Protein degradation by the ubiquitin-proteasome pathway and organ fibrosis.

Abnormal and exaggerated deposition of extracellular matrix proteins is the common feature of fibrotic diseases. The resulting fibrosis disrupts the normal architecture of the affected organs and finally leads to their dysfunction and failure. At present, there are no effective therapies for fibrotic diseases. Protein degradation via the ubiquitin-proteasome system is the major pathway for non-lysosomal proteolysis and controls many critical cellular functions including cell-cycle progression, deoxyribonucleic acid repair, growth and differentiation. Therefore, aberration of the system leads to dysregulation of cellular homeostasis and development of many diseases such as cancers, degenerative diseases and fibrotic diseases. Although the ubiquitin-proteasome system has mainly been investigated in the field of cancers so far and several anti-cancer drugs that modulate the activity of the system have been used clinically, the recent findings regarding the system and fibrosis can provide a rational basis for the discovery of novel therapy for fibrotic diseases. In this article, we discuss (i) the basic mechanism of the ubiquitin-proteasome system and (ii) the recent findings regarding the association between the system and pathological organ fibrosis. These examples indicate that the ubiquitin-proteasome system plays diverse roles in the progression of fibrotic diseases, and further studies of the system are expected to reveal new strategies for overcoming pathological fibrosis.

The effects of cigarette exposure on rat salivary proteins and salivary glands.

OBJECTIVE: Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on saliva and salivary glands (SGs). METHODS: Cigarette smoke-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (0 day), and 15 and 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. SGs were collected on 31 days. RESULTS: The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rates did not differ at 0, 15 or 30 days after the start of CS exposure. However, the amylase and peroxidase activities and total protein content in the saliva were significantly lower in 15-day CS-exposed rats than in 15-day control rats. Histological examination of the SGs of CS-exposed rats showed vacuolar degeneration, vasodilation and hyperemia. CONCLUSION: These results suggest that CS exposure has adverse impacts on salivary composition and SGs, which could aggravate the oral environment.

Ubiquitin-dependent degradation of SnoN and Ski is increased in renal fibrosis induced by obstructive injury.

Transforming growth factor-beta (TGF-beta) plays a critical role in the progression of renal fibrosis. The activity of TGF-beta is tightly controlled by various mechanisms, among which antagonizing Smad-mediated gene transcription by co-repressors represents one of the important components. We investigated the expression, degradation, and ubiquitination of Smad transcriptional co-repressors SnoN (ski-related novel gene N) and Ski (Sloan-Kettering Institute proto-oncogene) in renal fibrogenesis. We also studied the involvement of Smad-ubiquitination regulatory factor 2 (Smurf2) in ubiquitination of SnoN protein. The kidneys of mice with unilateral ureteral obstruction (UUO) and those of sham-operated mice were used. Renal lesions and the expression of TGF-beta1, type I collagen, SnoN, Ski, and Smurf2 were examined by immunohistochemistry, Western blot, and/or real-time reverse transcriptase-polymerase chain reaction. Degradation and ubiquitination of SnoN/Ski proteins were also investigated. The obstructed kidneys of UUO mice showed progressive tubulointerstitial fibrosis, high expression levels of TGF-beta1, type I collagen, SnoN and Ski mRNAs, and low levels of SnoN and Ski proteins. Both degradation and ubiquitination of SnoN/Ski proteins were markedly increased in the obstructed kidneys, in which Smurf2 expression was increased. Smurf2 immunodepletion in extracts of obstructed kidneys resulted in reduced ubiquitination of SnoN. Our results suggest that the reduction of SnoN/Ski proteins resulting from increased ubiquitin-dependent degradation is involved in the progression of tubulointerstitial fibrosis.

Focal segmental glomerulosclerosis in a case of panhypopituitarism: a possible role of growth hormone treatment.

