Further evidence for the association of MMP9 with nephropathy in type 2 diabetes and application of DNA pooling technology to candidate gene screening.

BACKGROUND: Diabetic nephropathy is characterised by extracellular matrix (ECM) expansion, a key modulator of which is TGF-b1. Glucose-stimulated transcriptional activation of the TGF-b1 gene is an important component of the pathogenesis of nephropathy, following which latent TGF-b1 protein is synthesised. Matrix metalloproteinase 9 (MMP9) remodels the ECM and has been implicated in TGF-b1 activation. The ECM glycosaminoglycan hyaluronan (HA) influences TGF-b1 generation and can modulate its signal transduction activity; renal HA is synthesised by HA synthases HAS2 and HAS3. METHODS: We report the first screening of the genes encoding HAS2 and HAS3 for sequence variants predisposing to nephropathy in UK type 2 diabetes patients, together with the MMP9 and TGF-b1 genes. Also for the first time, we used validated DNA pools to carry out association analyses of single nucleotide polymorphisms on nephropathic and non-nephropathic cohorts from a total of 199 type 2 diabetes patients, to increase the throughput and decrease the cost of genotype analysis. RESULTS: None of the 23 single nucleotide polymorphisms analysed in DNA pools were found to be associated with diabetic nephropathy. However, genotyping of alleles at the MMP9 promoter microsatellite locus D20S838 in individual genomic DNA samples supported previous evidence of association between this locus and diabetic nephropathy. CONCLUSIONS: The use of DNA pooling technology increased the throughput and decreased the cost of our association analysis of nephropathy in our type 2 diabetes sample, which demonstrated sufficient sensitivity to support previous positive findings of association with a microsatellite in the MMP9 promoter region.

Sp1 and Sp3 mediate constitutive transcription of the human hyaluronan synthase 2 gene.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-kappaB recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation.

The impact of an out-reach clinic on referral of patients with renal impairment.

BACKGROUND: Early diagnosis and prompt treatment of a number of renal diseases may delay renal failure, obviate the need for renal replacement therapy and reduce co-morbidity. The aim of this study was to examine the impact of out-reach renal clinics on patterns of referral of patients with renal impairment to a nephrologist. METHODS: The number of patients with renal impairment was determined as defined by serum creatinine levels >150 micromol/l in three centres within a single NHS trust over two separate 1-week periods. None of the centres studied has a local nephrologist, however one centre (hospital A) has renal out-reach clinics, another is geographically close to a renal unit (hospital B), while the third unit (hospital C) has no nephrology presence and is geographically furthest from the renal unit. In addition, retrospective as well as follow-up data on the renal function of all patients with renal impairment was collected. RESULTS: In hospital A, there was a lower proportion of patients with unreferred renal impairment than in the other two hospitals. Within the unreferred patient group there were significantly more patients whose renal function improved during the follow-up period. A considerable proportion of patients with documented deterioration in renal function remained unknown to nephrology services 6 months after initial presentation. Other than the presence of an onsite nephrology service, the only other factor found to be significantly different in those patients not referred to nephrologists was age: as in all three centres, those not referred were significantly older. CONCLUSION: Inequity of access to renal services is an important obstacle to early referral of patients with impaired renal function. Out-reach renal services provide a model which significantly improves referral patterns.

Peritoneal dialysis training: a multisensory approach.

Undertaking peritoneal dialysis (PD) therapy poses a challenge to all patients with renal failure. The potentially high risk of infection makes it essential that patients undertaking PD have adequate training and ongoing support. Over recent years, increasing numbers of elderly patients, patients with significant learning disabilities, and patients with marked comorbidities have been accepted onto renal replacement therapy programs. For those undertaking PD in particular, this has posed new educational challenges. The Community Dialysis Team recognized an area of weakness in their current training program for these patient groups. The degree of literacy skills as well as the volume of written material and the amount of medical terminology used did not result in a user-friendly training program. A collaborative approach involving various members of the multidisciplinary team designed an appropriate training program for patients with learning disabilities. The new program included (1) a photographic bag-exchange procedure; (2) the provision of simple, step-by-step instructions on audiotape; (3) a new assessment sheet where words were replaced with symbols; (4) a redesigned daily record sheet (used to monitor clinical parameters); and (5) a simple contact card. The quality of the new training program was assessed by a small pilot study evaluation. The reduction of training times and the satisfactory peritonitis rates suggest that the new multisensory training program could be successfully implemented. The use of pictorial aids and more symbols, with less focus on the written word, made PD training a viable option for many individuals, including elderly patients and those with learning disabilities. The increased use of pictorial aids and symbols may also be helpful in training patients where there is a language barrier as well as the pediatric population.

