Proteoglycans contribute to the functional integrity of the glomerular endothelial cell surface layer and are regulated in diabetic kidney disease

All capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.publishedVersio

Role of Glomerular Proteoglycans in IgA Nephropathy

Mesangial matrix expansion is a prominent feature of the most common form of glomerulonephritis, IgA nephropathy (IgAN). To find molecular markers and improve the understanding of the disease, the gene and protein expression of proteoglycans were investigated in biopsies from IgAN patients and correlated to clinical and morphological data. We collected and microdissected renal biopsies from IgAN patients (n = 19) and from healthy kidney donors (n = 14). Patients were followed for an average time of 4 years and blood pressure was according to target guidelines. Distinct patterns of gene expression were seen in glomerular and tubulo-interstitial cells. Three of the proteoglycans investigated were found to be of special interest and upregulated in glomeruli: perlecan, decorin and biglycan. Perlecan gene expression negatively correlated to albumin excretion and progress of the disease. Abundant decorin protein expression was found in sclerotic glomeruli, but not in unaffected glomeruli from IgAN patients or in controls. Transforming growth factor beta (TGF-β), known to interact with perlecan, decorin and biglycan, were upregulated both on gene and protein level in the glomeruli. This study provides further insight into the molecular mechanisms involved in mesangial matrix expansion in IgAN. We conclude that perlecan is a possible prognostic marker for patients with IgAN. In addition, the up-regulation of biglycan and decorin, as well as TGF-β itself, indicate that regulation of TGF-β, and other profibrotic markers plays a role in IgAN pathology

Pharmacokinetic Properties of the Nephrotoxin Orellanine in Rats

Orellanine is a nephrotoxin found in mushrooms of the Cortinarius family. Accidental intake of this substance may cause renal failure. Orellanine is specific for proximal tubular cells and could, therefore, potentially be used as treatment for metastatic renal cancer, which originates from these cells. However, more information is needed about the distribution and elimination of orellanine from the body to understand its potential use for therapy. In this study, 5 mg/kg orellanine (unlabeled and 3H-labeled) was injected intravenously in rats (Wistar and Sprague Dawley). Distribution was measured (Wistar rats, n = 10, n = 12) using radioluminography and the highest amount of orellanine was found in the kidney cortex and bladder at all time-points investigated. The pharmacokinetic properties of orellanine was investigated using LC-MS/MS and β-scintillation to measure the amount of orellanine in plasma. Three groups of rats were investigated: control rats with intact kidneys (n = 10) and two groups with bilateral renal artery ligation (n = 7) where animals in one of these groups were treated with peritoneal dialysis (n = 8). Using LC-MS/MS, the half-life of orellanine was found to be 109 ± 6 min in the controls. In the groups with ligated renal arteries, orellanine had a half-life of 756 ± 98 min without and 238 ± 28 min with dialysis. Thus, orellanine was almost exclusively eliminated by glomerular filtration as well as by peritoneal dialysis

Serum levels of galactose-deficient IgA are elevated in patients with IgA nephropathy but do not correlate to disease activity or progression

