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.

Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

AIM: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. MAIN METHODS: We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. KEY FINDINGS: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. SIGNIFICANCE: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

High-protein diet accelerates diabetes and kidney disease in the BTBRob/ob mouse.

There is a need for improved animal models that better translate to human kidney disease to predict outcome of pharmacological effects in the patient. The diabetic BTBRob/ob mouse model mimics key features of early diabetic nephropathy in humans, but with chronic injury limited to glomeruli. To explore if we could induce an accelerated and more advanced disease phenotype that closer translates to human disease, we challenged BTBRob/ob mice with a high-protein diet (HPD; 30%) and followed the progression of metabolic and renal changes up to 20 wk of age. Animals on the HPD showed enhanced metabolic derangements, evidenced by further increased levels of glucose, HbA1C, cholesterol, and alanine aminotransferase. The urinary albumin-to-creatinine ratio was markedly increased with a 53-fold change compared with lean controls, whereas BTBRob/ob mice on the standard diet only presented an 8-fold change. HPD resulted in more advanced mesangial expansion already at 14 wk of age compared with BTBRob/ob mice on the standard diet and also aggravated glomerular pathology as well as interstitial fibrosis. Gene expression analysis revealed that HPD triggered expression of markers of fibrosis and inflammation in the kidney and increased oxidative stress markers in urine. This study showed that HPD significantly aggravated renal injury in BTBRob/ob mice by further advancing albuminuria, glomerular, and tubulointerstitial pathology by 20 wk of age. This mouse model offers closer translation to humans and enables exploration of new end points for pharmacological efficacy studies that also holds promise to shorten study length.

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

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 in vitro 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 in vitro 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.

The fungal nephrotoxin orellanine simultaneously increases oxidative stress and down-regulates cellular defenses.

Confusion of various nephrotoxic Cortinarius species with edible mushrooms occurs every year throughout Europe and North America. The toxin, orellanine (OR), accumulates selectively in renal tubular epithelium with ensuing renal failure after several days as the only clinical manifestation. This study was performed to clarify the mechanisms behind the kidney damage. Sprague-Dawley rats, 100 g bw, received various doses of purified OR ip (0-5 mg/kg bw). One week later, renal function (GFR) was determined (51Cr-EDTA), ascorbyl radicals in venous blood were analyzed using electron spin resonance, and oxidative protein damage was evaluated immunohistochemically. One OR-treated group (3.5 mg/kg) simultaneously received superoxide dismutase (SOD) targeted to tubular epithelium (HC-SOD; 10 mg/kg ip daily for 5 days). RT-PCR was used for analysis of mRNA expression of genes related to oxidative stress. OR caused a dose-dependent decrease in GFR, paralleled by increased levels of ascorbyl radicals and oxidative protein damage. Antioxidant treatment with HC-SOD decreased renal function even more and also increased tissue damage and mortality. Renal mRNA levels for key components in the antioxidative defense were strongly decreased, whereas those for several cytokines were increased. The data strongly suggest that OR nephrotoxicity in vivo is mediated by oxidative stress, including a virtual shutdown of important antioxidative enzymes. We interpret the unexpected effect of HC-SOD in terms of unbalanced SOD and catalase levels in the presence of OR, leading to massive generation of *OH and cell death.