Panhypopituitarism manifests various symptoms including growth failure, hypothyroidism, adrenal insufficiency and hypogonadism. Dwarfism is an important problem in children with this condition, and long-term treatment with recombinant human growth hormone (GH) is usually required. We report a 24-year-old man with panhypopituitarism complicated by focal segmental glomerulosclerosis (FSGS). The patient had been treated with GH for hypopituitary dwarfism from 3 years of age. Proteinuria was initially noticed at 15 years of age and persisted despite cessation of GH supplementation at 18 years of age. A renal biopsy specimen showed glomerular hypertrophy and limited glomerulosclerosis, compatible with FSGS. To our knowledge, this is the first reported case of panhypopituitarism complicated by FSGS. Our case suggests that GH treatment for dwarfism may induce irreversible glomerular disease.

Pseudohyperkalemia occurring in a patient with chronic renal failure and polycythemia vera without severe leukocytosis or thrombocytosis.

Pseudohyperkalemia is defined as a serum potassium concentration 0.4 mEq/l greater than the plasma concentration. The basis of this phenomenon is the release of intracellular potassium from platelets, leukocytes, or erythrocytes, commonly in the setting of extreme leukocytosis (> 10 x 10(4)/microl) or thrombocytosis (> 60 x 10(4)/microl). We report a case of pseudohyperkalemia in a patient with chronic renal failure and polycythemia vera without the finding of severe leukocytosis or thrombocytosis (white blood cell count 1.88 x 10(4)/microl and platelet count 37.9 x 10(4)/microl, respectively). The serum potassium concentration was 8.2 mEq/l, while the plasma potassium level was 6.4 mEq/l in a sample obtained simultaneously. The concentrations of platelet factor IV and beta-thromboglobulin, known to be markers of platelet activation, were greater than 100 ng/ml and 200 ng/ml, respectively, indicating that platelet activation may have been related to the development of pseudohyperkalemia in this patient. These findings suggest that pseudohyperkalemia should be considered when hyperkalemia is seen in a patient with chronic renal failure and myeloproliferative disorders.

Cytokines and cell cycle regulation in the fibrous progression of crescent formation in antiglomerular basement membrane nephritis of WKY rats.

Cytokines may regulate cell proliferation by cell-cycle-regulatory proteins, in which cyclin-dependent kinase inhibitors (CDKI) inhibit cell proliferation. We investigated whether CDKI p21 or p27, both of which are potentially regulated by transforming growth factor (TGF)-beta, a key cytokine in fibrogenesis, are involved together with TGF-beta and/or platelet-derived growth factor (PDGF) in the fibrous progression of glomerular crescent formation and examined the sequential change in the cell type and the cellular background of myofibroblasts in crescent formation. Crescentic glomerulonephritis (GN) was induced by i.v. injection of rabbit antirat glomerular basement membrane antiserum in WKY rats. Animals were killed 1, 2, 3 and 4 weeks after the induction of GN, and their kidneys were processed for immunohistochemical examination. After 1 week more than 85% of glomeruli showed cellular crescents, which became fibrocellular with decreased cellularity by 4 weeks. ED 1-positive macrophages were components of crescent cells in about 44% at 1-2 weeks, and this proportion declined markedly afterwards. Alpha smooth muscle actin (alpha SMA, a marker for myofibroblasts)-positive cells were found in Bowman's epithelial cells (BEP) and in some crescent cells at 1 week, becoming major components of crescent cells by 4 weeks (about 40%). It was 2 weeks before invasion of alpha SMA-positive interstitial cells into glomeruli was evident. PDGF-B and PDGF receptor beta-positive cells, indicating possible targets for PDGF, were found in BEP adjoining crescent formation almost exclusively from 1 to 2 weeks. By contrast, both TGF-beta receptor types I- and II-positive cells, indicating possible effectors for TGF-beta, were found in BEP and crescent formation, and the percentage of these in the crescent formation did not change until 4 weeks (about 32%). Cells with positive immunostaining for proliferating cell nuclear antigen and cyclin A, markers for cell proliferation, in the crescent formation peaked in number and proportion at 1-2 weeks, then decreased. In contrast, cells with positive immunostaining for p21 and p27, CDKI, were sparse at 1 week, and then increased markedly in number and in proportion, peaking at 3 (39.6%) or 2-3 weeks (about 25-30%), respectively. The present study demonstrates that restrained expression or a transient increase in p21 and p27 may be associated with proliferation or with inhibited proliferation of crescent cells, most of which are macrophages and myofibroblasts. The action, of PDGF and TGF-beta may contribute to the recruitment of myofibroblasts into the crescent. The action of TGF-beta on crescent cells might be linked to the expression of p21 and/or p27.

