Essential fatty acid deficiency ameliorates acute renal dysfunction in the rat after the administration of the aminonucleoside of puromycin.

The administration of the aminonucleoside of puromycin (PAN) to rats causes the nephrotic syndrome that is associated with an acute decline in renal function, and an interstitial infiltrate. We examined whether essential fatty acid deficiency (EFAD), which inhibits macrophage infiltration in glomerulonephritis, affects PAN-induced renal dysfunction. Both control and EFAD rats developed proteinuria that resolved over 28 d. After PAN administration, there was a prominent infiltration of macrophages in rats fed a normal diet. The infiltrate was prevented by the EFAD diet. The absence of a macrophage interstitial infiltrate was associated with a significantly higher Cin in the EFAD rats than in controls at 7 d (5.21 +/- 1.19 versus 0.39 +/- 0.08, P less than 0.002 ml/min/kg BW). In addition, CPAH fell to less than 10 ml/min/kg BW by day 7 in controls, but remained the same as normal in the EFAD. After administration of PAN to control rats, there was no increase in urinary thromboxane excretion or an increase in glomerular thromboxane production. Furthermore, the effect of EFAD could not be mimicked by the administration of a thromboxane synthase inhibitor. Irradiation-induced leukopenia in rats on a normal diet markedly improved glomerular filtration and renal blood flow in acutely nephrotic rats. EFAD prevents the interstitial cellular infiltrate and the renal ischemia associated with experimental nephrosis. The recruitment of mononuclear cells into the kidney following PAN directly contributes to the decline in renal function.

Computerized histomorphometric assessment of protocol renal transplant biopsy specimens for surrogate markers of chronic rejection.

BACKGROUND: Chronic transplant rejection has emerged as the commonest cause of long-term renal allograft failure, and early identification of those grafts at risk could allow the targeting of specific therapies aimed at delaying this process. This study explores the usefulness of quantitative immunohistochemistry in defining biopsy-based surrogate markers of allograft damage. METHODS: A consecutive series of 52 renal transplant recipients immunosuppressed with cyclosporine were studied. Needle core transplant biopsies were performed at 1, 3, and 6 months postoperatively. Immunostaining for collagen III, and smooth muscle actin, tenascin, and infiltrating leukocytes was performed using an indirect immunoperoxidase technique. The interstitial area stained (%) was measured using a semiautomatic image analysis system. The results were related to glomerular filtration rates (GFR) measured at 6, 12, and 24 months after transplantation using rank correlation coefficients. RESULTS: The area fraction of immunostained collagen III correlated with 6-month GFR (r=-0.42, P=0.005) and was predictive of 12-month GFR (r=-0.32, P=0.03). An area fraction of immunostained collagen III of >40% at 6 months was associated with a significantly lower GFR at 24 months, compared with a percentage area of < or =40% (31+/-4 versus 45+/-4 ml/min/1.73 m2, P=0.01). Furthermore, a collagen III of >40% at 6 months identified patients who were at risk of progressive deterioration in graft function. CONCLUSIONS: Grafts with poorer long-term function can be predicted using 6-month protocol biopsy specimens immunostained for collagen III. This should prove to be a useful ad interim surrogate marker of allograft damage in studies addressing the effects of new immunosuppressive agents on the development of chronic rejection.

Semiautomatic quantitation of macrophages in human renal biopsy specimens in proteinuric states.

