Advances in Urine Microscopy.

Urine microscopy is an important tool for the diagnosis and management of several conditions affecting the kidneys and urinary tract. In this review, we describe the automated instruments, based either on flow cytometry or digitized microscopy, that are currently in use in large clinical laboratories. These tools allow the examination of large numbers of samples in short periods. We also discuss manual urinary microscopy commonly performed by nephrologists, which we encourage. After discussing the advantages of phase contrast microscopy over bright field microscopy, we describe the advancements of urine microscopy in various clinical conditions. These include persistent isolated microscopic hematuria (which can be classified as glomerular or nonglomerular on the basis of urinary erythrocyte morphology), drug- and toxin-related cystalluria (which can be a clue for the diagnosis of acute kidney injury associated with intrarenal crystal precipitation), and some inherited conditions (eg, adenine phosphoribosyltransferase deficiency, which is associated with 2,8-dihydroxyadenine crystalluria, and Fabry disease, which is characterized by unique urinary lamellated fatty particles). Finally, we describe the utility of identifying "decoy cells" and atypical malignant cells, which can be easily done with phase contrast microscopy in unfixed samples.

Lipiduria--with special relevance to Fabry disease.

Examination of the urine under the microscope using polarised light is invaluable for detecting and identifying lipid particles. Attention to the shape of these Maltese cross bearing bodies can distinguish conventional fat particles from Fabry bodies with great sensitivity and specificity across a wide phenotypic spectrum. This could be a cheap and rapid tool for screening subjects suspected of having Fabry disease for renal involvement. It remains to be seen whether there is value in integrating polarised light into automated urine microscopy machines, but potentially this could greatly help the pathologist or nephrologist in identifying unusual urinary particles, and broaden the capacity for larger scale screening.

Animal models of chronic kidney disease: useful but not perfect.

Animal models of chronic kidney disease (CKD) approximate the human condition and are keys to understanding its pathogenesis and to developing rational treatment strategies. The ethical use of animals requires a detailed understanding of the strengths and limitations of each species and the disease model, and the way in which findings can be translated from animals to humans. While not perfect, the careful use of animal experiments offers the opportunity to examine individual mechanisms in an accelerated time frame.

The anti-fibrotic hormone relaxin is not reno-protective, despite being active, in an experimental model of type 1 diabetes.

The end-point of diabetic renal disease is the accumulation of excess collagen (fibrosis/sclerosis). A number of studies have shown that the hormone relaxin (RLX) ameliorates progression of renal and non-renal fibrosis. This study assessed the anti-fibrotic potential of RLX in streptozotocin (STZ)-treated transgenic mRen-2 rats, an accelerated model of type 1 diabetes. Eight-week old hyperglycaemic (STZ-treated at week-6) and normoglycaemic (STZ-untreated) animals were treated with or without recombinant human gene-2 (H2) RLX for 4-weeks (by osmotic mini-pumps) and assessed for various parameters at 12-weeks of age. Hyperglycaemic mRen-2 rats had elevated kidney weight/body weight ratio, glomerular filtration rate (GFR), albumin excretion rate (AER), interstitial collagen I and glomerulosclerosis (all p<0.05 vs non-diabetic controls). H2 RLX infusion had no effect on any of these parameters. Increased MMP-2 levels in RLX-treated rats demonstrated that the hormone was administered and active in this model. The inability of H2 RLX to slow glomerulopathy in diabetic mRen-2 rats could be in part due to the absence of its receptor, RXFP1, in rat mesangial cells, a primary mediator of diabetic glomerulosclerosis and/or the lack of any effect on TGF-ß1/Smad2 signalling, a well described mediator of RLX activity. These findings highlight the cell specific actions of RLX, the dissociation of anti-fibrogenic (collagen synthesis) and antifibrolytic (MMP mediated collagen degradation) properties, and the central involvement of TGF-ß1 in its actions.

Summary of KDIGO guideline. What do we really know about management of blood pressure in patients with chronic kidney disease?

The Kidney Disease: Improving Global Outcomes Clinical Practice Guideline for management of blood pressure (BP) in chronic kidney disease (CKD) supersedes the 2004 Kidney Disease Quality Outcomes Initiative document on this topic. The new guideline has been designed to assist clinical decision making in patients with CKD who are not receiving dialysis. The recommendations in the guideline acknowledge that no single BP target is optimal for all CKD patients and encourage individualization of treatment depending on age, the severity of albuminuria, and comorbidities. In general, the available evidence indicates that in CKD patients without albuminuria the target BP should be ≤140 mm Hg systolic and ≤90 mm Hg diastolic. However, in most patients with an albumin excretion rate of ≥30 mg/24 h (i.e., those with both micro- and macroalbuminuria), a lower target of ≤130 mm Hg systolic and ≤80 mm Hg diastolic is suggested. In achieving BP control, the value of lifestyle changes and the need for multiple pharmacological agents is acknowledged. Use of agents that block the renin-angiotensin-aldosterone system is recommended or suggested in all patients with an albumin excretion rate of ≥30 mg/24 h. Recommendations are almost identical in CKD patients with and without diabetes. Special considerations relevant to children and those of older age and those who have received a kidney transplant are included. Ongoing controversies in BP management in the context of CKD are highlighted along with key areas for future research.

