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.
•Diabetic kidney disease (DKD) is a disease of high unmet therapeutic need.•Several drugs are in clinical development for treatment of DKD.•Rodent models of DKD show variable human ...translatability.•Selection of a preclinical rodent model of DKD should be based on drug target.•Rodent models of advanced DKD improve the ability to predict clinical outcomes.
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.
IntroductionDiabetic 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 analysisIn 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 disseminationThe 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 numberNCT04916132.
Introduction: Emerging treatments of diabetic kidney disease (DKD) include SGLT2 inhibitors and GLP-1 receptor agonists that are nephroprotective beyond their blood glucose lowering effects. To ...confirm the translatability of a mouse model for drug discovery in DKD, we tested the efficacy of an ACE inhibitor and a SGLT2 inhibitor in the reninAAV UNx db/db mouse model.
Methods: Female db/db mice were injected with a renin-encoding adeno-associated virus construct (reninAAV) to induce hypertension and uninephrectomized (UNx). At 12 weeks of age, dosing with vehicle, lisinopril, empagliflozin, or the combination (combo) was initiated. Plasma and urine markers were measured after 12 weeks of dosing and terminal kidney samples were collected for 3D light sheet microscopy and 2D histology.
Results: In reninAAV UNx db/db mice, treatment with empagliflozin and combo reduced fed BG and HbA1c compared to vehicle. Treatment with lisinopril and combo reduced urine ACR and KIM1-to-creatinine compared to vehicle, while treatment with empagliflozin alone worsened urine ACR. Glomerular hypertrophy as assessed by 3D imaging was reduced in combo treated reninAAV UNx db/db mice compared to vehicle, while treatment with empagliflozin alone worsened glomerular hypertrophy. The total number of glomeruli per kidney was unaffected by treatments. Compared to vehicle treatment, lisinopril and combo treatment reduced the fraction of score 3+4 glomeruli, and glomerulosclerosis index (GSI) was reduced by with lisinopril and combo treatment. Treatment with empagliflozin alone worsened GSI. Morphometric analyses showed that lisinopril and combo treatment reduced kidney CD11b and KIM1 load.
Conclusion: Responses to the combination treatment with lisinopril and empagliflozin showed improvement of urine and histological markers of DKD. Together, these data confirm the translatability of the reninAAV UNx db/db mouse model of DKD.
Disclosure
M. V. Østergaard: None. M. Christensen: None. T. Secher: Employee; Self; Gubra, Employee; Spouse/Partner; Novo Nordisk, Stock/Shareholder; Self; Gubra, Stock/Shareholder; Spouse/Partner; Novo Nordisk. J. L. Skytte: None. U. Roostalu: Employee; Self; Gubra. C. G. Salinas: Employee; Self; Gubra, Employee; Spouse/Partner; Novo Nordisk, Stock/Shareholder; Spouse/Partner; Novo Nordisk. F. E. Sembach: None. L. N. Fink: Employee; Self; Gubra, Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra, Employee; Self; Gubra, Stock/Shareholder; Self; Gubra.
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.
Introduction: Diabetic nephropathy (DN) is a long-term complication that occurs in ∼40% of diabetes patients and is a leading cause of end-stage renal disease. In a newly established mouse model of ...progressive DN, we investigated the effects of hypertension on kidney injury.
Methods: Female db/db mice were uninephrectomized (UNx) at 8 weeks of age and injected i.v. with a Renin adeno-associated virus (AAV) construct at different doses to induce hypertension, while a LacZAAV construct was used as negative control. Sham-operated db/+ mice served as healthy controls. Blood pressure and glomerular filtration rate (GFR) was measured at 22 weeks of age, while urine albumin to creatinine ratio (ACR) was measured before termination and kidney tissue sampling 24 weeks of age.