Hypercalcemia in a patient with B-cell acute lymphoblastic leukemia: a role of proinflammatory cytokine.

The complication of hypercalcemia is reported to occur only in 2.5-4.8% of patients with acute lymphoblastic leukemia (ALL). We herein report a 53-year-old female patient with early B-cell ALL, complicated with extreme hypercalcemia (15.2 mg/dL). Bone X-ray revealed osteolytic changes in many locations. Serum 1,25(OH)2vitaminD3 and parathyroid hormone (PTH) levels were suppressed below normal ranges on admission. The circulating parathyroid hormone-related protein (PTHrP) value was within a normal range (< 1.1 pmol/L). Serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and soluble IL-2 receptor were increased to 72 pg/ml, 25.3 pg/ml, and 1469 U/ml, respectively. Following the induction chemotherapy, the serum calcium level was promptly normalized accompanied with decreases in serum TNF-alpha, IL-6 and soluble IL-2 receptor values to 34 pg/ml, 6.35 pg/ml, and 737 U/ml, respectively. Serum PTHrP values remained within detectable levels. To our knowledge, this is the first case of B-cell ALL in a patient who developed hypercalcemia with elevated concentrations of TNF-alpha, IL-6, and soluble IL-2 receptor, but not related to PTHrP. High circulating proinflammatory cytokines may have contributed to development of ALL-induced osteolysis and hypercalcemia in the present case.

Tuberculous arthritis mimicking neoplasm in a hemodialysis patient.

Hemodialysis patients are known to develop the complication of extrapulmonary tuberculosis more frequently than the general population. Tuberculous arthritis is a rare form of extrapulmonary tuberculosis and is reported to occur in approximately 1% of cases in nonuremic patients. Only 3 cases in dialysis patients, who were not proven by a bacterial culture or had died before treatment, have been reported. We report herein a culture-proven case of tuberculous arthritis developing at the sternoclavicular joint, which initially mimicked an apparent neoplasm in a hemodialysis patient. A favorable outcome was obtained after antituberculous therapy. Tuberculosis must be considered one of the most significant diagnoses in hemodialysis patients who present with a tumor-like lesion.

New insights on the pathogenesis of hypercalcemia in primary hyperparathyroidism.

The pathogenesis of hypercalcemia in primary hyperparathyroidism is attributed to increased calcium release from bone, increased calcium reabsorption in renal distal tubules, and increased intestinal calcium absorption. However, it remains unclear which factor is the main process. We encountered a 56-year-old woman with myasthenia gravis, in whom hypercalcemia and elevated serum parathyroid hormone (PTH) level were observed. Diagnosis of primary hyperparathyroidism was made. Treatment with methylprednisolone for myasthenia gravis was associated with a marked decrease in both biochemical markers of bone formation and resorption without any changes in endogenous cAMP and serum levels of calcium, PTH, and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. These findings suggest that the possible pathogenesis of hypercalcemia in primary hyperparathyroidism may be attributed to the increased calcium reabsorption in the kidney and the increased intestinal calcium absorption as a result of stimulated production of 1,25-(OH)2D3 in the kidney. It thus follows that the renal tubular effect rather than the skeletal effect of the PTH excess may play a pivotal role in the development of hypercalcemia in primary hyperparathyroidism.

Vitamin D deficiency is implicated in reduced serum albumin concentrations in patients with end-stage renal disease.

