Acquired resistance to rechallenge injury in rats that recovered from mild renal damage induced by uranyl acetate: accelerated proliferation and hepatocyte growth factor/c-Met axis.

BACKGROUND: Rats that recovered from mild proximal tubule (PT) injury without renal dysfunction by subtoxic insult, developed partial resistance to subsequent nephrotoxic insult. This partial resistance was associated with reduced renal dysfunction and accelerated PT cell proliferation compared with vehicle treatment as the first insult. Here we assessed the role and potential mechanisms of accelerated PT proliferation in this acquired resistance model. METHODS: Rats at 14 days after recovering from prior mild renal damage induced by 0.2 mg/kg uranyl acetate (UA) (subtoxic dose) were rechallenged with 4 mg/kg UA (nephrotoxic dose) to establish the acquired resistance model. Cell cycle was inhibited by colchicine to examine the contribution of accelerated PT cell proliferation evaluated by in vivo bromodeoxyuridine (BrdU) labeling on acquired resistance to subsequent nephrotoxic insult. Hepatocyte growth factor (HGF)/c-Met axis and other related factors of cell cycle were analyzed. RESULTS: The acquired resistance to rechallenge injury with nephrotoxic dose of UA in rats recovered from mild renal injury was associated with an earlier increase in BrdU-positive PT cells, accelerated upregulation of HGF mRNA, c-Met mRNA/protein, cyclin D1, phospho-Rb and an earlier phenotypic change of PT cells. Colchicine inhibited PT cell proliferation, reduced the upregulated cyclin D1 and phospho-Rb in the kidney, completely abolishing acquired resistance. CONCLUSIONS: Cell cycle progression with upregulated renal HGF/c-Met axis may contribute to the accelerated recovery from acute renal failure in rats that recovered from prior mild renal damage, followed by nephrotoxic insult, resulting in partial acquired resistance.

A case with significant proteinuria caused by secreted protein from urothelial carcinoma.

58-year-old female was admitted to our hospital complaining isolated proteinuria of 1.7 g/day. Abdominal echography showed right-sided unilateral hydronephrosis, and computed tomography pointed out a tumor of the right renal pelvis, suggesting cancer of renal pelvis. The right nephroureterectomy was carried out. Pathological diagnosis was urothelial carcinoma. Renal tissue revealed no apparent glomerulopathy with tubular atrophy, interstitial fibrosis, and mildly-to-moderately interstitial mononuclear cell infiltration. Immunofluorescence study showed no deposition of immunoreactanct, and electron microscopy showed almost normal glomerulus without electron dense deposit. Proteinuria disappeared within 6 days after the operation. Moderate amount of proteinuria in our patient was probably caused by secreted protein from urothelial carcinoma. This condition is rare but should be taken into account in patients with even moderate amount of proteinuria.

Siblings with Alport's syndrome showing unique staining patterns for alpha5(IV) and alpha6(IV) chains of collagen type IV.

Here we report two brothers with electron-microscopically diagnosed Alport's syndrome (AS) who showed normal staining patterns for the alpha1(IV)-alpha4(IV) chains of collagen type IV, but abnormal expression of the alpha5(IV) and alpha6(IV) chains. Both patients had microscopic hematuria and mild proteinuria from around 10 years old, and had renal biopsies at 23 (older) and 26 (younger) years old due to increased proteinuria (0.5-0.8 g/day) with normal renal function. A skin biopsy of the patients' mother showed similar abnormal staining patterns for the alpha5(IV) and alpha6(IV) chains in the skin basement membranes. Both of them showed slow progression of renal dysfunction and no extrarenal manifestations. The existences of incomplete alpha3,alpha4,alpha5(IV) molecules in the glomerular basement membrane (GBM) and inadequately formed alpha5,alpha5,alpha6(IV) molecules are suggested for these patients. A missense mutation of the COL4A5 gene may present in this family as possible X-linked inheritance and a mild form of AS.

