Michael S. Forbes

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.

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-β, 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.

Cytochemical staining procedures selective for sarcotubular systems of muscle: Modifications and applications

The diaminobenzidine (DAB)-ferrocyanide cytochemical procedure for staining of myocardial sarcoplasmic reticulum (SR), first described by Waugh and his co-workers, has been subjected to further experimentation and modification. We have found, on the basis of studies utilizing inhibitory substances and conditions, that the staining reaction in the SR apparently is not attributable to endogenous peroxidase activity within the lumina of the SR tubules and saccules (nor, for that matter, to the activity of any species of enzyme). Perfusion of the vascular system with DAB-H2O2-Tris solution is unnecessary. In fact, exposure of tissues to DAB itself is superfluous, since mere postfixation in osmium—ferrocyanide solution serves to produce SR staining in muscle cells that have been fixed in an aldehyde solution containing calcium (or other divalent or trivalent cations), but lacking phosphate. This technique is applicable to both cardiac and skeletal muscle cells of mammals and lower vertebrates; it has been applied with success to mammalian vascular and nonvascular smooth muscle as well. Some degree of filling (or staining) of the system of extracellular spaces [ECS, including, in mammalian myocardium, the T-axial tubular system (TAx)] often occurs, which appears to be the result of colloid formation by the osmium ferrocyanide. SR staining in skeletal muscle usually is inseparable from T-system filling, although filling of T tubules may occur in the absence of SR staining. Choice of concentration or type of aldehyde(s) in the fixative solution does not appreciably alter the staining; however, exposure of tissues to aldehyde fixative which either does not contain calcium or contains phosphate brings about filling of the ECS-TAx without SR staining, thereby mimicking in effect the results obtained by more complicated tracer techniques.

Co-involvement of mitochondria and endoplasmic reticulum in regulation of apoptosis: changes in cytochrome c, Bcl-2 and Bax in the hippocampus of aluminum-treated rabbits.

Neurodegenerative diseases, including Alzheimers disease, are characterized by a progressive and selective loss of neurons. Apoptosis under mitochondrial control has been implicated in this neuronal death process, involving the release of cytochrome c into the cytoplasm and initiation of the apoptosis cascade. However, a growing body of evidence suggests an active role for the endoplasmic reticulum in regulating apoptosis, either independent of mitochondrial, or in concert with mitochondrial-initiated pathways. Members of the Bcl-2 family of proteins have been shown to either inhibit apoptosis, as is the case with Bcl-2, or to promote it, in the case of Bax. Investigations in our laboratory have focused on neuronal injury resulting from the intracisternal administration of aluminum maltolate to New Zealand white rabbits, an animal system relevant to a study of human disease in that it reflects many of the histological and biochemical changes associated with Alzheimers disease. Here we report that treatment of young adult rabbits with aluminum maltolate induces both cytochrome c translocation into brain cytosol, and caspase-3 activation. Furthermore, as assessed by Western blot analysis, these effects are accompanied by a decrease in Bcl-2 and an increase in Bax reactivity in the endoplasmic reticulum.

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 Bowmans 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 Bowmans 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.

AB(1-42) and aluminum induce stress in the endoplasmic reticulum in rabbit hippocampus, involving nuclear translocation of gadd 153 and NF-kB

Abstract Apoptosis may represent a prominent form of neuronal death in chronic neurodegenerative disorders, such as Alzheimer’s disease. Although apoptosis under mitochondrial control has received considerable attention, mechanisms used within the endoplasmic reticulum (ER) and nucleus in mediating apoptotic signals are not well understood. A growing body of evidence is emerging from different studies which suggests an active role for the ER in regulating apoptosis. Disturbances of ER function have been shown to trigger two different apoptotic pathways; one involves cross-talk with mitochondria and is regulated by the antiapoptotic Bcl-2, and the second is characterized by the activation of caspase-12. Also, stress in the ER has been suggested to result in the activation of a number of proteins, such as gadd 153 and NF-κ, and in the downregulation of the antiapoptotic protein, Bcl-2. In the present study, the intracisternal injection in aged rabbits of either the neurotoxin aluminum maltolate or of Aβ(1-42), has been found to induce nuclear translocation of gadd 153 and the inducible transcription factor, NF-κB. Translocation of these two proteins is accompanied by decreased levels of Bcl-2 in both the ER and the nucleus. Aluminum maltolate, but not Aβ, induces caspase-12 activation which is a mediator of ER-specific apoptosis; this is the first report of the in vivo activation of caspase-12. These findings indicate that the ER may play a role in regulating apoptosis in vivo, and could be of significance in the pathology of neurodegeneration and related disorders.

Ultrastructure of lizard ventricular muscle

The ultrastructure of the ventricular myocardium of the lizard Anolis carolinensis was examined using two aldehyde fixatives, including glutaraldehyde. The lanthanum hydroxide method of Revel and Karnovsky also was used to trace those spaces continuous with the interstitial space. The extracellular gap at the intercalated discs varies from about 150 to 800 A. Desmosomes are present at the discs and at lateral cell borders. However, semitight or gap junctions are only rarely observed at the intercalated discs, lanthanum colloid readily penetrating throughout the entire intercalated disc cleft. Longitudinally running structures are seen, continuous with the interstitial space as evidenced by lanthanum penetration, which may be narrow intercellular clefts at regions close to discs. The numerous micropinocytotic vesicles which occur in association with the sarcolemma also become filled with lanthanum. Transverse tubules are not observed, and lanthanum does not penetrate intracellularly. However, the sacroplasmic reticulum is well developed. Two types of close neuromuscular junctions are observed with respect to the synaptic vesicles: those containing larger diameter vesicles with electron-dense cores and those containing smaller vesicles with transparent cores. Cross sections of myofibrils reveal that 7 to 10 thin filaments surround each thick filament. In conclusion, semitight junctions are essentially absent at the intercalated discs and T tubules are lacking in the myocardial cells of this animal.

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.

Membrane systems in skeletal muscle of the lizard Anolis carolinensis

The iliotibialis muscle of the iguanid lizard Anolis carolinensis is composed of fibers that display highly developed membrane systems. One of these systems is composed of the so-called “transverse” tubules, which may branch in both the horizontal and vertical planes and which usually are connected to the surface sarcolemma via intermediary passages composed of caveolar vesicles. The remaining membrane system comprises the various elements of the sarcoplasmic reticulum (SR). Three reasonably distinct yet confluent conformations of SR can be found: (1) highly developed “conventional” SR which surrounds each myofibril and is segmented into specialized regions according to the divisions of the sarcomeres, such as junctional SR at the A–I levels, “network” SR massed over the I/Z/I band complexes, and fenestrated collars at the M lines; (2) subsarcolemmal SR whose junctional SR cisternae form peripheral couplings with the inner surface of the external cell membrane; and (3) extensive masses of proliferated SR that form roughly cylindrical bodies oriented longitudinally within intermyofibrillar spaces. These last (“columnar SR”) are found at all depths of the fibers and may be collected into less discrete aggregates that lie just underneath the sarcolemma, including the soleplate regions of motor end plates. The degree of development of the Anolis skeletal muscle membrane systems may reflect the requirements for rapid movement of the fibers.