Mortality in dialysis patients may not be associated with ESA dose: a 2-year prospective observational study.

BACKGROUND: Anaemia of chronic kidney disease increases the risk of death and adverse events, but can be managed using erythropoiesis stimulating agents (ESAs). However, recent evidence suggests that targeting a higher haemoglobin concentration ([Hb]) increases mortality risk, and both higher [Hb] targets and ESA doses have been implicated. Nonetheless, a causative role has not been demonstrated, and this potential relationship requires further appraisal in such a complex patient group. METHODS: The relationship between the haematopoietic response to ESAs and patient survival in 302 stable, prevalent dialysis patients was explored in a prospective, single-centre study. Clinical and laboratory parameters influencing mortality and ESA resistance were analysed. Patients were stratified into 5 groups, according to their [Hb] and ESA dosage, and were followed for 2 years. RESULTS: Little difference in co-morbidities between groups was identified. 73 patients died and 36 were transplanted. Initial analysis suggested a direct relationship between mortality and ESA dosage. However, Cox proportional hazards multivariate analysis demonstrated mortality risk was associated only with age (adjusted HR per year: 1.061, 95% CI 1.031-1.092), dialysis duration (adjusted HR: 1.010, 95% CI 1.004-1.016), peripheral vascular disease (adjusted HR: 1.967, 95% CI 1.083-3.576) and CRP (adjusted HR: 1.024, 95% CI 1.011-1.039). Mortality was increased in patients poorly responsive to ESAs (55.5%). CONCLUSION: ESA dose does not appear to contribute substantially to mortality risk in dialysis patients. Instead, age and co-morbidities appear to be the critical determinants. A poor response to ESAs is a marker of overall poor health status.

Engaging medical staff in clinical governance: introducing new technologies and clinical practice into public hospitals.

INTRODUCTION: To enhance patient care, medical staff at major tertiary teaching hospitals are encouraged to innovate through introducing new technologies and clinical practices. However, such introduction must be safe, efficient, effective and appropriate for patients and the organisation, and actively lead by engage medical staff. METHOD: This study outlines the development, implementation and evaluation of a framework for introducing new technologies and clinical practice to a major tertiary health service. Evaluation includes survey of medical Heads of Units (HOUs) for framework's effectiveness, and comparison of level of medical staff engagement against a best-practice model. RESULTS: Over 2-year period: 19 applications, 7 approved. Successful external funding of $1.993 million achieved. Survey of HOUs in June 2009: response rate 59% (25 of 42 HOUs), with 11 of 25 respondents utilised the committee. Of those 14 of 25 who had not utilised the committee, low awareness of the committee's existence (2 respondents). Most elements of the best-practice model for engaging medical staff were achieved. Recommendations include improvements to committee process and raising profile with medical staff. DISCUSSION: This study demonstrates an effective and successful clinical governance process for introducing new technologies and clinical practice into a major tertiary teaching hospital, supported by moderate levels of medical staff engagement.

Long-term mineralocorticoid receptor blockade ameliorates progression of experimental diabetic renal disease.

The final end point of diabetic renal disease is the accumulation of excess collagen. A number of studies have shown that aldosterone antagonism ameliorates progression of renal fibrosis. This study was designed to examine the effect of the mineralocorticoid receptor blocker eplerenone (EPL) on progression in streptozotocin (STZ)-treated spontaneously hypertensive rats (SHR), an accelerated model of Type I diabetes. STZ-treated SHRs with a blood glucose >18 mmol/L were randomly divided into treatment (100 mg/kg/day EPL) and non-treatment groups. Sham-injected SHR animals were used as a control. Functional parameters were monitored for 16 weeks, with structural parameters assessed at completion. Both hyperglycaemic groups developed progressive albuminuria, but the increase was ameliorated by EPL from Week 12. STZ-SHRs had elevated kidney weight/body weight ratio, glomerular size, glomerular macrophages (ED-1-positive cells), tissue transforming growth factor beta 1 (TGFß1) concentrations and glomerular collagen IV staining (all P < 0.05 versus control animals). EPL reduced glomerular volume, TGFß1 expression and glomerular collagen IV without changing glomerular macrophage infiltration. The ability of EPL to ameliorate these functional and structural changes in hyperglycaemic SHRs suggest that EPL has a renoprotective role in diabetic renal disease.