Acquired resistance to rechallenge injury in rats recovered from subclinical renal damage with uranyl acetate--Importance of proliferative activity of tubular cells.

Animals recovered from acute renal failure are resistant to subsequent insult. We investigated whether rats recovered from mild proximal tubule (PT) injury without renal dysfunction (subclinical renal damage) acquire the same resistance. Rats 14 days after recovering from subclinical renal damage, which was induced by 0.2 mg/kg of uranyl acetate (UA) (sub-toxic dose), were rechallenged with 4 mg/kg of UA (nephrotoxic dose). Fate of PT cells and renal function were examined in response to nephrotoxic dose of UA. All divided cells after sub-toxic dose of UA insult were labeled with bromodeoxyuridine (BrdU) for 14 days then the number of PT cells with or without BrdU-labeling was counted following nephrotoxic dose of UA insult. Rats recovered from subclinical renal damage gained resistance to nephrotoxic dose of UA with reduced renal dysfunction, less severity of peak damage (necrotic and TUNEL+ apoptotic cells) and accelerated PT cell proliferation, but with earlier peak of PT damage. The decrease in number of PT cells in the early phase of rechallenge injury with nephrotoxic UA was more in rats pretreated with sub-toxic dose of UA than vehicle pretreated rats. The exaggerated loss of PT cells was mainly caused by the exaggerated loss of BrdU+ divided cells. In contrast, accelerated cell proliferation in rats recovered from sub-toxic dose of UA was observed mainly in BrdU- non-divided cells. The findings suggest that rats recovered from subclinical renal damage showed partial acquired resistance to nephrotoxic insult. Accelerated recovery with increased proliferative activity of non-divided PT cells after subclinical renal damage may mainly contribute to acquired resistance.

Rapid deterioration of renal function in a patient with multiple myeloma associated with amyloid and light chain depositions.

Here we report a 75-year-old man with multiple myeloma who developed acute deterioration of renal function. Systemic AL amyloid deposition was found in the stomach, duodenum and brachial artery. A small amount of proteinuria without significant abnormal urinary sediments, increased excretion of urinary low-molecular-weight proteins and Bence Jones protein were observed. Significant renal Ga-67 uptake suggested acute tubulointerstitial lesions. Renal necropsy after sudden death 40 days after introduction of hemodialysis revealed mesangial expansion with glomerular basement membrane thickening, tubular basement membrane thickening with or without tubular atrophy and massive tubulointerstitial fibrosis. Slight amyloid depositions in the mesangium and vessels, and massive granular electron-dense deposits and deposition of monoclonal light chain lambda in renal basement membranes and vessels were found, indicating the rare condition of coexistence of amyloidosis and light chain deposition disease(LCDD). The rapid progression of renal failure may have been caused by massive deposition of monoclonal light chains in our patient.

Cell division and phenotypic regression of proximal tubular cells in response to uranyl acetate insult in rats.

BACKGROUND: We examined whether dedifferentiation is necessary for cell division of proximal tubule (PT) cells after acute PT injury. METHODS: Rats were injected with a low (0.2 mg/kg) or high (4 mg/kg) dose of uranyl acetate (UA) to induce acute PT injury. Proliferating PT cells were labelled with bromodeoxyuridine (BrdU) before sacrifice. Renal tissues were examined by double labelling of BrdU and megalin, aquaporin 1 (AQP1), Na(+)-K(+)ATPase or vimentin, and by immunoelectron microscopy for BrdU+ cells. RESULTS: Under normal conditions, BrdU+ PT cells were positive for the PT phenotype (megalin-, AQP1- and Na(+)-K(+)ATPase positive and vimentine negative, a mesenchymal marker). Low-dose UA induced focal PT injury, and BrdU+ initially proliferating PT cells were found in the proximal three quarters of the S3 segment of nephron as early as 12 h, which maintained the PT phenotype and were vimentin negative. Proliferating PT cells showed low expression of the PT cell protein phenotype from Day 2 to Day 5 with vimentin expression from Day 2. High-dose UA induced severe PT injury in the proximal three quarters of the S3 segment by Day 5. BrdU+ initially proliferating PT cells, which were found in distal areas of the S3 segment as early as Day 2, showed low expression of the PT protein phenotype but were vimentin positive. Immunoelectron microscopy showed mature PT morphology for BrdU+ PT cells in control rats. BrdU+ initially proliferating PT cells showed a relatively mature phenotype after low-dose UA in- sult but an immature phenotype after high-dose UA insult. CONCLUSIONS: PT cells can initiate cell division without de- differentiation after mild PT injury by low-dose UA insult.

