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

Functional and molecular aspects of the glomerular barrier

The kidneys are crucial for maintaining a normal salt-water balance in the body, which is a prerequisite for life. They do that by filtering 180 liters of plasma per day across the highly permselective glomerular barrier and reabsorbing 99 % of the filtrate. Proteinuria is a hallmark of renal disease and reflects damage to one or more of the components of the barrier, namely fenestrated endothelial cells, basement membrane, and podocytes. For several years, the basement membrane was considered to be the main barrier. Recently, research has focused on the podocytes, while the endothelium has received much less attention. The glomerular endothelial cells are covered however by a thick negatively charged cell surface coat, a glycocalyx, which most likely has selective properties. The glycocalyx is composed of plasma proteins, glycoproteins and proteoglycans. Proteoglycans (PG) are negatively charged molecules with a protein-core to which glycosaminoglycan (GAG) chains are attached. In this thesis, we have made a detailed analysis of the biosynthesis of PG and GAG by human and bovine glomerular endothelial cells, by human podocytes and by rat glomeruli in vivo. These cells were shown to express the following core proteins: syndecan, glypican, versican, perlecan, biglycan and decorin. When treating the cells with puromycin amino nucleoside (PAN), a drug known to induce nephrotic syndrome, we could see dramatic down-regulation of PGs, mainly versican, and of enzymes involved in the synthesis and modification of the glycosaminoglycan chains. Indeed, when analyzing the cell media after PAN treatment, a significant decrease in the amount of sulfate groups and the chain length was detected. These effects of PAN on glomerular cells were confirmed in a study on rats, in which molecular and physiological measurements were used to study the course of proteinuria for seven days. In these animals, PAN decreased the expression of nephrin, podocin and VEGF as well as with certain PGs and GAG enzymes. The marked changes of PG and GAG induced by PAN are likely to reduce both size and charge selectivity of the glomerular barrier

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

Renal Endothelial Single-Cell Transcriptomics Reveals Spatiotemporal Regulation and Divergent Roles of Differential Gene Transcription and Alternative Splicing in Murine Diabetic Nephropathy

Endothelial cell (EC) injury is a crucial contributor to the progression of diabetic kidney disease (DKD), but the specific EC populations and mechanisms involved remain elusive. Kidney ECs (n = 5464) were collected at three timepoints from diabetic BTBRob/ob mice and non-diabetic littermates. Their heterogeneity, transcriptional changes, and alternative splicing during DKD progression were mapped using SmartSeq2 single-cell RNA sequencing (scRNAseq) and elucidated through pathway, network, and gene ontology enrichment analyses. We identified 13 distinct transcriptional EC phenotypes corresponding to different kidney vessel subtypes, confirmed through in situ hybridization and immunofluorescence. EC subtypes along nephrons displayed extensive zonation related to their functions. Differential gene expression analyses in peritubular and glomerular ECs in DKD underlined the regulation of DKD-relevant pathways including EIF2 signaling, oxidative phosphorylation, and IGF1 signaling. Importantly, this revealed the differential alteration of these pathways between the two EC subtypes and changes during disease progression. Furthermore, glomerular and peritubular ECs also displayed aberrant and dynamic alterations in alternative splicing (AS), which is strongly associated with DNA repair. Strikingly, genes displaying differential transcription or alternative splicing participate in divergent biological processes. Our study reveals the spatiotemporal regulation of gene transcription and AS linked to DKD progression, providing insight into pathomechanisms and clues to novel therapeutic targets for DKD treatment.De två första författarna delar förstaförfattarskapetDe två sista författarna delar sistaförfattarskapet</p

Preclinical pharmacology of AZD9977: A novel mineralocorticoid receptor modulator separating organ protection from effects on electrolyte excretion

<div><p>Excess mineralocorticoid receptor (MR) activation promotes target organ dysfunction, vascular injury and fibrosis. MR antagonists like eplerenone are used for treating heart failure, but their use is limited due to the compound class-inherent hyperkalemia risk. Here we present evidence that AZD9977, a first-in-class MR modulator shows cardio-renal protection despite a mechanism-based reduced liability to cause hyperkalemia. AZD9977 <i>in vitro</i> potency and binding mode to MR were characterized using reporter gene, binding, cofactor recruitment assays and X-ray crystallopgraphy. Organ protection was studied in uni-nephrectomised db/db mice and uni-nephrectomised rats administered aldosterone and high salt. Acute effects of single compound doses on urinary electrolyte excretion were tested in rats on a low salt diet. AZD9977 and eplerenone showed similar human MR <i>in vitro</i> potencies. Unlike eplerenone, AZD9977 is a partial MR antagonist due to its unique interaction pattern with MR, which results in a distinct recruitment of co-factor peptides when compared to eplerenone. AZD9977 dose dependently reduced albuminuria and improved kidney histopathology similar to eplerenone in db/db uni-nephrectomised mice and uni-nephrectomised rats. In acute testing, AZD9977 did not affect urinary Na⁺/K⁺ ratio, while eplerenone increased the Na⁺/K⁺ ratio dose dependently. AZD9977 is a selective MR modulator, retaining organ protection without acute effect on urinary electrolyte excretion. This predicts a reduced hyperkalemia risk and AZD9977 therefore has the potential to deliver a safe, efficacious treatment to patients prone to hyperkalemia.</p></div

AZD9977 and eplerenone activities in reporter gene assays for human, mouse and rat MR.

<p>AZD9977 and eplerenone activities in reporter gene assays for human, mouse and rat MR.</p

AZD9977 does not change urinary Na⁺/K⁺ ratio in rat urine electrolyte secretion model.

<p>a) Salt deprived rats were treated with increasing doses of eplerenone (a) or AZD9977 (b), urine collection for 8 hours after dose. Average +/- SEM; <i>n</i> = 8; *p<0.05 compared to vehicle. c) Salt deprived rats were treated with 10 or 30 mg kg^-1 eplerenone in absence or presence of 100 mg kg^-1 AZD9977, urine collection for 8 hours after dose. Average +/- SEM; <i>n</i> = 8, *p<0.05 vs vehicle. ^#p<0.05 vs eplerenone treatment in absence of AZD9977.</p

AZD9977 and eplerenone activities in reporter gene assays.

<p>Concentration response curves of AZD9977 and eplerenone tested in a reporter gene assay in (a) presence or (b) absence of 0.1 nM aldosterone. <i>n</i> = 4, average ± SD.</p

AZD9977 and eplerenone protect against aldosterone and high salt induced renal injury.

<p>Uni-nephrectomised rats with an aldosterone mini-pump and on a high salt diet were treated for 4 weeks with AZD9977 or eplerenone as food admixtures. Doses in mg kg^-1 d^-1 are indicated under the bars. (a) UACR was measured in urine collected for 24h at day 28. (b) UACR vs free steady average plasma drug exposure normalized to rat <i>in vitro</i> IC₅₀ (C_uss-average/<i>in vitro</i> IC₅₀). Drug exposure levels correspond to the exposure levels achieved at the studied doses (a). c) Renal pathology scores (renal fibrosis and glomerular nephritis). d) Histological sections stained with PAS.Representative photomicrographs of glomeruli/tubular sections from rats treated with vehicle (V), AZD9977 at 10 (AZ 10), 30 (AZ 30) or 100 mg kg^-1 d^-1 (AZ 100) or eplerenone at 10 (EP 10) or 30 mg kg^-1 d^-1 (EP 30). The scale bar corresponds to 200 μm original size. Average +/- SEM; <i>n</i> = 7–8; *p<0.05 compared to vehicle.</p