The mortality rate in hemodialysis patients remains extremely high, and reduced serum albumin concentration resulting from malnutrition is the strongest predictor of mortality and morbidity. Several inflammatory cytokines involved in malnutrition, including interleukin-1, interleukin-6, and tumor necrosis factor-alpha, are modulated by 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], of which synthesis is impaired in end-stage renal disease. We evaluated whether 1,25-(OH)(2)D(3) deficiency might be involved in reduced serum albumin concentrations. Fifty-one predialysis uremic patients about to begin hemodialysis therapy were divided into groups with serum 1,25-(OH)(2)D(3) concentrations less than 18 pg/mL (low-D(3) group; n = 39) and concentrations of 18 pg/mL or greater (normal-D(3) group; n = 12). Serum albumin concentrations before the initiation of hemodialysis treatment were compared between the two groups. Furthermore, the effect of supplementation with active forms of vitamin D during 4 months of hemodialysis treatment on serum albumin concentrations was retrospectively evaluated in the low-D(3) group. Serum albumin concentrations in the low-D(3) group were significantly less than those in the normal-D(3) group (3.58 +/- 0. 50 versus 3.82 +/- 0.10 g/dL; P = 0.034). Considering all patients, a significant positive correlation between serum concentrations of albumin and 1,25-(OH)(2)D(3) was noted (r = 0.417; P = 0.0023). Supplementation with active forms of vitamin D significantly increased serum albumin concentrations in the low-D(3) group from 3. 61 +/- 0.12 to 3.79 +/- 0.13 g/dL (P = 0.0067). These findings indicate that reductions in serum albumin concentrations may be attributed, at least in part, to vitamin D deficiency in patients with end-stage renal disease.

Atypical Fabry's disease presenting with cholesterol crystal embolization.

We describe a 65-year-old man who presented with pulmonary hemorrhage and progressive renal insufficiency three months after resection surgery for an abdominal aortic aneurysm. Intensive treatment with corticosteroids and hemodialysis were not effective, and the patient died. Postmortem examination of the kidneys revealed widespread cholesterol clefts within the renal arterioles and a number of lamellar inclusion bodies were observed by electron microscopy. The diagnosis of Fabry's disease was made by the absence of plasma alpha-galactosidase A activity. This was a very rare case of subclinical Fabry's disease coexistent with cholesterol crystal embolization, mimicking pulmonary-renal syndrome.

A case with acute renal failure complicated by Waldenström's macroglobulinemia and cryoglobulinemia.

We encountered a 53-year-old man associated with acute renal failure caused by Waldenström's macroglobulinemia and type I cryoglobulinemia. Treatment with prednisolone and cyclophosphamide induced a rapid recovery from acute renal failure. Renal histology revealed endocapillary proliferation and lobular formation with scattered subendothelial, amorphous and periodic acid-Schiff (PAS)-positive materials in the glomerular capillaries which were positive for IgM on immunofluorescence study. Although the exact mechanism for pathophysiology of acute renal failure remains unknown, treatment with prednisolone and cyclophosphamide could induce a rapid recovery from acute renal failure accompanied by Waldenström's macroglobulinemia and type I cryoglobulinemia.

Role of apoptosis in uranyl acetate-induced acute renal failure and acquired resistance to uranyl acetate.