Explanting is an ex vivo model of renal epithelial-mesenchymal transition.

Recognised by their de novo expression of alpha-smooth muscle actin (SMA), recruitment of myofibroblasts is key to the pathogenesis of fibrosis in chronic kidney disease. Increasingly, we realise that epithelial-mesenchymal transition (EMT) may be an important source of these cells. In this study we describe a novel model of renal EMT. Rat kidney explants were finely diced on gelatin-coated Petri dishes and cultured in serum-supplemented media. Morphology and immunocytochemistry were used to identify mesenchymal (vimentin+, α-smooth muscle actin (SMA)+, desmin+), epithelial (cytokeratin+), and endothelial (RECA+) cells at various time points. Cell outgrowths were all epithelial in origin (cytokeratin+) at day 3. By day 10, 50 ± 12% (mean ± SE) of cytokeratin+ cells double-labelled for SMA, indicating EMT. Lectin staining established a proximal tubule origin. By day 17, cultures consisted only of myofibroblasts (SMA+/cytokeratin-). Explanting is a reproducible ex vivo model of EMT. The ability to modify this change in phenotype provides a useful tool to study the regulation and mechanisms of renal tubulointerstitial fibrosis.

Targeted urine microscopy in Anderson-Fabry disease: a cheap, sensitive and specific diagnostic technique.

BACKGROUND: Anderson-Fabry disease (AFD) is an X-linked lysosomal storage disorder resulting from the deficiency of trihexosylceramide α-galactosidase (α-Gal A). The diagnosis is often missed or delayed, and specific diagnostic tests (serum α-Gal A activity, genotyping or biopsy) are expensive and not widely available. We evaluated the diagnostic potential of urine microscopy in AFD. METHODS: We studied 35 male and female AFD patients across a wide phenotypic spectrum and 21 controls with other renal diseases. Fresh urine sediment was examined under phase-contrast microscopy using polarized light for Maltese cross (MC) particles, anti-CD77 antibody to detect globotriaosylceramide (GL3, the substrate of α-Gal A), and anti-podocalyxin antibody to assess podocyte excretion. RESULTS: Characteristic MC 2 particles and anti-CD77 binding within vacuolated urinary epithelial cells were both detected in AFD with high sensitivity and specificity (MC 2 detection sensitivity 100%, 95% confidence interval (CI) 85.4-100%, specificity 100%, CI 80.8-100%; anti-CD77-binding sensitivity 97.1%, CI 83.3-99.9, specificity 100%, CI 80.8-100%). Albuminuria (urinary albumin-to-creatinine ratio, ACR) correlated with quantitative particle excretion--in low, intermediate and high MC excretors, and median ACR was 1.6, 6.9 and 20.0 mg/µmol, respectively (analysis of variance P = 0.017). Podocyte staining was positive in ~50% of all AFD patients and was similar in those with and without clinical Fabry nephropathy (FN), whether or not treated with enzyme replacement. CONCLUSIONS: Targeted urinary microscopy is a non-invasive, inexpensive, accessible and rapid diagnostic technique, especially applicable where serum α-Gal A activity and genotyping are not affordable or available. As the number of urinary MC 2 particles increases with rising albuminuria, the technique may also be useful in assessing FN burden.

Lateral lumbar X-ray assessment of abdominal aortic calcification in Australian haemodialysis patients.

AIM: Vascular calcification is prevalent in patients with chronic kidney disease. Abdominal aortic calcification (AAC) can be detected by X-ray, although AAC is less well documented in anatomical distribution and severity compared with coronary calcification. Using simple radiological imaging we aimed to assess AAC and determine associations in prevalent Australian haemodialysis (HD) patients. METHODS: Lateral lumbar X-ray of the abdominal aorta was used to determine AAC, which is related to the severity of calcific deposits at lumbar vertebral segments L1 to L4. Two radiologists determined AAC scores, by semi-quantitative measurement using a validated 24-point scale, on HD patients from seven satellite dialysis centres. Regression analysis was used to determine associations between AAC and patient characteristics. RESULTS: Lateral lumbar X-ray was obtained in 132 patients. Median age of patients was 69 years (range 29-90), 60% were male, 36% diabetic, median duration of HD 38 months (range 6-230). Calcification (AAC score ≥ 1) was present in 94.4% with mean AAC score 11.0 ± 6.4 (median 12). Independent predictors for the presence and severity of calcification were age (P = 0.03), duration of dialysis (P = 0.04) and a history of cardiovascular disease (P = 0.009). There was no significant association between AAC and the presence of diabetes or time-averaged serum markers of mineral metabolism, lipid status and C-reactive protein. CONCLUSIONS: AAC detected by lateral lumbar X-ray is highly prevalent in our cohort of Australian HD patients and is associated with cardiovascular disease, increasing age and duration of HD. This semi-quantitative method of determining vascular calcification is widely available and inexpensive and may assist cardiovascular risk stratification.

