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.

Effects of glucose dialysate on extracellular matrix production by human peritoneal mesothelial cells (HPMC): the role of TGF-beta.

BACKGROUND: Dialysate glucose has been implicated in the loss of peritoneal membrane function seen in long-term CAPD patients. METHODS: In order to investigate this in vitro, human peritoneal mesothelial cells (HPMC) were cultured in a 50:50 mix of dialysis solution and M199 for 12 h. The dialysate was laboratory manufactured and designed to be identical in composition to PD4 (LAB). The final glucose concentration ranged between 5 and 40 mmol/l. Experiments were conducted in the presence and absence of an anti-transforming growth factor-beta (TGF-beta) antibody. Cell viability was measured by lactate dehydrogenase (LDH) release. Fibronectin (FN) and TGF-beta protein were measured by ELISA, and FN gene expression was measured by Northern analysis. Separately, the effects of recombinant TGF-beta(1) added to M199: dialysate at 5 mmol/l glucose were investigated. RESULTS: Forty millimoles per litre d-glucose LAB caused a decrease in cell viability, as evidenced by an increase in LDH release (6.0+/-1.3 vs 2.6+/-0.7%). This effect was dependent on osmolality. Forty millimoles per litre d-glucose LAB stimulated a 15.4+/-4.6% increase in FN, a 46.5+/-18.3% increase in TGF-beta protein (both P<0.05), and 1.4+/-0.09-fold increase in FN mRNA compared with 5 mmol/l d-glucose LAB. Exogenous TGF-beta 0-1 ng/ml induced a dose-dependent increase in FN protein (280+/-45% increase at TGF-beta 1 ng/ml, P<0.0001), and FN mRNA levels (10.0+/-1.8-fold at TGF-beta 1 ng/ml). The increase in FN in response to 40 mmol/l glucose was significantly reduced by anti-TGF-beta antibody to levels not different from control (93.8+/-6.6%, P<0.05 vs no Ab). CONCLUSIONS: These data suggest that the pro-fibrotic effect of glucose dialysate on HPMC is mediated through stimulation of TGF-beta, which promotes FN gene expression and protein production.

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.

Role of diuretics in the preservation of residual renal function in patients on continuous ambulatory peritoneal dialysis.

BACKGROUND: Patients on continuous ambulatory peritoneal dialysis (CAPD) are dependent on residual renal function for solute and water clearances, and this declines with time on dialysis. Loop diuretics have been postulated to slow this decline. METHODS: Sixty-one patients new to dialysis were randomly assigned to either furosemide 250 mg every day or no furosemide at the time of CAPD training and were followed prospectively. Urine volume (UV), urea clearance (C(Urea)), and creatinine clearance on cimetidine (C(Cr)) were measured at randomization at six months and at one year. Patients underwent a standard four-hour peritoneum equilibrium test, and total body water was measured by bioelectrical impedance. Results were expressed on an intention-to-treat basis. RESULTS: UV, C(Cr), and C(Urea) were similar at randomization (1020 +/- 104 vs. 1040 +/- 130 mL/24 hours, 4.95 +/- 0.51 vs. 4.07 +/- 0.40 mL/min/1.73 m2, 0.91 +/- 0.09 vs. 0.84 +/- 0.08, diuretic vs. control). UV in the diuretic-treated group increased, whereas in the control group, it declined (+176 vs. -200 mL/24 hours at 6 months and +48.8 vs. -305 mL/24 hours at 1 year, P < 0.05). C(Cr) and C(Urea) declined at a constant rate and were unaffected by diuretic administration (0.12 +/- 0.05 vs. 0.071 +/- 0.04 mL/min/1.73 m2/month, 0.020 +/- 0.01 vs. 0.019 +/- 0.01 per month). Urinary sodium excretion increased in the diuretic group and declined in the control group (+0.72 +/- 0.85 vs. -2.56 +/- 1.31 mmol/24 hours/month, P = 0.04). Body weight rose in both groups (4.3 vs. 3.0 kg), but the percentage of total body weight rose in the control group and remained constant in the diuretic group (52 +/- 2.4 vs. 64 +/- 6.6%, P = 0.10). CONCLUSIONS: Long-term furosemide produces a significant increase in UV over 12 months when on CAPD and may result in clinically significant improvement in fluid balance. However, furosemide has no effect on preserving residual renal function.