BACKGROUND: We have previously reported that animals recovering from uranyl acetate (UA)-induced acute renal failure (ARF) were resistant to subsequent insult. Recent evidence suggests that apoptosis participates in tubular damage. We investigated the role of apoptosis in UA-induced ARF and attenuation of ARF in acquired resistance to UA in rats. METHODS: ARF was induced by an intravenous injection of UA (5 mg/kg) in rats. Rats of group 1 were injected with UA and followed for 28 days. Group 2 rats were injected with a second dose of UA (5 mg/kg) 14 days after the first injection and were followed for 14 days. All rats received an intraperitoneal injection of bromodeoxyuridine (BrdU) one hour before sacrifice. Using kidneys, histologic examination and immunohistochemical detection of proliferating cell nuclear antigen (PCNA), BrdU, Bcl-2, and Bax were performed. To detect apoptosis, electron microscopy, analysis of DNA fragmentation, and the TUNEL methods were adopted. RESULTS: UA increased the number of damaged renal tubules and serum creatinine, which peaked at 5 days in group 1, but both returned to baseline values by 14 days. Apoptosis was confirmed by electron microscopy and the "ladder" pattern of DNA fragments on gel electrophoresis. The number of apoptotic tubular cells evaluated by the TUNEL method showed two peaks at days 5 and 14 in group 1. The second peak of TUNEL-positive cells was preceded by an increased number of BrdU-positive nuclei, PCNA-positive nuclei, and total number of tubular epithelial cells. Renal damage after the second UA injection was markedly reduced. The peak number of apoptotic cells in group 2 was significantly less than that in group 1. CONCLUSIONS: Two peak levels of apoptotic cells occurred in UA-induced ARF. The first peak might play a role in UA-induced tubular damage, while the second one might represent the removal of excess regenerating cells during the recovery phase. Modulation of apoptotic cell death might be involved in the acquired resistance to rechallenge injury by UA.

Immunoelectron microscopic study on type I, II and III TGF-beta receptors on visceral glomerular epithelial cells in relation to glomerular basement membrane alterations in proteinuric rats.

BACKGROUND: Transforming growth factor (TGF)-beta is a regulator of extracellular matrix accumulation. Both TGF-beta receptors, type I (TbetaRI) and type II (TbetaRII), may be required for signal transduction in the TGF-beta pathway. The aim of this study was to investigate the relationship between the TGF-beta pathways and glomerular basement membrane (GBM) accumulation in vivo. METHODS: We examined TbetaRI, II, and III protein expression on visceral glomerular epithelial cells (GEP) in relation to GBM alterations in passive Heymann nephritis (PHN), anti-GBM nephritis and anti-thymocyte serum (ATS) nephritis. Renal tissues were examined by pre-embedding immunoelectron microscopy 3, 7 and 14 days after induction of nephritis in rats. RESULTS: In normal control rats TbetaRI was not detected on GEP, TbetaRII expression was very occasionally found on GEP and TbetaRIII was seen in the cytoplasm of the GEP. TbetaRI, TbetaRII, and TbetaRIII were constitutively expressed on glomerular endothelial cells. By day 3 of anti-GBM nephritis and PHN, expression of TbetaRI, TbetaRII, and TbetaRIII was still similar to that of normal control rats, and GBM alterations in both models were not prominent except for deposit formation in PHN. From day 7 onwards, in both models, expression of TbetaRI and TbetaRII on GEP increased in association with GBM thickening. Expression of TbetaRIII in the cytoplasm of the GEP was increased, with occasional positive staining being seen on the urinary surface of the GEP from day 7 onwards. On the other hand, at day 3 of ATS nephritis, increased expression of TbetaRI and TbetaRII on GEP was noted, but from day 7 onwards, expression of TbetaR II on GEP dramatically decreased. Expression of TbetaRIII in the cytoplasm of the GEP also transiently increased at day 3. GBM thickening was not noted in ATS nephritis. CONCLUSIONS: The results suggest that persistent upregulation of expression of TbetaRI, TbetaRII and possibly TbetaRIII on GEP may contribute to GBM matrix accumulation in vivo.

Attenuation of cisplatin-induced acute renal failure is associated with less apoptotic cell death.