Analysis of intra-GBM microstructures in a SLE case with glomerulopathy associated with podocytic infolding.

BACKGROUND: Systemically podocytic infolding into the GBM which causes nonargyrophilic holes in the GBM in association with intra-GBM microstructures has been considered as a new pathological entity. However, its pathomechanisms are largely unknown. METHODS: We analyzed intra-GBM microstructures in an SLE patient with glomerulopathy associated with podocytic infolding by immunoelectron microscopy for vimentin (a marker for both podocyte and endothelium) and C5b-9 and by 3D reconstruction of transmission electron microscopy (TEM) images by computer tomography method. RESULTS: Immunofluorescent study showed immunoglobulin deposition in a diffuse, capillary pattern; however, electron-dense deposits like stage 3 membranous nephropathy could be found only in some capillary loops by TEM in spite of the systemic existence of podocytic infolding and the intra-GBM microstructures. Three-dimensional reconstructed images of the TEM images revealed that some of the intra-GBM microstructures made connections with the podocyte. The clustered microstructures underneath the podocyte and their surroundings looked as a whole like the degraded part of podocyte in 3D reconstructed images. Immunoelectron microscopy showed that vimentin was positive in most intra-GBM microstructures. C5b-9 was positive along the entire epithelial side of the GBM and in some microstructures, suggesting that the podocytes may be attacked by C5b-9 and that the microstructures may contain C5b-9 bound cellular membranes. CONCLUSION: Intra-GBM microstructures may be originated mainly from the podocyte. Podotyte and GBM injuries caused by C5b-9 attack to podocytes might contribute in part to podocytic infolding and intra-GBM microstructures in this case.

A distinct population of tubular cells in the distal S3 segment contributes to S3 segment regeneration in rats following acute renal failure induced by uranyl acetate.

BACKGROUND: We previously reported that early regenerating cells found at the distal area of the S3 segment of the nephron (designated as target cells) were label-retaining cells in uranyl acetate (UA)-induced acute renal failure (ARF) in rats. In this study, we examined the contribution of these target cells to S3 segment repair after UA treatment. We also discriminated target cells from bromodeoxyuridine (BrdU)-label-retaining cells labeled under normal conditions and examined their capacity to proliferate following a second insult and their resistance to 5-fluorouracil (5-FU). METHODS: Target cells were labeled and tracked using a (3)H-thymidine pulse/chase approach after UA (4 mg/kg) injection to rats. Normal rats were labeled with BrdU, and cells positive for BrdU and Ki67 were analyzed after UA treatment. The kinetics of target cells was examined after a second dose of UA and treatment with 5-FU. RESULTS: The target cells were strongly labeled by (3)H-thymidine and were predominantly found in the distal quarter of the S3 segment until 40 weeks after generating 'label-diluted' cells throughout the S3 segment. Cells labeled with BrdU under normal conditions did not express Ki67 after UA treatment but target cells were Ki67-positive. The target cells underwent further proliferation following the second treatment with UA and were transiently arrested by 5-FU treatment at G0/G1 after UA. CONCLUSIONS: The target cells are slow-cycling cells, resistant to 5-FU treatment and have the capacity to undergo further proliferation following a second insult with UA. These data suggest the presence of a distinct population of cells that can regenerate the S3 segment in UA-induced ARF.

