Genome-wide Association Studies Identify Genetic Loci Associated with Albuminuria in Diabetes

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and associate with increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes mellitus and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (p=2.4*10(-10)). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. SNPs at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in average UACR per minor allele was 21% for HS6ST1 and 13% for RAB38/CTSC (p=6.3*10(-7) and 5.8*10(-7), respectively). Experiments using streptozotocin-treated diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout vs. control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared to controls. The loci identified here confirm known and highlight novel pathways influencing albuminuria.

Podocyte EphB4 signaling helps recovery from glomerular injury

Eph receptor tyrosine kinases and their ligands (ephrins) have a pivotal role in the homeostasis of many adult organs and are widely expressed in the kidney. Glomerular diseases beginning with mesangiolysis can recover, with podocytes having a critical role in this healing process. We studied here the role of Eph signaling in glomerular disease recovery following mesangiolytic Thy1.1 nephritis in rats. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and were upregulated during Thy1.1 nephritis, with EphB4 strongly phosphorylated around day 9. Treatment with NPV-BHG712, an inhibitor of EphB4 phosphorylation, did not cause glomerular changes in control animals. Nephritic animals treated with vehicle did not have morphological evidence of podocyte injury or loss; however, application of this inhibitor to nephritic rats induced glomerular microaneurysms, podocyte damage, and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis (an alternative to sprouting angiogenesis), indicating a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. Thus, our results identify EphB4 signaling as a pathway allowing podocytes to survive transient capillary collapse during glomerular disease.

Single application of low-dose mycophenolate mofetil-OX7-immunoliposomes ameliorates experimental mesangial proliferative glomerulonephritis

IgA nephropathy, one of the most frequent forms of glomerulonephritis, characterized by mesangial hypercellularity and glomerular extracellular matrix (ECM) expansion, often leads to end-stage renal disease over a prolonged period. We investigated whether antiproliferative treatment in a single low dose specifically targeted to the glomerular mesangium by immunoliposomes (ILs) results in an amelioration of mesangial proliferative glomerulonephritis in rats (anti-Thy1.1 nephritis). Mycophenolate mofetil (MMF) containing ILs was generated that targets the Thy1.1 antigen (OX-7) in rat mesangial cells. Treatment benefit of a single intravenous dose of these ILs given 2 days after disease induction was investigated by stereology, immunohistochemistry, and functional analyses (creatinine, albuminuria) until day +9 and was compared among untreated and free MMF-treated rats using six male Wistar rats per group. MMF-loaded OX7-IL prevented creatinine increase and albuminuria. Stereological analyses of MMF OX7-IL-treated animals yielded 30% reduction of mesangial cells on day +9 and a 40% reduction of glomerular ECM volume on day +5, compared with all of the other nephritic animals. Furthermore, at days +5 and +9 we observed decreased ECM content and decreased glomerular volume (day +5) in the MMF-OX7-IL-treated group compared with the nephritic group treated with free MMF. In conclusion, MMF-OX7-IL-based directed drug delivery represents a novel approach for treating mesangial cell-mediated forms of glomerulonephritis.

[Microalbuminuria--a new cardiovascular risk factor?]

Microalbuminuria is an established risk factor for renal disease, especially in the diabetic population. Recent studies have shown that microalbuminuria has also a highly relevant predictive value for cardiovascular morbidity and mortality. From normal to overt proteinuria levels, albuminuria shows a continuous marked increase in cardiovascular risk. This association is independent of other "classical" cardiovascular risk factors such as hypertension, hyperlipidemia or smoking. Furthermore it has a predictive value not only for patients with diabetic or renal disease, but also for hypertensive individuals or the general population. Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have been shown to display not only reno--but also cardioprotective effects. Their unique ability to lower albuminuria by 40% is related to a significant risk reduction in cardiovascular mortality. New clinical trials are needed to define "normal" albuminuria levels and how low we should go.