Anemia and diabetes in the absence of nephropathy.

OBJECTIVE: Patients with diabetes commonly have a greater degree of anemia for their level of renal impairment than those presenting with other causes of renal failure. To clarify the contribution and differing roles of diabetes and nephropathy in the development of anemia in diabetic patients, we examined the hematologic and hematinic parameters of diabetic patients without nephropathy. RESEARCH DESIGN AND METHODS: The study group was comprised of 62 patients with type 2 diabetes who had been followed for a median of 7 years. For the study, these patients had additional samples taken during their annual routine blood testing for the measurement of extra parameters, including serum ferritin, serum erythropoietin (Epo) levels, and the percentage of reticulocytes. These measurements were combined with the routine parameters Hb, hematocrit, HbA(1c), and glomerular filtration rate. RESULTS: In all, 8 of the 45 male patients (17.8%) and 2 of the 17 female patients (11.8%) were classified as anemic (Hb <13g/dl and <11.5g/dl, respectively). Although only a small number of the patients had anemia as defined by normal values, a retrospective analysis of individual patients over time revealed a sustained though small decrease in Hb from initial presentation. A statistically significant difference in Epo levels (P = 0.016 by Kruskal-Wallis test) was observed from the group with the lowest (Hb < or =11.5) to that with the highest (Hb > or =14.5) Hb values, with a median Epo value of 37 (interquartile range 24-42) vs. 13 (9-15) IU/l, respectively. In contrast, there was no evidence of an increased reticulocyte response to higher levels of Epo (r = 0.134 [Pearsons], P = 0.36). Reticulocyte counts ranged from 44 (38-57) to 76.5 (56-83) in the lowest and highest Hb groups, respectively. CONCLUSIONS: Although only a small number of subjects in the group were overtly anemic, all subjects had an ongoing, small but significant decrease in Hb since presentation. This study of diabetic patients without nephropathy shows an expected increase in Epo production in response to lowering levels of Hb but without the expected reticulocyte response.

The Euro-Balance Trial: the effect of a new biocompatible peritoneal dialysis fluid (balance) on the peritoneal membrane.

