Rationale and design of a prospective, clinical study of kidney biopsies in people with type 2 diabetes and severely increased albuminuria (the PRIMETIME 2 study).

INTRODUCTION: Diabetic kidney disease is a severe complication of diabetes. The diagnosis is based on clinical characteristics such as persistently elevated albuminuria, hypertension and decline in kidney function, although this definition is not specific to kidney disease caused by diabetes. The only way to establish an accurate diagnosis-diabetic nephropathy-is by performing a kidney biopsy. The histological presentation of diabetic nephropathy can be associated with a heterogeneous range of histological features with many pathophysiological factors involved demonstrating the complexity of the condition. Current treatment strategies aim to slow disease progression and are not specific to the underlying pathological processes.This study will investigate the prevalence of diabetic nephropathy in individuals with type 2 diabetes (T2D) and severely elevated albuminuria. The deep molecular characterisation of the kidney biopsy and biological specimens may pave the way for improved diagnostic accuracy and a better understanding of the pathological processes involved and may also reveal new targets for individualised treatment. METHODS AND ANALYSIS: In the PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy 2 study, research kidney biopsies will be performed in 300 participants with T2D, urine albumin/creatinine ratio ≥700 mg/g and estimated glomerular filtration ratio >30 mL/min/1.73 m2. Cutting-edge molecular technologies will be applied to the kidney, blood, urine, faeces and saliva samples for comprehensive multi-omics profiling. The associated disease course and clinical outcomes will be assessed by annual follow-up for 20 years. ETHICS AND DISSEMINATION: The Danish Regional Committee on Health Research Ethics and the Knowledge Center on Data Protection (in the Capital Region of Denmark) have granted approval for the study. The results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04916132.

Integrative transcriptomic profiling of a mouse model of hypertension-accelerated diabetic kidney disease.

The current understanding of molecular mechanisms driving diabetic kidney disease (DKD) is limited, partly due to the complex structure of the kidney. To identify genes and signalling pathways involved in the progression of DKD, we compared kidney cortical versus glomerular transcriptome profiles in uninephrectomized (UNx) db/db mouse models of early-stage (UNx only) and advanced [UNxplus adeno-associated virus-mediated renin-1 overexpression (UNx-Renin)] DKD using RNAseq. Compared to normoglycemic db/m mice, db/db UNx and db/db UNx-Renin mice showed marked changes in their kidney cortical and glomerular gene expression profiles. UNx-Renin mice displayed more marked perturbations in gene components associated with the activation of the immune system and enhanced extracellular matrix remodelling, supporting histological hallmarks of progressive DKD in this model. Single-nucleus RNAseq enabled the linking of transcriptome profiles to specific kidney cell types. In conclusion, integration of RNAseq at the cortical, glomerular and single-nucleus level provides an enhanced resolution of molecular signalling pathways associated with disease progression in preclinical models of DKD, and may thus be advantageous for identifying novel therapeutic targets in DKD.

Rodent models of diabetic kidney disease: human translatability and preclinical validity.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD). Except for SGLT2 inhibitors and GLP-1R agonists, there have been few changes in DKD treatment over the past 25 years, when multifactorial intervention was introduced in patients with type 2 diabetes mellitus (T2DM). The unmet clinical need is partly due to the lack of animal models that replicate clinical features of human DKD, which has raised concern about the utility of these models in preclinical drug discovery. In this review, we performed a comprehensive analysis of rodent models of DKD to compare treatment efficacy from preclinical testing with outcome from clinical trials. We also investigated whether rodent models are predictive for clinical outcomes of therapeutic agents in human DKD.

Liraglutide Improves the Kidney Function in a Murine Model of Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is a global health burden, and the current treatment options only slow down the disease progression. GLP-1 receptor agonists (GLP-1 RA) have shown a renal protective effect in models of CKD; however, the mechanism behind the beneficial effect is not understood. In this study, we investigate the effect of the GLP-1 RA liraglutide in the nephrotoxic serum nephritis (NTN) CKD model. Moreover, we compare the gene expression pattern of liraglutide-treated mice to the gene expression pattern of mice treated with the angiotensin converting enzyme inhibitor, enalapril. METHODS: The effect of liraglutide was tested in the NTN model by evaluating the glomerular filtration rate (GFR), albuminuria, mesangial expansion, renal fibrosis, and renal inflammation. Furthermore, the regulation of selected genes involved in CKD and in glomerular, cortical tubulointerstitial, and whole kidney structures was analyzed using a gene expression array on samples following laser capture microdissection. RESULTS: Treatment with liraglutide improved CKD hallmarks including GFR, albuminuria, mesangial expansion, renal inflammation, and renal fibrosis. The gene expression revealed that both liraglutide and enalapril reversed the regulation of several fibrosis and inflammation associated genes, which are also regulated in human CKD patients. Furthermore, liraglutide and enalapril both regulated genes in the kidney involved in blood pressure control. CONCLUSIONS: Treatment with liraglutide improved the kidney function and diminished renal lesions in NTN-induced mice. Both liraglutide and enalapril reversed the regulation of genes involved in CKD and regulated genes involved in blood pressure control.

Automated Image Analyses of Glomerular Hypertrophy in a Mouse Model of Diabetic Nephropathy.