Turnover of human tubular cells exposed to proteins in vivo and in vitro.

BACKGROUND: The cause of tubulointerstitial pathology in glomerular disease is uncertain. Proteinuria may be a causative factor and has been shown to increase the turnover of tubular cells in a rat model of proteinuria. We investigated whether increased tubular cell proliferation occurs in human proteinuric renal disease. A human cell culture model was used to investigate the effects of proteins on tubular cell turnover further. METHODS: Tubular proliferation in renal biopsies from patients with membranous nephropathy (MN) and minimal change nephropathy (MCN) was assessed by in situ hybridization for histone mRNA. Proliferation was measured in polarized human tubular cell cultures using tritiated thymidine, following addition of proteins to the apical medium. Toxicity was assessed by lactate dehydrogenase (LDH) release and monolayer permeability to inulin. RESULTS: Increased tubular cell histone mRNA expression occurred in biopsies in MN (3.0-fold increase, P < 0.002) and MCN (3.6-fold increase, P < 0.02). Serum proteins added to the medium on human tubular cell cultures increased thymidine uptake (1.3-fold, P < 0.005), LDH release (1.5-fold, P < 0.001), and monolayer permeability (1.7-fold, P < 0.005). The effects were reproduced by a fraction of molecular weight 40 to 100 kD, but not by pure albumin or transferrin. CONCLUSIONS: Proliferation of tubular cells is associated with proteinuria in vivo and is caused by proteins in cell culture. Toxicity of proteins to tubular cells and increased cell turnover may contribute to tubulointerstitial pathology in glomerular disease.

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.

Influence of allograft size to recipient body-weight ratio on the long-term outcome of renal transplantation.

BACKGROUND: The critical nephron mass needed to meet the metabolic demands of an individual depends on the body-weight. This study evaluated the effect of the kidney transplant ultrasonographic size to recipient body-weight ratio (Tx/W) on the outcome of kidney transplantation. METHODS: A consecutive series of 104 cadaveric renal transplants was studied. Transplant cross-sectional area (TXSA) was measured ultrasonographically in the first week after transplantation as an index of renal size. A 'nephron dose' index (Tx/W) was calculated by dividing TXSA by recipient weight and was used to define three groups of patients, with high (more than 0.45), medium (0.3-0.45) or low (less than 0.3) Tx/W ratios. Isotope glomerular filtration rate (GFR) measurements were made at 1, 6 and 12 months after transplantation. RESULTS: The serum creatinine level was significantly lower in the first 5 years after transplantation in patients with a high Tx/W ratio than in those with a medium or low ratio. GFR measurements were marginally higher in the groups with a high and medium Tx/W ratio compared with the low Tx/W group. A statistically significant association between Tx/W ratio and graft survival was not found. CONCLUSION: The renal transplant size to recipient weight ratio was an important determinant of long-term renal allograft function in this study. Extreme mismatching between allograft and recipient size should be avoided where possible, but the findings presented require confirmation in larger studies before clear recommendations can be made about size matching and kidney allocation.

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.

Secretion of chemokines and cytokines by human tubular epithelial cells in response to proteins.