AIMS: To develop and validate a rapid and economical semiautomated approach to the measurement of immunostainable tissue components which is applicable to routine diagnostic practice. To apply this approach to the measurement of macrophages in renal biopsy specimens in nephrotic states, as protein in the renal tubules may induce macrophage infiltration, and the morphology of macrophages in tissue sections does not lend itself to cell counting. METHODS: Macrophages were identified by immunostaining with a pan-macrophage marker, followed by digital image capture and analysis using a macro procedure written for the freeware image analysis program NIH-Image. RESULTS: The method was rapid, robust and accurate to within the limits imposed by sampling error inherent in the use of small needle biopsy specimens. Very few macrophages are found in normal kidney (mean volume fraction (+/- 95% confidence limits) 0.04% (0.02%)) but infiltration of macrophages was detected in minimal change nephropathy (0.29% (0.12%)) and in membranous glomerulonephritis (0.42% (0.11%)). A statistically significant correlation was found between macrophage volume fraction and weight of proteinuria in minimal change nephropathy but not in membranous glomerulonephritis. Correlations were found in both diseases between macrophage volume fraction and serum creatinine at time of biopsy. CONCLUSIONS: The equipment is inexpensive and measurement takes less than one minute per biopsy specimen. The results indicate that macrophage infiltration is part of the pathological process in minimal change nephropathy and membranous glomerulonephritis. The correlation with creatinine at time of biopsy suggests that renal impairment in minimal change nephropathy may result from infiltration by immunologically active cells and not merely from haemodynamic changes in nephrons. However, the correlation is not close, indicating that the relation between macrophage infiltration and disease severity is not a simple one.

Peritoneal dialysis fill volume: can the patient tell the difference?

OBJECTIVE: Patients on continuous ambulatory peritoneal dialysis (CAPD) must receive an increased dialysis dose as they lose residual renal function so that total clearances are optimized. The dialysis dose may be increased by increasing the exchange volume. Patients on CAPD are often reluctant to use a greater exchange volume, fearing increased pain and discomfort and an altered body image. To assess patient perception of various fill volumes, we studied 12 stable patients currently treated with 2-L exchanges who had no surgical contraindication to larger fill volumes. METHOD: After an overnight dwell, patients received a 2-L, 2.5-L, or 3-L exchange of Baxter PD4 (Baxter Healthcare SA, Castlebar, Ireland) for 3 hours in a randomized crossover design. Patients and staff were both blinded to the fill volume. At the beginning and end of the exchange, intraperitoneal hydrostatic pressure (IPP) in the supine position was measured, and the patient's perception of the exchange was evaluated using the validated McGill Pain Questionnaire (MPG). RESULTS: Initial IPP increased with increasing fill volume (12.5 +/- 3.7 cmH2O vs 16.1 +/- 4.2 cmH2O vs 18.7 +/- 3.6 cmH2O for 2, 2.5 L, and 3L, respectively). For all fill volumes, IPP had fallen by the end of the 3-hour dwell, at which time it was similar to that after an overnight 2-L exchange. The pain rating index by was generally low for all exchange volumes and did not correlate with IPP. Minor degrees of discomfort were reported by 4, 2, and 1 patients with 3-L, 2.5-L, and 2-L exchanges respectively. CONCLUSION: Our study suggests that, despite an increased IPP, larger exchange volumes are generally well tolerated by patients, with only a minority of patients feeling mild discomfort.

Comparison of surgical duration of canine ovariectomy and ovariohysterectomy in a veterinary teaching hospital.

OBJECTIVE: To prospectively evaluate ovariectomy and ovariohysterectomy via midline coeliotomy when being employed by supervised final year veterinary students for the purpose of routine canine neutering. METHODS: One hundred and eight female dogs of various breeds, presented to a veterinary teaching hospital for neutering, were randomly allocated to one of two surgery groups, ovariectomy or ovariohysterectomy. The specified procedure was performed by a supervised final year veterinary student. If the duration of surgery exceeded 2 hours or if major surgical or anaesthetic complications occurred, the supervising surgeon intervened to complete the procedure. RESULTS: Data analysed included age, weight, time from first incision to start of closure, duration of closure, total surgical time and length of incision. Fifty-four dogs underwent each procedure. There was no significant difference between the two surgery groups for any of the measured variables. CLINICAL SIGNIFICANCE: Ovariectomy is not associated with shorter surgical times or smaller abdominal incisions than ovariohysterectomy when employed by inexperienced surgeons. As no major complications novel to ovariectomy occurred in this cohort of dogs, this study adds support to the existing literature indicating that ovariectomy is an acceptable alternative to ovariohysterectomy for canine neutering.