Abstract Introduction IgA nephropathy (IgAN) is the most common glomerulonephritis globally. Because of the heterogeneity of the disease prognostic biomarkers are highly needed. Aim To investigate associations between galactose-deficient IgA1 (Gd-IgA1) concentrations in plasma and urine and disease activity and progression in patients with IgAN. Methods Serum and urine samples were collected at the time of kidney biopsy (baseline) in patients with IgAN (n = 40) and analysed for Gd-IgA1. Patients with chronic kidney disease (CKD) without IgAN (n = 21) and healthy controls (n = 19) were examined as controls. In 19 patients with IgAN, analyses of Gd-IgA1 were repeated after a median follow up time of approximately 10 years. Results Serum Gd-IgA1 and Gd-IgA1:IgA were significantly elevated at the time of kidney biopsy in patients with IgAN compared to patients with non-IgAN CKD and healthy controls (p < 0.001). Urinary Gd-IgA1:creatinine was significantly elevated in patients with IgAN compared to patients with non-IgAN CKD. Neither serum Gd-IgA1, nor serum Gd-IgA1:IgA, correlated significantly to estimated GFR, urine albumin:creatinine (UACR), or blood pressure, at baseline. Serum Gd-IgA1 and Gd-IgA1:IgA at time of biopsy did not correlate significantly to annual changes in eGFR or UACR during follow up. In patients with IgAN, serum Gd-IgA1 decreased significantly over time during approximately 10 years of follow up (Δ-20 ± 85%, p = 0.027). Urinary Gd-IgA1:creatinine showed a strong positive correlation to UACR in patients with IgAN and likely reflected unspecific glomerular barrier injury. Conclusion Although serum Gd-IgA1 and the Gd-IgA1:IgA ratio were significantly elevated in patients with IgAN at the time of kidney biopsy they were not related to disease activity or progression in this patient cohort

Relative gene expression of proteoglycan core proteins and enzymes in glomeruli (A) and tubulo-interstitial compartment (B) of biopsies from IgAN patients compared to controls.

<p>Controls values are defined as 1, and higher values indicate up-regulation while lower values indicate down-regulation. Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001.</p

PAS staining of sclerotic glomeruli from a representative IgAN patient (A), and the consecutive section stained for decorin (B) shows overlap of the sclerotic area and decorin staining.

<p>PAS staining of glomeruli without sclerosis (C) and consecutive section showing no staining for decorin (D) in the same patient. Decorin is abundantly expressed in sclerotic glomeruli both in controls and patients with IgAN, but not in non-sclerotic glomeruli. Magnification ×63.</p

Melanocortin 1 Receptor Agonists Reduce Proteinuria

Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. Recent reports suggest that treatment with adrenocorticotropic hormone (ACTH) reduces proteinuria, but the mechanism of action is unknown. Here, we identified gene expression of the melanocortin receptor MC1R in podocytes, glomerular endothelial cells, mesangial cells, and tubular epithelial cells. Podocytes expressed most MC1R protein, which colocalized with synaptopodin but not with an endothelial-specific lectin. We treated rats with passive Heymann nephritis (PHN) with MS05, a specific MC1R agonist, which significantly reduced proteinuria compared with untreated PHN rats (P < 0.01). Furthermore, treatment with MC1R agonists improved podocyte morphology and reduced oxidative stress. In summary, podocytes express MC1R, and MC1R agonism reduces proteinuria, improves glomerular morphology, and reduces oxidative stress in nephrotic rats with PHN. These data may explain the proteinuria-reducing effects of ACTH observed in patients with membranous nephropathy, and MC1R agonists may provide a new therapeutic option for these patients

Protein expression of TGF-β in sections from control (A), patient with IgAN (B) and negative control (C).

<p>IgAN sections stained significantly stronger for TGF-β, and the average arbitrary unit score for IgAN (n = 83 glomeruli, 4.51±0.25) was higher than control (n = 19 glomeruli, 2.89±0.28) P<0.05 (D). In (B) there is some auto-fluorescence of red blood cells, this phenomenon can also be seen in the negative control, and were not included in the analysis of staining intensity of TGF-β. Magnification ×63.</p

The relative gene expression of perlecan in the glomeruli from patients with IgAN at time of the biopsy correlates inversely to the patient's albumin excretion in urine, n = 17, r = −0.58, p<0.05 (A) and disease progression, n = 18, r = −0.52, p<0.05 (C).

<p>A high expression of perlecan at the time of the biopsy correlates to a lower albumin excretion and slower progression of the disease, implicating that the relative gene expression of perlecan can be used as a prognostic molecular marker in IgAN. The relative gene expression of nephrin did not correlate to urinary albumin excretion, n = 17 (B), but correlated inversely to disease progression, n = 18, r = −0.47, P<0.05 (D). This suggests that even the smallest changes in gene expression of nephrin may be important for disease progression in IgAN.</p