Improving CKD-MBD management in haemodialysis patients: barrier analysis for implementing better practice.

BACKGROUND: Although clinical guidelines exist for optimal levels of serum markers of chronic kidney disease mineral and bone disorder (CKD-MBD), target parameters are not achieved in many haemodialysis (HD) patients. The reason for this evidence-practice gap is unclear and more information from patients and healthcare professionals is required to improve knowledge transfer. We aimed to determine potential barriers by surveying HD patients and staff about awareness and management of CKD-MBD. METHODS: A total of 136 prevalent HD patients, 25 nephrologists and 58 dialysis nurses/technicians were surveyed. Three separate questionnaires included issues of knowledge and awareness of CKD-MBD and factors limiting management (including compliance, medications and general understanding). RESULTS: Of patients surveyed, 84% had heard of phosphate, but 42% were unsure of high phosphate foods and 46% unaware of consequences of elevated phosphate. Twenty-seven percent and thirty-five percent of patients, respectively, had difficulty taking or forgetting to take phosphate binders. Seventy-four percent of patients wanted to know more about CKD-MBD (40% via written material). Of nephrologists surveyed, 76% thought non-compliance with phosphate binders was the main reason for poor control of phosphate (predominantly related to poor patient understanding); 84% thought patients wanted to know more but only 28% provided written material on CKD-MBD. Of dialysis staff surveyed, 63% thought non-compliance with binders explained poor control, the main reason being lack of patient understanding; 88% thought patients wanted to know more but only 17% provided written education. CONCLUSIONS: Implementation of an intensive educational programme, with a multi-faceted approach, for HD patients may promote better control of CKD-MBD and improve achievement of target levels.

A challenge to chronic kidney disease in Asia: The report of the second Asian Forum of Chronic Kidney Disease Initiative.

BACKGROUND: The Asian Forum of Chronic Kidney Disease Initiative started in 2007 in Hamamatsu, Japan when delegates from 16 countries joined together to facilitate collaboration in studying chronic kidney disease (CKD) in the Asia-Pacific region. Based on the outcome of the first meeting, the second meeting was organized as a consensus conference to frame the most relevant issues, and develop research recommendations and action plan. PROCEEDINGS: The meeting was held on 4 May 2008 as a pre-conference meeting to the 11th Asian Pacific Congress of Nephrology in Kuala Lumpur. This meeting consisted of three sessions: Session I was dedicated to the estimation of glomerular filtration rate and the standardization of serum creatinine measurements. Session II discussed specific considerations in the aetiology of and risk factors for end-stage renal disease in Asia. We concluded that there were regional specific problems that might lead to a very high prevalence of end-stage renal disease. Session III discussed the issue of facilitation of coordination and integration of the CKD initiative between developed and developing countries in the Asia-Pacific region. CONCLUSION: The following action plans were formulated: (i) validating the existing global estimated glomerular filtration rate equation or creating a new one using serum creatinine standardized by a central laboratory; (ii) establishing a pan-Asian CKD registry to facilitate risk analysis of CKD and its comorbidities; (iii) adapting existing clinical practice guidelines for CKD detection and management to address specific problems in this region; and (iv) working closely with other international professional organizations to promote manpower development and education in different aspects of CKD in developing countries.

Tissue preparation for histochemistry: fixation, embedding, and antigen retrieval for light microscopy.

A number of techniques have been developed to use chemical, immunological, and molecular biology assays in histological material. Collectively termed histochemistry, these techniques have allowed us to better understand tissue and organ biology in situ. Success with each of these methods is dependent on the adequate preparation of material. In this article, we describe the basic steps required to prepare tissue for routine histochemical analysis.Histochemical techniques routinely use frozen and paraffin-embedded tissue as a basis for cellular and morphological analysis. Freezing tissue results in less alteration to epitopes and therefore may offer improved staining characteristics compared to techniques based on paraffin embedding. As in conventional histology, the use of fixation and embedding in more rigid media such as wax offers a number of potential advantages related to improved structural detail. Improvements in morphology may however be offset by a loss of antigens. The careful application of antigen retrieval techniques may overcome these deficiencies.