Thyroid fine needle cytology complicated by recurrent laryngeal nerve palsy and unnecessary radical surgery.

Fine needle aspiration cytology (FNAC) is an important tool in the investigation of thyroid nodules and has few reported complications. We present the first report of recurrent laryngeal nerve palsy arising as a complication of thyroid nodule FNAC. This complication led to inaccurate diagnosis and unnecessarily radical surgery, with consequent increased morbidity.

Cholesterol feeding activates macrophages to upregulate rat mesangial cell fibronectin production.

BACKGROUND: Cholesterol feeding has been shown to accelerate the development of glomerulosclerosis in many experimental renal diseases, possibly by promoting the infiltration of macrophages into the glomerulus. METHODS: In order to assess whether hyperlipidaemia could directly modulate macrophage function to promote glomerulosclerosis, confluent quiescent mesangial cells were exposed to resident (r) or elicited (e) macrophages, from either control (C) or cholesterol-fed (HC) rats or the conditioned media derived from the various macrophage preparations. RESULTS: All macrophage preparations stimulated mesangial cell fibronectin accumulation over medium alone, but eHC macrophages stimulated significantly greater fibronectin levels. Similarly, all macrophage conditioned media (MPCM) stimulated mesangial cell fibronectin production over medium alone and again the effect was greatest with MPCM derived from eHC macrophages. Proliferation studies using [(3)H]thymidine incorporation demonstrated that all conditioned media, with the exception of rC, stimulated significant mesangial cell proliferation over control levels. TGF-beta and PDGF, pro-fibrogenic growth factors known to be associated with macrophage infiltration, could not be detected in the MPCMs per se. However, they were detected in the culture supernatants of mesangial cells exposed to MPCMs and again secretion was greatest from mesangial cells exposed to eHC-MCPM. CONCLUSION: Monocytes are systemically activated by high serum cholesterol levels so that following maturation to macrophages they elaborate soluble factors that can stimulate mesangial cell fibronectin production, cell proliferation, and growth factor secretion. Hypercholesterolaemia may therefore accelerate glomerulosclerosis not only by increasing macrophage number, but also by upregulating the ability of macrophages to induce pro-sclerotic responses in glomerular mesangial cells.

The role of proteinuria in the progression of chronic renal failure.

The cause of the relentless progression of chronic renal failure of diverse origins remains unknown and is likely to be multifactorial. Numerous studies have now demonstrated a correlation between the degree of proteinuria and the rate progression of renal failure, which has led to the hypothesis that proteinuria may be an independent mediator of progression rather than simply being a marker of glomerular dysfunction. This article reviews the evidence underlying this hypothesis and the mechanisms by which particular proteins may cause renal pathology. The abnormal filtration of proteins across the glomerular basement membrane will bring them into contact with the mesangium and with the tubular cells. There is evidence to support a role of lipoproteins on mesangial cell function, which ultimately could contribute to glomerular sclerosis. The proximal tubular cells reabsorb proteins from the tubular fluid, which leaves them particularly vulnerable to any adverse effects proteins may have. It has been postulated that the sheer amount of protein to be metabolized by these cells may overwhelm the lysosomes and result in leakage of cytotoxic enzymes into the cells. In addition, the increased metabolism of proteins may result in production of ammonia, which can mediate inflammation through activation of complement. Specific proteins that have been shown to be cytotoxic are transferrin/iron, low-density lipoprotein, and complement components, all of which appear in the urine in proteinuric states. Other specific proteins have been shown to stimulate production of cytokines, chemoattractants, and matrix proteins by tubular cells and thus may stimulate interstitial inflammation and scarring. The mechanisms by which the presence of proteins in the tubular fluid alters tubular cell biology is yet to be determined.

