotubular "neck." In summary, targeted deletion of eNOS in mice leads to progressive focal renal abnormalities, including glomerular hypoplasia, and tubular cell death, leading to separation of glomeruli from tubules and tubular disruption. These abnormalities begin developing during the normal up-regulation of eNOS in the maturing kidney and are similar to those of a variety of chronic renal disorders. Endogenous renal eNOS production therefore seems critical for the maintenance of nephron maturation and integrity.