BACKGROUND: Although peritoneal dialysis (PD) is a widely accepted form of renal replacement therapy (RRT), concerns remain regarding the bioincompatible nature of standard PD fluid. In order to evaluate whether a newly formulated fluid of neutral pH, and containing low levels of glucose degradation products (GDP), resulted in improved in vivo biocompatibility, it was compared in a clinical study to a standard PD fluid. METHODS: In a multicenter, open, randomized, prospective study with a crossover design and parallel arms, a conventional, acidic, lactate-buffered fluid (SPDF) was compared with a pH neutral, lactate-buffered, low GDP fluid (balance). Overnight effluent was collected and assayed for cancer antigen 125 (CA125), hyaluronic acid (HA), procollagen peptide (PICP), vascular endothelial growth factor (VEGF), and tumor necrosis factor alpha (TNFalpha). Serum samples were assayed for circulating advanced glycosylation end products (AGE), N(epsilon)-(carboxymethyl)lysine (CML), and imidazolone. Clinical end points were residual renal function (RRF), adequacy of dialysis, ultrafiltration, and peritoneal membrane function. Eighty-six patients were randomized to either group I starting with SPDF for 12 weeks (Phase I), then switching to "balance" for 12 weeks (Phase II), or group II, which was treated vice versa. Seventy-one patients completed the study with data suitable for entry into the per protocol analysis. Effluent and serum samples, together with peritoneal function tests and adequacy measurements, were undertaken at study centers on three occasions during the study: after the four-week run-in period, after Phase I, and again after Phase II. RESULTS: In patients treated with balance there were significantly higher effluent levels of CA125 and PICP in both arms of the study. Conversely, levels of HA were lower in patients exposed to balance, while there was no change in the levels of either VEGF or TNFalpha. Serum CML and imidazolone levels fell significantly in balance-treated patients. Renal urea and creatinine clearances were higher in both treatment arms after patients were exposed to balance. Urine volume was higher in patients exposed to balance. In contrast, peritoneal ultrafiltration was higher in patients on SPDF. When anuric patients were analyzed as a subgroup, there was no significant difference in peritoneal transport characteristics or in ultrafiltration on either fluid. There were no changes in peritonitis incidence on either solution. CONCLUSION: This study indicates that the use of balance, a neutral pH, low GDP fluid, is accompanied by a significant improvement in effluent markers of peritoneal membrane integrity and significantly decreased circulating AGE levels. Clinical parameters suggest an improvement in residual renal function on balance, with an accompanying decrease in peritoneal ultrafiltration. It would appear that balance solution results in an improvement in local peritoneal homeostasis, as well as having a positive impact on systemic parameters, including circulating AGE and residual renal function.

Identification and analysis of the promoter region of the human hyaluronan synthase 2 gene.

Hyaluronan (HA) is a linear glycosaminoglycan of the vertebrate extracellular matrix that is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2 and -3. The regulation of HA synthesis has been implicated in a variety of extracellular matrix-mediated and pathological processes, including renal fibrosis. We have recently described the genomic structures of each of the human HAS genes. In the present study, we analyzed the HAS2 promoter region. In 5'-rapid amplification of cDNA ends analysis of purified mRNA from human renal epithelial proximal tubular cells, we detected an extended sequence for HAS2 exon 1, relocating the transcription initiation site 130 nucleotides upstream of the reference HAS2 mRNA sequence, GenBank accession number NM_005328. A luciferase reporter gene assay of nested fragments spanning the 5' terminus of NM_005328 demonstrated the constitutive promoter activity of sequences directly upstream of the repositioned transcription initiation site but not of the newly designated exonic nucleotides. Using reverse transcription-PCR, expression of this extended HAS2 mRNA was demonstrated in a variety of human cell types, and orthologous sequences were detected in mouse and rat kidney. Alignment of human, murine, and equine genomic DNA sequences upstream of the repositioned HAS2 exon 1 provided evidence for the evolutionary conservation of specific transcription factor binding sites. The location of the HAS2 promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts as well as in developmental models in which HAS2 null animals have an embryonic lethal phenotype.

Identification and management of diabetic nephropathy in the diabetes clinic.

OBJECTIVE: To examine the prevalence and management of diabetic nephropathy in a diabetes clinic. RESEARCH DESIGN AND METHODS: Characteristics of nephropaths identified by existing screening practice (phase I, albuminuria >20 mg/l in three separate urine samples), were compared with those identified by a nurse-led management program (phase II, in which screening for nephropathy was based on albumin-to-creatinine ratio in a single random urine specimen). RESULTS: In phase I, 644 patients attended a diabetes clinic over a 6-month period. Microalbuminuria results were available for 485 patients (75%). A total of 115 patients were identified as nephropaths (prevalence 17.8%). Of these patients, 91% had type 2 diabetes. During phase II, prospective analysis of urinary albumin-to-creatinine ratio was carried out in 880 patients over 8 months. A total of 174 patients were identified as nephropaths (prevalence 20%). Of these, 134 patients had been identified by existing screening protocols. Forty had no previous record of microalbuminuria and were therefore newly identified by prospective screening. Systolic blood pressure guidelines were met in only 31% of all known nephropaths and 26.5% of newly diagnosed nephropaths. Diastolic blood pressure guidelines were met in 36% of all known and 38% of newly diagnosed nephropaths. In the patient group of known nephropaths from phases I and II, 62% were prescribed ACE inhibitors (ACEIs) or angiotensin II receptor (AIIR) antagonists. In the newly identified nephropathy patient cohort from phase II, 48% used ACEIs or AIIR antagonists. CONCLUSIONS: Introduction of a nurse-led management program significantly improved detection of nephropathy. We are currently evaluating its impact on clinical management.