Cytokine interactions promote synergistic fibronectin accumulation by mesangial cells.

BACKGROUND: The development of glomerulosclerosis has been associated with the presence of the cytokines transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-1 beta (IL-1 beta), at some stage in the glomerulus. To better understand the role of these cytokines in the scarring process their effect on rat mesangial cell fibronectin production was investigated. METHODS: Mesangial cells were exposed to 10 ng/ml of either TGF-beta 1, TNF-alpha, or IL-1 beta or to TGF-beta 1 in combination with TNF-alpha or IL-1 beta. Tissue culture supernatants and cell lysates were assayed for fibronectin. Supernatants were also assayed for TGF-beta 1. Northern blot analyses probing for fibronectin, transin, TIMP-1 and TGF-beta 1 were carried out on RNA extracted from mesangial cells exposed to individual and combinations of cytokines. RESULTS: Individually these cytokines were only able to induce modest increases in fibronectin protein levels. However, when mesangial cells were exposed to TGF-beta 1 in combination with either TNF-alpha or IL-1 beta then fibronectin levels were synergistically up-regulated approximately fivefold over unstimulated levels. Northern analysis demonstrated that fibronectin mRNA levels in the combination were also synergistically increased. In contrast, rat transin gene expression in the combinations was reduced to well below levels induced by TNF-alpha and IL-1 beta individually. In addition, synergistic up-regulation of both TGF-beta 1 protein and message by the cytokine combinations was also observed. TGF-beta 1: TNF-alpha and TGF-beta 1: IL-1 beta induced additive increases in TIMP-1 (tissue inhibitor of metalloproteinases-1) mRNA levels. CONCLUSIONS: These data illustrate that complex interactions can occur between cytokines within the glomerulus modulating both matrix synthetic and degradation pathways. These could initiate the scarring process and the development of glomerulosclerosis.

Macrophages promote prosclerotic responses in cultured rat mesangial cells: a mechanism for the initiation of glomerulosclerosis.

Glomerulosclerosis is the final outcome of a number of different causes of glomerular injury, during which the structures of the glomerulus are obliterated by extracellular matrix. Accumulating evidence suggests that infiltrating macrophages play a pivotal role in the progression to glomerulosclerosis. The present study defines the role played by macrophages at both cellular and molecular levels in the initiation of the sclerotic process in cultured rat mesangial cells. Macrophage-conditioned medium (MPCM) generated from thioglycollate-elicited, lipopolysaccharide-stimulated macrophages upregulated mesangial cell fibronectin production in a dose- and time-dependent manner, independently of cell proliferation. Immunoprecipitation of metabolically labeled 35S-fibronectin confirmed that the matrix protein was synthesized de novo. The genes for fibronectin and the matrix proteins laminin and collagen IV were also found to be upregulated 2.86 +/- 0.24-, 4.94 +/- 0.17-, and 3.03 +/- 0.31-fold over controls, respectively (P < 0.001). Macrophage modulation of matrix turnover was suggested by an upregulation of both transin and tissue inhibitor of metalloproteinase-1 gene transcription. Transforming growth factor (TGF) beta1, platelet-derived growth factor, tumor necrosis factor (TNF) alpha, or interleukin (IL)-1beta could not be detected in the MPCM per se; however, TGFbeta1 and platelet-derived growth factor AB were found to be secreted into mesangial cell culture supernatants. Secretion was augmented 1.69 +/- 0.16- and 2.28 +/- 0.28-fold, respectively (both P < 0.001), in response to MPCM. Northern blot analysis demonstrated that protein secretion had been preceded by upregulation of the genes for these cytokines (2.2 +/- 0.4-fold [P < 0.001] and 5.7 +/- 1.2-fold [P < 0.004], respectively). Incubation of MPCM with either neutralizing antibody or the growth factor receptor antagonist suramin demonstrated that TGFbeta1 played a significant, although minor, role in MPCM-stimulated fibronectin production. In conclusion, this study provides compelling evidence for a direct role of macrophages in the progression to glomerulosclerosis.