Results: GFR measurements indicated hyperfiltration in all AAV-injected UNx db/db mice compared to db/+ mice, while ReninAAV tended to dose-dependently decrease GFR compared to LacZAAV in UNx db/db mice. Urine ACR was worsened by ReninAAV-induced hypertension compared to LacZAAV controls. 3D kidney imaging demonstrated increased glomerular volume in LacZAAV UNx db/db mice compared to db/+ mice with no further effect in ReninAAV groups. Automized AI-based glomerulosclerosis scoring showed ReninAAV dose-dependent increases in glomerulosclerosis compared to LacZAAV controls. RNA sequencing revealed upregulated gene expression markers of kidney fibrogenesis (Col1a1, Col4, Fn1) and tubular injury (Ngal and Kim-1), as well as downregulation of proximal tubular markers (Megalin, Aqp1) in ReninAAV UNx db/db mice compared to LacZAAV controls.
Conclusion: ReninAAV-induced hypertension in female UNx db/db mice aggravates albuminuria and glomerulosclerosis paralleled by increased expression of genes associated with tubular injury renal fibrosis, thus confirming that ReninAAV UNx db/db mice is a reliable model of DN with features of late stage human disease.
Disclosure
M.V. Østergaard: None. I.R. Sørensen: None. A.A. Pedersen: None. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Gubra. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. F.E. Sembach: Employee; Self; Gubra. M.R. Madsen: None. K. Fosgerau: None. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra. Employee; Self; Gubra. Stock/Shareholder; Self; Gubra.
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.
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. We find that male and female UNx db/db mice developed similar hallmarks of DN pathology.
Glomerular hypertrophy is a hallmark of kidney injury in metabolically induced renal diseases such as obesity-associated glomerulopathies and diabetic nephropathy (DN).
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.
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
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.
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.
Diabetic nephropathy (DN) is a serious complication of diabetes and the leading cause of end-stage renal disease. To reproducibly test novel therapies, there is a need for precise quantification of ...DN pathology in rodent models. We compared the accelerated development of DN in male and female uninephrectomized db/db mice using advanced imaging techniques. Unilateral nephrectomy (UNx) was performed in 7-8 weeks old male and female db/db mice. Sham-operated db/+ mice served as controls. Kidneys were preserved for histology and stereology. In a separate study, UNx was performed in 18 weeks old male db/db mice. These mice and control db/+ mice were injected with lectin-594 prior to termination to visualize glomerular morphology by 3D light sheet microscopy. All mice were terminated at 24 weeks of age. Male and female db/db UNx mice showed comparable increases in albuminuria. Total glomerular volume was markedly higher in male and female db/db UNx mice compared to db/+ controls (mean males; 4.42 vs. 2.71 mm3; p<0.001; females; 5.04 vs. 2.69 mm3; p<0.001). Tubulointerstitial collagen III immuno-staining was significantly increased only in female db/db UNx mice compared to db/+ mice. In contrast, total glomerular collagen IV levels were increased in male db/db UNx mice compared to db/+ controls (mean 1.30 vs. 0.80 mm3; p<0.01), and female db/db UNx mice displayed increased glomerular collagen IV compared to male db/db UNx mice (mean 1.80 vs. 1.30 mm3; p<0.05). 3D imaging at 24 weeks following UNx at 18 weeks of age confirmed expansion in glomerular volume (median db/+ 96.000 vs. db/db 145.000 µm3). Moreover, a marked increase was seen in cortical lectin-594 staining, indicating glomerular leakage in db/db UNx mice.
In conclusion, male and female db/db UNx mice displayed comparable features of DN and both constitute valid models for DN. The use of whole-kidney 3D imaging provides a novel avenue for assessment of individual glomerular volume and permeability.
Disclosure
L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. F.E. Sembach: Consultant; Self; Novo Nordisk A/S. Employee; Self; Gubra. U. Roostalu: Employee; Self; Gubra. T. Johansen: Employee; Self; Gubra. J.L. Skytte: Employee; Self; Gubra. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Gubra. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. K. Fosgerau: Stock/Shareholder; Self; Gubra. N. Vrang: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. J. Jelsing: Stock/Shareholder; Self; Gubra. J. Hecksher-Sørensen: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. T.X. Pedersen: Employee; Self; Gubra.
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