Association of serum adiponectin levels with all-cause mortality in hemodialysis patients.

OBJECTIVE: Adiponectin (ADPN) has been shown to protect against cardiovascular disease for the general population with problematic metabolic syndrome. However, it remains unclear whether ADPN is associated with mortality in patients on maintenance hemodialysis (HD). METHODS, PATIENTS OR MATERIALS: We selected 85 HD patients [51 men/34 women; mean age, 64+/-2 years; underlying kidney diseases, diabetic nephropathy in 36 patients (42.3%), chronic glomerulonephritis in 29 (34.1%), hypertensive nephrosclerosis in 10 (11.8%), and others in 10 (11.8%)] who survived for more than 3 months after the start of HD. We first measured serum ADPN levels and prospectively followed patients for the next 3 years. RESULTS: We were able to follow 74 of 85 patients; 59 survived, and 15 died. Serum log-transformed ADPN levels were negatively correlated with BMI (r=-0.43, p<0.01). Despite a similar BMI (20.7+/-0.8 vs. 20.3+/-0.4 kg/m(2)), the expired patients had significantly higher ADPN compared with the surviving patients (20.5 microg/ml [14.0-23.5] vs. 14.2 microg/ml [9.7-21.3], p<0.05). Cox-hazards multivariate regression analysis adjusted for conventional case-mix features (age, sex, and underlying kidney disease) revealed that serum ADPN became a significant determinant of all-cause mortality. There was a 10.3% risk increment for each 1-microg/ml increase in ADPN during the follow-up. Kaplan-Meier analysis revealed that patients with higher ADPN levels (> or =15 microg/ml) had a significantly lower survival rate compared with those with lower ADPN levels (<15 microg/ml) (76 vs. 92%, p<0.05). CONCLUSION: These results indicated that high rather than low ADPN independently predict total mortality in HD patients.

Immunohistochemical study on caveolin-1alpha in regenerating process of tubular cells in gentamicin-induced acute tubular injury in rats.

Caveolin-1, a principal component of caveolae, modulates growth signaling, endocytosis, and intracellular transport. We examined the expression of caveolin-1alpha and its relation to cell cycle and caveolin-interacting growth factor receptors in regenerating proximal tubules (PTs) after gentamicin-induced acute renal failure in rats. Caveolin-1alpha appeared in regenerating PTs as early as day 4 after last gentamicin, peaked at days 6 to 8, and showed cytoplasmic pattern after day 8. Immunoelectron microscopy revealed caveolin-1alpha-positive caveolae on the cell membrane and in cytoplasms in regenerating PTs at days 4 to 8 and caveolin-positivity confined to cytoplasms after day 10. The number of PT cells with proliferation markers peaked at day 6 and decreased afterwards as expression of cyclin-dependent kinase inhibitors increased. Platelet-derived growth factor receptor-beta (PDGFR-beta) and epidermal growth factor receptor (EGFR) were colocalized with caveolin-1alpha in proliferating PTs as early as day 4. Phosphorylated EGFR increased at day 8 and afterwards when caveolins dissociated from EGFR or decreased. In case of PDGFR-beta, phosphorylation seemed to be associated with the increase and association of caveolins to the receptors. Our results suggest that transient expression of caveolin-1alpha in early regenerating PTs might contribute to the regenerating process of PTs through modulating growth factor receptors.

Kinetics and characterization of initially regenerating proximal tubules in S3 segment in response to various degrees of acute tubular injury.