Transforming growth factor-beta suppresses macrophage-induced mesangial cell fibronectin expression.

BACKGROUND: We have previously shown that macrophages are able to promote prosclerotic responses in rat mesangial cells. Th2-type cytokines, including interleukin-10 (IL-10), IL-13, and IL-4 as well as transforming growth factor-beta (TGF-beta), are known to have suppressive effects on various aspects of macrophage function. In the current study, we investigated the effect of TGF-beta pretreatment on the ability of macrophages to induce fibronectin expression. RESULTS: Conditioned medium from TGF-beta pretreated macrophages (MPCM(TGF)) induced lower fibronectin levels in mesangial cells in both the secreted and cell-associated forms, compared with conditioned medium from standard macrophages (MPCM) (5.5 +/- 0.2 vs. 3.4 +/- 0.3 and 4.05 +/- 0.45 vs. 2.3 +/- 0.2-fold increase over medium alone for MPCM versus MPCM(TGF) in supernatants and cell lysates, respectively). Northern blot analysis demonstrated that fibronectin message was marginally reduced to 0.88 +/- 0.04 (P < 0.03 vs. MPCM, N = 3) of MPCM-induced levels. However, mesangial cell transin mRNA levels induced in response to MPCM(TGF) were 2.29 +/- 0.47-fold greater than those induced by standard MPCM (P = 0.03 vs. MPCM, N = 4). TIMP-1 mRNA levels were also increased in response to MPCM(TGF), but only by 1.43 +/- 0.1-fold (P = 0.02 vs. MPCM, N = 5). Casein-FITC digestion studies confirmed that MPCM(TGF) stimulated more mesangial cell caseinolytic activity than did MPCM. In addition, MPCM-mediated up-regulation of mesangial cell TGF-beta mRNA and protein expression was significantly reduced in response to conditioned medium from macrophages pretreated with TGF-beta. CONCLUSION: This study suggests that TGF-beta is able to regulate negatively the profibrotic effects of macrophages on mesangial cells by both enhancing matrix degradation and reducing synthesis.

Combined orthodontic-orthognathic surgical treatment of a Class II, Division I malocclusion.

This case report shows the need to extract four first premolars in addition to orthognathic surgery, even though the initial treatment plan involved a nonextraction strategy. The extractions were necessary to reduce maxillary dental protrusion and proclination and also to recover from the mandibular incisor proclination that occurred as a consequence of leveling the mandibular arch.

Early increase in glomerular leucocyte number after a reduction in renal mass: implications for the pathogenesis of glomerulosclerosis.

1. Adult female Wistar rats underwent uninephrectomy (n = 8) through a flank incision, or a sham operation (n = 7). One to two weeks later the kidney was perfused in situ and glomeruli were isolated from cortical tissue by sequential sieving, and partially digested. Glomerular leucocytes were labelled with a mouse monoclonal antibody against leucocyte common antigen followed by a fluorescein-labelled anti-mouse immunoglobulin to allow counting. 2. In a further group of animals 24 h albumin excretion and glomerular size were measured 2 weeks after either uninephrectomy (n = 6) or sham operation (n = 6). 3. Glomerular leucocyte number was significantly increased in uninephrectomized animals (15.7 +/- 0.9 versus 8.9 +/- 0.4, P < 0.001), with some glomeruli having leucocyte numbers comparable with those seen in glomerulonephritis. 4. Albuminuria was not increased 2 weeks after uninephrectomy (233 +/- 35 versus 170 +/- 42 micrograms/24 h, not significant), and glomerular size was unchanged. Light microscopical appearance was normal. 5. An increase in glomerular leucocyte number is an early response in what was previously considered a non-immunological lesion. It precedes the development of renal scarring and may be important in the pathogenesis of this process.