BACKGROUND: Chronic interstitial scarring contributes to the progression of renal failure in glomerular disease but its cause is unknown. The development of proteinuria could stimulate tubular cells to release cytokines, chemoattractants and matrix proteins into the interstitium, thus contributing to interstitial disease. METHODS: Polarized human tubular epithelial cells were grown on permeable supports and exposed to serum proteins on their apical surface. The release of tumour necrosis factor alpha(TNFalpha), platelet derived growth factor (PDGF) and monocyte chemoattractant protein-1 (MCP-1) by the cells was measured using immunoassays. RESULTS: Under control conditions there was polarized release of PDGF-AB with predominant basolateral secretion (basolateral to apical ratio 4.7+/-1.6). MCP-1 release was less polarized (ratio 1. 7+/-0.5). TNFalpha was not detected. Exposure of the cells to normal human serum proteins on their apical side increased basolateral release of PDGF-AB (1.7+/-0.4 fold) and MCP-1 (2.4+/-0.2 fold). Fractionation of the serum showed that this effect on human tubular epithelial cells was reproduced by a fraction of molecular weight 40-100 kDa. The predominant proteins in this fraction were albumin and transferrin but these purified proteins alone did not alter secretion of PDGF-AB or MCP-1. CONCLUSION: This data demonstrates that human tubular cells exposed to proteins, which would be filtered in glomerular disease, produce inflammatory mediators with the potential to stimulate inflammation and scarring in the interstitium of the kidney.

Effects of acidosis on leptin secretion from 3T3-L1 adipocytes and on serum leptin in the uraemic rat.

Marked hyperleptinaemia and metabolic acidosis are common findings in patients with chronic renal failure. In animal models, both leptin administration and acidosis reduce food intake. However, leptin causes loss of body fat, while acidosis induces muscle wasting. Whether a low pH and leptin production are related has not been studied. Leptin secretion was measured in cultured 3T3-L1 adipocytes exposed to acid or control pH for up to 96 h. In addition, serum leptin was compared between acidotic and bicarbonate-treated uraemic Wistar rats using the remnant model. Leptin levels in the culture medium were decreased at an acid pH of 7.1 compared with a control pH of 7.5 at 96 h (562+/-78 and 831+/-103 pg.48 h(-1). well(-1) respectively; mean+/-S.E.M.; P=0.037). Similarly, serum leptin in uraemic rats was found to be lower in the acidotic group than in the bicarbonate-treated group, although this observation fell just short of statistical significance (1273+/-171 compared with 2059+/-376 pg/ml; P=0.07). In conclusion, acidosis decreases leptin secretion from cultured adipocytes. Accordingly, acidotic uraemic rats seem to exhibit lower serum leptin levels than their bicarbonate-supplemented counterparts. This study is the first report providing a link between acidosis and leptin levels.

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.

Proteinuria induces tubular cell turnover: A potential mechanism for tubular atrophy.

BACKGROUND: Proteinuria and tubular atrophy have both been closely linked with progressive renal failure. We hypothesized that apoptosis may be induced by tubular cell exposure to heavy proteinuria, potentially leading to tubular atrophy. Apoptosis was studied in a rat model of "pure" proteinuria, which does not induce renal impairment, namely protein-overload proteinuria. METHODS: Adult female Lewis rats underwent intraperitoneal injection of 2 g of bovine serum albumin (BSA, N = 16) or sham saline injections (controls, N = 8) daily for seven days. Apoptosis was assessed at day 7 in tissue sections using in situ end labeling (ISEL) and electron microscopy. ISEL-positive nuclei (apoptotic particles) were counted in blinded fashion using image analysis with NIH Image. Cell proliferation was assessed by detection of mRNA for histone by in situ hybridization, followed by counting of positive cells using NIH Image. RESULTS: Animals injected with saline showed very low levels of apoptosis on image analysis. BSA-injected rats had heavy proteinuria and showed both cortical and medullary apoptosis on ISEL. This was predominantly seen in the tubules and, to a lesser extent, in the interstitial compartment. Overall, the animals injected with BSA showed a significant 30-fold increase in the number of cortical apoptotic particles. Electron microscopy of tubular cells in a BSA-injected animal showed a progression of ultrastructural changes consistent with tubular cell apoptosis. The BSA-injected animals also displayed a significant increase in proximal tubular cell proliferation. This increased proliferation was less marked than the degree of apoptosis. CONCLUSION: Protein-overload proteinuria in rats induces tubular cell apoptosis. This effect is only partially balanced by proliferation and potentially provides a direct mechanism whereby heavy proteinuria can induce tubular atrophy and progressive renal failure.