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Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.

Angiotensin II (Ang II) activates at least two receptors, AT1 and AT2, with the majority of its effects-such as vasoconstriction, inflammation, and matrix deposition-mediated by the AT1 receptor. It is thought that the AT2 receptor counteracts these processes; however, recent studies have found proinflammatory and hypertrophic effects of this receptor subtype. To identify the physiological roles of the AT2 receptor in chronic kidney disease, we performed renal ablation in AT2 receptor knockout and wild-type mice. Renal injury caused a greater impairment of renal function, glomerular injury, albuminuria, and mortality in the knockout mice than in the wild-type mice. There was increased fibronectin expression and inflammation in the knockout mice, as shown by augmented monocyte/macrophage infiltration and higher chemokine monocyte chemotactic protein-1 (MCP-1) and RANTES expression in the remnant kidney. The higher mortality and renal morbidity of the knockout mice was not due to differences in systemic blood pressure, glomerular volume, AT1 receptor, renin, or endothelial nitric oxide synthase expression. Whether activation of the AT2 receptor will have therapeutic benefit in chronic kidney disease will require further study.

The design and operation of a separation unit based on frontal chromatography is described. The important feature of the design is the array column → detector → column which allows process monitoring below the detection limits of the monitor. By using “fraternal twin columns” an in-process calibration of the detector is achieved reducing the waste production. The design contains provisions for on-line destructive and non-destructive monitoring. Tests of the unit prove its versatility with respect to decontamination processes. Due to its compactness, the unit is transportable, if not portable, and the module construction allows easy posting and set-up in areas with restricted access. The unit is capable of processing up to 5 m 3 solution per year depending on the chemical system used.

A frontal chromatographic unit was devised consisting of a column-detector-column array. The unit is either equipped with identical columns (identical twins) or with columns of varying length (fraternal twins). Due to the finite nature of the columns, a prerun is formed at the column walls following the same regularities as the main stream. These regularities are used for the identification of the process termination below the detection limits of the monitor. For the implementation, a clear preference is given to the employment of fraternal twins, as the feed assay can be integrated into the separation process.

Angiotensin II (ANG II) inhibits proliferation and induces differentiation through AT2 receptors. However, target genes involved in this process are not well characterized. We studied PC12 cells, a rat pheochromocytoma cell line exclusively expressing AT2 receptors. ANG II attenuated proliferation of PC12 cells without concomitant induction of apoptosis. To identify potential novel genes involved in the antimitogenic actions of ANG II, we performed differential display analysis of PC12 cells after challenge with 10(-7) M ANG II for 6 hours. One identified gene selected for further study that was down-regulated by ANG II in PC12 cells was SM-20. This gene has been previously isolated from vascular smooth muscle cells treated with mitogens by differential hybridization. Recent findings show a homology of SM-20 with enzymes involved in the regulation of hypoxia inducible factor 1. ANG II suppressed mRNA expression of SM-20 in PC12 cells after only 30 minutes, as detected by Northern blotting. This effect was antagonized by an AT2 receptor blocker, but not by losartan. A rabbit polyclonal antibody was generated against a peptide sequence of SM-20 and detected a major band of the predicted size of 40 kd and a second 33-kd band that likely represents a processed form present in mitochondria. Immunohistochemistry revealed a granular staining of the cytoplasm of PC12 cells compatible with a previously described mitochondrial localization of SM-20 protein. Western blots confirmed the down-regulation of SM-20 protein in PC12 cells subsequent to incubation with ANG II. SM-20 transcripts were also reduced by ANG II acting on AT2 receptors in rat glomerular endothelial cells that express both AT1 and AT2 receptors. SM-20 antisense, but not sense, phosphothioate-modified oligonucleotides reduced base-line proliferation of PC12 cells. In contrast, inducible overexpression of SM-20 using the ecdysone system prevented the antiproliferative effects of ANG II in PC12 cells. In summary, our study identified SM-20 as an essential component of ANG II's growth-suppressive effects mediated through AT2 receptors. This gene apparently plays an important role in the regulatory processes determining whether a cell should undergo differentiation, apoptosis, or proliferation.