Microalbuminuria in diabetes mellitus: efficacy of a new screening method in comparison with timed overnight urine collection

Diabetic nephropathy and end-stage renal failure are still a major cause of mortality amongst patients with diabetes mellitus (DM). In this study, we evaluated the Clinitek-Microalbumin (CM) screening test strip for the detection of microalbuminuria (MA) in a random morning spot urine in comparison with the quantitative assessment of albuminuria in the timed overnight urine collection ("gold standard"). One hundred thirty-four children, adolescents, and young adults with insulin-dependent DM Type 1 were studied at 222 outpatient visits. Because of urinary tract infection and/or haematuria, the data of 13 visits were excluded. Finally, 165 timed overnight urine were collected in the remaining 209 visits (79% sample per visit rate). Ten (6.1%) patients presented MA of > or =15 microg/min. In comparison however, 200 spot urine could be screened (96% sample/visit rate) yielding a significant increase in compliance and screening rate (P<.001, McNemar test). Furthermore, at 156 occasions, the gold standard and CM could be directly compared. The sensitivity and the specificity for CM in the spot urine (cut-off > or =30 mg albumin/l) were 0.89 [95% confidence interval (CI) 0.56-0.99] and 0.73 (CI 0.66-0.80), respectively. The positive and negative predictive value were 0.17 (CI 0.08-0.30) and 0.99 (CI 0.95-1.00), respectively. Considering CM albumin-to-creatinine ratio, the results were poorer than with the albumin concentration alone. Using CM instead of quantitative assessment of albuminuria is not cost-effective (35 US dollars versus 60 US dollars/patient/year). In conclusion, to exclude MA, the CM used in the random spot urine is reliable and easy to handle, but positive screening results of > or =30 mg albumin/l must be confirmed by analyses in the timed overnight collected urine. Although the screening compliance is improved, in terms of analysing random morning spot urine for MA, we cannot recommend CM in a paediatric diabetic outpatient setting because the specificity is far too low.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

Effect of large doses of parenteral vitamin D on glycaemic control and calcium/phosphate metabolism in patients with stable type 2 diabetes mellitus: a randomised, placebo-controlled, prospective pilot study.

OBJECTIVE Vitamin D (D₃) status is reported to correlate negatively with insulin production and insulin sensitivity in patients with type 2 diabetes mellitus (T2DM). However, few placebo-controlled intervention data are available. We aimed to assess the effect of large doses of parenteral D3 on glycosylated haemoglobin (HbA(₁c)) and estimates of insulin action (homeostasis model assessment insulin resistance: HOMA-IR) in patients with stable T2DM. MATERIALS AND METHODS We performed a prospective, randomised, double-blind, placebo-controlled pilot study at a single university care setting in Switzerland. Fifty-five patients of both genders with T2DM of more than 10 years were enrolled and randomised to either 300,000 IU D₃ or placebo, intramuscularly. The primary endpoint was the intergroup difference in HbA(₁c) levels. Secondary endpoints were: changes in insulin sensitivity, albuminuria, calcium/phosphate metabolism, activity of the renin-aldosterone axis and changes in 24-hour ambulatory blood pressure values. RESULTS After 6 months of D₃ supply, there was a significant intergroup difference in the change in HbA(₁c) levels (relative change [mean ± standard deviation] +2.9% ± 1.5% in the D₃ group vs +6.9% ± 2.1% the in placebo group, p = 0.041) as HOMA-IR decreased by 12.8% ± 5.6% in the D₃ group and increased by 10% ± 5.4% in the placebo group (intergroup difference, p = 0.032). Twenty-four-hour urinary albumin excretion decreased in the D₃ group from 200 ± 41 to 126 ± 39, p = 0.021). There was no significant intergroup difference for the other secondary endpoints. CONCLUSIONS D₃ improved insulin sensitivity (based on HOMA-IR) and affected the course of HbA(₁c) positively compared with placebo in patients with T2DM.