Peritoneal dialysis: changes to the structure of the peritoneal membrane and potential for biocompatible solutions.

BACKGROUND: Peritoneal dialysis (PD) is now established as a viable and successful alternative to hemodialysis (HD) for patients starting on renal replacement therapy. A number of studies have confirmed that equivalent adequacy and fluid balance are provided at least for the first four to five years of renal replacement therapy (RRT). Loss of peritoneal membrane function remains a major factor leading to treatment failure in a significant number of patients on PD. Numerous studies have suggested a relationship between these changes in function and structural changes in the membrane. A careful analysis of peritoneal biopsies from PD patients would allow the clear identification of those changes unique to PD, in addition to indicating possible correlations with glucose exposure as well as other functional parameters. METHODS: We systematically examined peritoneal biopsies from 13 normal individuals, 29 uremic predialysis patients, 55 HD patients, and 157 patients on long-term PD. Well-oriented specimens were stained with toluidine blue and examined by a blinded pathologist. Limited clinical data has allowed a preliminary analysis of structure-function relationships. RESULTS: The median thickness of the submesothelial compact collagenous zone was 40 microm in normal individuals, 150 microm in uremic patients, 150 microm in patients on HD, and 2550 microm in patients on PD (P < 0.001 for all vs. normal individuals). Compact zone thickness increased significantly with duration of PD therapy (0 to 24 months, 180 microm;>97 months, 600 microm). Vascular changes comprised progressive subendothelial hyalinization of postcapillary venules, with luminal narrowing or obliteration. These changes were present in uremic patients and increased significantly with PD duration (P = 0.0001). CONCLUSIONS: These data indicate that morphologic changes in the postcapillary venules and the submesothelial compact zone of PD patients begin during the uremic phase of their illness. This is then worsened by time spent on PD. The relationships with glucose exposure or glucose degradation products have yet to be established.

Morphologic changes in the peritoneal membrane of patients with renal disease.

This study examined the morphologic features of the parietal peritoneal membranes of 130 patients undergoing peritoneal dialysis (PD) and compared them with the features of the peritoneal membranes of normal individuals, uremic predialysis patients, and patients undergoing hemodialysis. The median thickness of the submesothelial compact collagenous zone was 50 microm for normal subjects, 140 microm for uremic patients, 150 microm for patients undergoing hemodialysis, and 270 microm for patients undergoing PD (P < 0.001 for all versus normal subjects). Compact zone thickness increased significantly with the duration of PD therapy [0 to 24 mo, 180 microm (n = 58); 25 to 48 mo, 240 microm (n = 24); 49 to 72 mo, 300 microm (n = 13); 73 to 96 mo, 750 microm (n = 16); >97 mo, 700 microm (n = 19)]. Vascular changes included progressive subendothelial hyalinization, with luminal narrowing or obliteration. These changes were absent in samples from normal subjects but were present in 28% of samples from uremic patients and 56% of biopsies from patients undergoing PD. In the PD group, the prevalence of vasculopathy increased significantly with therapy duration (P = 0.0001). The density of blood vessels per unit length of peritoneum was significantly higher for patients with membrane failure and was correlated with the degree of fibrosis (P = 0.01). For the first time, a comprehensive cross-sectional analysis of the morphologic changes in the peritoneal membranes of patients undergoing PD is provided. The infrequency of fibrosis in the absence of vasculopathy suggests that vasculopathy may predispose patients to the development of fibrosis. This study provides a sufficiently large cohort of samples to allow structure-function relationships to be established, as well as providing a repository of tissue for further studies.