Systemic hypertension is a major risk factor that determines the rate of progression of kidney disease. The underlying mechanisms, however, are incompletely understood. To gain insight into these mechanisms, the present study was undertaken to characterize the effects of renovascular hypertension on the course of anti-thymocyte antibody-induced glomerulonephritis. Glomerulonephritis was induced in rats 6 weeks after the initiation of two-kidney, one-clip hypertension, when blood pressure was already increased. Structure and function of the clipped and the nonclipped kidney were examined 5 days later. Glomerular filtration rate (GFR) was measured by inulin clearance. The induction of nephritis did not alter the blood pressure in either hypertensive rats or normotensive controls. Albuminuria increased slightly in normotensive rats after the induction of nephritis, whereas no significant differences were found between hypertensive rats with or without nephritis. No significant differences were found for the GFR values of normotensive controls and nephritic animals or for values in the clipped kidney with or without nephritis. However, the GFR of the nonclipped kidney was significantly reduced in nephritic animals as compared with all other groups. Morphologic evaluation revealed that hypertensive rats with nephritis exhibited a combination of characteristics of nephritis and hypertensive glomerular injury. Histologic findings of nephritis, such as glomerular binding of rabbit IgG and glomerular proliferation and mesangial matrix expansion, were similar after the induction of nephritis in controls and in the clipped and nonclipped kidneys of hypertensive animals. However, intraglomerular microaneurysms were significantly more often found in the nonclipped kidneys after the induction of nephritis. Hypertension-induced deterioration of glomerular function was not associated with marked morphologic deterioration but rather with a combination of the characteristics of nephritis and hypertensive glomerular injury. (J Lab Clin Med 1999;134:292-303)

Monocyte chemoattractant protein-1 mediates collagen deposition in experimental glomerulonephritis by transforming growth factor-βbgr;. Background Monocyte chemoattractant protein-1 (MCP-1) plays a significant role in the recruitment of monocytes/macrophages in experimental glomerulonephritis (GN). Because recent evidence points to possible profibrogenic effects of leukocyte-derived factors in GN, this study was designed to evaluate the role of the chemokine MCP-1 in the fibrogenesis of experimental GN. Methods Rats with an anti-thy-1–induced GN were treated with a neutralizing antiserum against MCP-1. Glomerular collagen type IV, as a marker of glomerular matrix deposition, was assessed by Northern and Western blotting and immunohistology. Transforming growth factor-βbgr; (TGF-βbgr;), an important mediator of this matrix expansion, was studied by Northern and Western blotting. Results The induction of GN resulted in a significant increase of glomerular collagen type IV deposition and TGF-βbgr; synthesis. The neutralization of MCP-1 significantly reduced the enhanced collagen type IV protein synthesis and deposition without affecting collagen mRNA expression. However, both the enhanced transcription and protein synthesis of TGF-βbgr; were inhibited by anti–MCP-1 antiserum in nephritic animals. Conclusions In this model of GN, MCP-1 has a fibrogenic effect through the stimulation of TGF-βbgr;. MCP-1 is thus not only important for the recruitment of inflammatory cells, but also mediates glomerular matrix accumulation.

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C. Background Angiotensin II infusion has been shown to cause hypertension and endothelial dysfunction and to increase superoxide (O -· 2 ) production in vascular tissue, mainly via an activation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidase, the most significant O -· 2 source in endothelial and/or smooth muscle cells. With these studies, we sought to determine whether endothelial dysfunction in renovascular hypertension is secondary to an activation of these oxidases. Methods Endothelial function in aortas from rats with two kidney-one clip (2K-1C) hypertension and age-matched controls was assessed using isometric tension studies in organ chambers. Changes in vascular O -· 2 production were measured using lucigenin-enhanced chemiluminescence and electron spin resonance spectroscopy. Results In hypertensive animals, relaxation to endothelium-dependent (acetylcholine) and endothelium-independent nitrovasodilators (nitroglycerin) was impaired. Constriction to a direct activator of protein kinase C (PKC) phorbol ester 12,13 dibutyrate (PDBu) was enhanced, and vascular O -· 2 was significantly increased compared with controls. Vascular O -· 2 was normalized by the PKC inhibitor calphostin C, by the inhibitor of flavin-dependent oxidases, diphenylene iodonium, and recombinant heparin-binding superoxide dismutase, whereas inhibitors of the xanthine oxidase (oxypurinol), nitric oxide synthase (N G -nitro-L-arginine) and mitochondrial NADH dehydrogenase (rotenone) were ineffective. Studies of vascular homogenates demonstrated that the major source of O -· 2 was a NAD(P)H-dependent oxidase. Incubation of intact tissue with PDBu markedly increased O -· 2 , the increase being significantly stronger in vessels from hypertensive animals as compared with vessels from controls. Endothelial dysfunction was improved by preincubation of vascular tissue with superoxide dismutase and calphostin C. Conclusions We therefore conclude that renovascular hypertension in 2K-1C rats is associated with increased vascular O -· 2 leading to impaired vasodilator responses to endogenous and exogenous nitrovasodilators. Increased vascular O -· 2 is likely secondary to a PKC-mediated activation of a membrane-associated NAD(P)H-dependent oxidase.