BACKGROUND: We examined kinetics and characterization of regenerating proximal tubule (PT) cells after various degrees of tubular injury in S3 segments of PT and assessed label-retaining slow cycling cells in S3. METHODS: PT injury was induced by different doses of uranyl acetate (UA) injection into rats, and initially regenerating PTs were identified by in vivo bromodeoxyuridine (BrdU)-labelling before sacrifice or were examined on vimentin positivity. Next, the 3H-thymidine pulse/chase approach was applied to the early regenerating PTs identified by BrdU-labelling after UA injection. RESULTS: Low-dose UA induced focal PT depletion and initial BrdU positivity in the proximal three-quarters of the S3 segment of PT. Autoradiography showed the increased number of label-retaining and label-diluted cells in the proximal three-quarters of S3 in rats treated with low-dose UA compared to normal rats. High-dose UA induced almost complete PT depletion in the proximal three-quarters of S3 and less PT depletion in the distal quarter of S3 and initial BrdU+ cells were restrictedly found in the distal quarter of S3. Label-retaining and label-diluted cells were increasingly found in the entire S3 at day 7, but only label-retaining cells remained in similar numbers in the distal quarter of S3 until day 42. Initially regenerating PT cells with any doses of UA expressed vimentin, suggesting dedifferentiated PT cells. CONCLUSIONS: Initially regenerating cells after PT injury in S3 are dedifferentiated pre-existing PT cells, which may scatter throughout S3 and be responsible for focal repair of S3. Some initially regenerating PT cells in the distal S3 showed persistent label-retaining cells at day 42 after high-dose UA insult and contributed to renewal of the entire S3, thus they might be slow cycling cells with responsibility for S3 repair.

High dose chemotherapy and stem cell support in a patient of light- and heavy-chain deposition disease with abnormal marrow cell surface antigens and no monoclonal protein.

A 53-year-old man with nephrotic syndrome and severe renal failure was diagnosed with light- and heavy-chain deposition disease (LHCDD) by renal biopsy. The patient had no monoclonal protein and mild marrow plasmacytosis (6%), but marrow plasma cells expressed CD19(-)CD56+ and predominant monoclonal kappa-chain, indicating plasma cell dyscrasia. Conventional chemotherapy was ineffective and did not improve renal failure. High dose chemotherapy/peripheral blood stem cell transplantation (HDC/PBSCT) was introduced even after hemodialysis to eliminate aberrant clone and normalization of bone marrow cell surface markers. Immunophenotypic analysis of marrow cells facilitates clinical decision making regarding the use of HDC/PBSCT for LHCDD patients without monoclonal protein.

Transient myofibroblast differentiation of interstitial fibroblastic cells relevant to tubular dilatation in uranyl acetate-induced acute renal failure in rats.

To investigate the mechanisms of myofibroblast differentiation of interstitial fibroblastic cells (FCs) in rats with uranyl acetate-induced acute renal failure (ARF), we examined the relationship between the expression of alpha-smooth muscle actin (alpha-SMA), myofibroblast phenotype and tubular dilatation as well as cell shape and adhesion of FCs. Peritubular alpha-SMA-positive myofibroblasts appeared after induction of ARF and extended along the damaged, dilated proximal tubules and then almost disappeared after proximal tubular recovery. The perimeter of proximal tubules correlated with fractional areas stained for alpha-SMA (P<0.001). Most alpha-SMA-positive cells did not incorporate [3H]-thymidine, indicating a low proliferative activity. Transmission electron microscopy showed that FCs increasingly attached to the tubular basement membrane by elongated cytoplasm-containing microfilament bundles, which formed abundant adherens and gap junctions from day 4 to day 7. Scanning electron microscopy showed hypertrophic FCs covering large areas of tubules after induction of ARF. Administration of chlorpromazine, which can inhibit cytoskeletal movement, after induction of ARF partially inhibited myofibroblast differentiation of FCs immunohistochemically and morphologically and resulted in more dilated proximal tubules in concert with aggravation of renal dysfunction and inhibition of regenerative repair at day 4 than vehicle-administered rats. Our results indicate that mechanical tension, judged by tubular dilatation, may contribute to the induction of alpha-SMA phenotype with increased stress fiber formation and intercellular junctions in FCs to support damaged nephron structures by adjusting tensional homeostasis in rats with uranyl acetate-induced ARF.