To clarify the pathophysiologic role of apoptosis in acute renal failure (ARF), we examined whether the attenuation of cisplatin-induced ARF is associated with the change in the degree of apoptotic cell death. The administration of cisplatin (CDDP) (6 mg/kg body weight) in rats induced ARF at day 5, as manifested by a significant increase in serum creatinine (Scr) and tubular damage. CDDP-induced apoptotic cell death was confirmed by electron microscopic examination, agarose gel electrophoresis, and increased cells positive for TaT-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) in the outer medulla of the kidney. Treatment with dimethylthiourea (DMTU)--a scavenger of hydroxyl radicals--or glycine abrogated CDDP-induced increases in Scr, the tubular damage score, and the number of TUNEL-positive cells. Pretreatment with uranyl acetate (UA) induced a significant expression of Bcl-2 in the kidney and ameliorated CDDP-induced increases in Scr, the tubular damage score, and TUNEL-positive cells in the outer stripe of the outer medulla. Our findings indicate (1) that the attenuation of CDDP-induced ARF was associated with less apoptotic cell death and (2) that the induction of the anti-apoptotic protein Bcl-2 attenuated apoptosis and tubular damage. Our results suggest that apoptotic cell death may play an important role in the development of cisplatin-induced ARF.

A patient with thrombotic microangiopathy accompanied by glomerular subendothelial electron dense deposits.

A 50-year-old woman presented with thrombocytopenia, microangiopathic hemolytic anemia, and nephrotic syndrome. Although a high level of circulating immune complexes, mild hypocomplementemia, and the antinuclear antibody also were present, the criteria for collagen disease were not fulfilled. Renal biopsy demonstrated a typical thrombotic microangiopathy (TMA) involving glomeruli. There also were electron-dense deposits located just beneath the original glomerular basement membrane in the dilated subendothelial space, thought to be immune complexes following positive IgG, C1q and C3 staining on an immunofluorescent study. Corticosteroid therapy rapidly ameliorated her hematologic abnormalities and proteinuria and normalized the immunologic data. These findings strongly suggest that the TMA in this patient was induced by immune complex-associated mechanisms.

Successful treatment of invasive thoracopulmonary mucormycosis in a patient with acute lymphoblastic leukemia.

BACKGROUND: Pulmonary mucormycosis associated with hematologic malignancy is an uncommon, but important opportunistic fungal pneumonia that is usually a fatal infection. Only a few survivors of pulmonary mucormycosis have been reported. METHODS: A case report of invasive thoracopulmonary mucormycosis during remission-induction therapy for acute lymphoblastic leukemia and a review of the literature are presented. RESULTS: The fungal lesions extended to both lungs, the left ribs, and intercostal muscles. Percutaneous needle biopsy and immunostaining of the fungal hyphae established the diagnosis of thoracopulmonary mucormycosis. The patient was treated with granulocyte-colony stimulating factor (G-CSF) and intravenous amphotericin B for 9 weeks and the lesions in the right lung disappeared. Left pneumonectomy and partial resection of the chest wall were later performed. The left lung was grossly necrotic and contained a large cavity and bronchopulmonary fistula. Thereafter, antileukemic therapy was resumed and completed without recurrence of mucormycosis or leukemia. CONCLUSIONS: In the management of mucormycosis, the addition of G-CSF to the conventional treatment may substantially improve outcome.

Light chain nephropathy with remarkable accumulation of multinucleated giant cells in the kidney.

A case of light chain deposition disease with multinucleated giant cell accumulation in the kidney is described. A 54-year-old man was admitted to out hospital due to moderate renal failure. Complicated with eosinophilic pneumonia two months after admission, his renal function abruptly deteriorated and hemodialysis was started. Three-day methylprednisolone pulse therapy, however, partially recovered his renal function and hemodialysis was discontinued. His renal biopsy specimen revealed kappa light chain deposition disease with nodular mesangial expansion and subendothelial electron dense deposits. The most characteristic finding in this patient was accumulation of foreign body type multinucleated giant cells around atrophic tubules, small arteries and obsolescent glomeruli, probably associated with light chain deposition. No increase in kappa light chain was detected in his serum or concentrated urine. Such disseminated giant cell reaction, other than intra-luminal infiltration in the tubules, has not been reported in the literature and might be related to the physicochemical or structural properties of the deposited protein.