Background: Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN). Methods: Using light sheet fluorescent microscopy (LSFM) and 3D image analysis, we tested algorithms for automated and unbiased quantification of total glomerular numbers and individual glomerular volume in the uninephrectomized (UNx) db/db mouse model of DN. Results: At 6 weeks after surgery, db/db and UNx db/db mice showed increased urine albumin-to-creatinine ratio (ACR) compared with db/+ control mice. Before euthanasia, glomeruli were labeled in vivo by injecting tomato lectin. Whole-kidney LSFM 3D image analysis revealed that mean glomerular volume was significantly increased in UNx db/db mice compared with db/+ mice. Moreover, analysis of individual glomerular volume showed a shift in volume distribution toward larger glomeruli and thereby demonstrated additive effects of diabetes and UNx on induction of glomerular hypertrophy. The automatized quantification showed no significant differences in glomerular numbers among db/+, db/db, and UNx db/db mice. These data correlated with glomerular numbers as quantified by subsequent stereologic quantification. Conclusions: Overall, LSFM coupled with automated 3D histomorphometric analysis was demonstrated to be advantageous for unbiased assessment of glomerular volume and numbers in mouse whole-kidney samples. Furthermore, we showed that injection of fluorescently labeled lectin and albumin can be used as markers of nephron segments in the mouse kidneys, thus enabling functional assessment of kidney physiology, pathology, and pharmacology in preclinical rodent models of kidney disease.

Impact of sex on diabetic nephropathy and the renal transcriptome in UNx db/db C57BLKS mice.

Diabetic nephropathy (DN) is associated with albuminuria and loss of kidney function and is the leading cause of end-stage renal disease. Despite evidence of sex-associated differences in the progression of DN in human patients, male mice are predominantly being used in preclinical DN research and drug development. Here, we compared renal changes in male and female uninephrectomized (UNx) db/db C57BLKS mice using immunohistochemistry and RNA sequencing. Male and female UNx db/db mice showed similar progression of type 2 diabetes, as assessed by obesity, hyperglycemia, and HbA1c. Progression of DN was also similar between sexes as assessed by kidney and glomerular hypertrophy as well as urine albumin-to-creatinine ratio being increased in UNx db/db compared with control mice. In contrast, kidney collagen III and glomerular collagen IV were increased only in female UNx db/db as compared with respective control mice but showed a similar tendency in male UNx db/db mice. Comparison of renal cortex transcriptomes by RNA sequencing revealed 66 genes differentially expressed (p < .01) in male versus female UNx db/db mice, of which 9 genes were located on the sex chromosomes. In conclusion, male and female UNx db/db mice developed similar hallmarks of DN pathology, suggesting no or weak sex differences in the functional and structural changes during DN progression.

Temporal Regulation of Glomerular and Cortical Tubulointerstitial Genes Involved in the Development of Nephrotoxic Serum Nephritis.

BACKGROUND/AIMS: Murine nephrotoxic nephritis (NTN) is a well-established model resembling chronic kidney disease. Investigating gene expression patterns separately in the glomerular and cortical tubulointerstitial structure could provide new knowledge about structure-specific changes in expression of genes in the NTN model. METHODS: Glomerular, cortical tubulointerstitial and whole kidney tissues from mice subjected to nephrotoxic serum (NTS) or phosphate buffered saline (PBS) were collected on day 7, 21 and 42 using laser microdissection (LMD). Total RNA was extracted and subjected to nCounter NanoString. Histology, immunohistochemistry, in situ hybridization and/or quantitative real time PCR (qRT PCR) were performed to confirm regulation of selected genes. RESULTS: LMD provided detailed information about genes that were regulated differently between structures over time. Some of the fibrotic and inflammatory genes (Col1a1, Col3a1 and Ccl2) were upregulated in both structures, whereas other genes such as Spp1 and Grem1 were differentially regulated suggesting spatial pathogenic mechanisms in the kidney. Downregulation of cortical tubulointerstitium genes involved in iron metabolism was detected along with iron accumulation. CONCLUSION: This study demonstrates several regulated genes in pathways important for the pathogenesis of the NTN model and that LMD identifies structure-specific changes in gene expression during disease development. Furthermore, this study shows the benefits of isolating glomeruli and cortical tubulointerstitium in order to identify gene regulation.

CCHamide-2 Is an Orexigenic Brain-Gut Peptide in Drosophila.

The neuroendocrine peptides CCHamide-1 and -2, encoded by the genes ccha1 and -2, are produced by endocrine cells in the midgut and by neurons in the brain of Drosophila melanogaster. Here, we used the CRISPR/Cas9 technique to disrupt the ccha1 and -2 genes and identify mutant phenotypes with a focus on ccha-2 mutants. We found that both larval and adult ccha2 mutants showed a significantly reduced food intake as measured in adult flies by the Capillary Feeding (CAFE) assay (up to 72% reduced food intake compared to wild-type). Locomotion tests in adult flies showed that ccha2 mutants had a significantly reduced locomotor activity especially around 8 a.m. and 8 p.m., where adult Drosophila normally feeds (up to 70% reduced locomotor activity compared to wild-type). Reduced larval feeding is normally coupled to a delayed larval development, a process that is mediated by insulin. Accordingly, we found that the ccha2 mutants had a remarkably delayed development, showing pupariation 70 hours after the pupariation time point of the wild-type. In contrast, the ccha-1 mutants were not developmentally delayed. We also found that the ccha2 mutants had up to 80% reduced mRNA concentrations coding for the Drosophila insulin-like-peptides-2 and -3, while these concentrations were unchanged for the ccha1 mutants. From these experiments we conclude that CCHamide-2 is an orexigenic peptide and an important factor for controlling developmental timing in Drosophila.