Therapeutic effects of lisinopril and empagliflozin in a mouse model of hypertension-accelerated diabetic kidney disease.

Hypertension is a critical comorbidity for progression of diabetic kidney disease (DKD). To facilitate the development of novel therapeutic interventions with the potential to control disease progression, there is a need to establish translational animal models that predict treatment effects in human DKD. The present study aimed to characterize renal disease and outcomes of standard of medical care in a model of advanced DKD facilitated by adeno-associated virus (AAV)-mediated renin overexpression in uninephrectomized (UNx) db/db mice. Five weeks after single AAV administration and 4 wk after UNx, female db/db UNx-ReninAAV mice received (PO, QD) vehicle, lisinopril (40 mg/kg), empagliflozin (20 mg/kg), or combination treatment for 12 wk (n = 17 mice/group). Untreated db/+ mice (n = 8) and vehicle-dosed db/db UNx-LacZAAV mice (n = 17) served as controls. End points included plasma, urine, and histomorphometric markers of kidney disease. Total glomerular numbers and individual glomerular volume were evaluated by whole kidney three-dimensional imaging analysis. db/db UNx-ReninAAV mice developed hallmarks of progressive DKD characterized by severe albuminuria, advanced glomerulosclerosis, and glomerular hypertrophy. Lisinopril significantly improved albuminuria, glomerulosclerosis, tubulointerstitial injury, and inflammation. Although empagliflozin alone had no therapeutic effect on renal endpoints, lisinopril and empagliflozin exerted synergistic effects on renal histological outcomes. In conclusion, the db/db UNx-ReninAAV mouse demonstrates good clinical translatability with respect to physiological and histological hallmarks of progressive DKD. The efficacy of standard of care to control hypertension and hyperglycemia provides a proof of concept for testing novel drug therapies in the model.NEW & NOTEWORTHY Translational animal models of diabetic kidney disease (DKD) are important tools in preclinical research and drug discovery. Here, we show that the standard of care to control hypertension (lisinopril) and hyperglycemia (empagliflozin) improves physiological and histopathological hallmarks of kidney disease in a mouse model of hypertension-accelerated progressive DKD. The findings substantiate hypertension and type 2 diabetes as essential factors in driving DKD progression and provide a proof of concept for probing novel drugs for potential nephroprotective efficacy in this model.

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor.

AIMS/HYPOTHESIS: Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. METHODS: Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. RESULTS: Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. CONCLUSIONS/INTERPRETATION: Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.

DBA2J db/db mice are susceptible to early albuminuria and glomerulosclerosis that correlate with systemic insulin resistance.

Diabetic nephropathy (DN) is the leading cause of kidney failure in the world. To understand important mechanisms underlying this condition, and to develop new therapies, good animal models are required. In mouse models of type 1 diabetes, the DBA/2J strain has been shown to be more susceptible to develop kidney disease than other common strains. We hypothesized this would also be the case in type 2 diabetes. We studied db/db and wild-type (wt) DBA/2J mice and compared these with the db/db BLKS/J mouse, which is currently the most widely used type 2 DN model. Mice were analyzed from age 6 to 12 wk for systemic insulin resistance, albuminuria, and glomerular histopathological and ultrastructural changes. Body weight and nonfasted blood glucose were increased by 8 wk in both genders, while systemic insulin resistance commenced by 6 wk in female and 8 wk in male db/db DBA/2J mice. The urinary albumin-to-creatinine ratio (ACR) was closely linked to systemic insulin resistance in both sexes and was increased ~50-fold by 12 wk of age in the db/db DBA/2J cohort. Glomerulosclerosis, foot process effacement, and glomerular basement membrane thickening were observed at 12 wk of age in db/db DBA/2J mice. Compared with db/db BLKS/J mice, db/db DBA/2J mice had significantly increased levels of urinary ACR, but similar glomerular histopathological and ultrastructural changes. The db/db DBA/2J mouse is a robust model of early-stage albuminuric DN, and its levels of albuminuria correlate closely with systemic insulin resistance. This mouse model will be helpful in defining early mechanisms of DN and ultimately the development of novel therapies.

α1,2-Fucosyllactose Does Not Improve Intestinal Function or Prevent Escherichia coli F18 Diarrhea in Newborn Pigs.

OBJECTIVES: Infectious diarrhea, a leading cause of morbidity and deaths, is less prevalent in breastfed infants compared with infants fed infant formula. The dominant human milk oligosaccharide (HMO), α-1,2-fucosyllactose (2'-FL), has structural homology to bacterial adhesion sites in the intestine and may in part explain the protective effects of human milk. We hypothesized that 2'-FL prevents diarrhea via competitive inhibition of pathogen adhesion in a pig model for sensitive newborn infants. METHODS: Intestinal cell studies were coupled with studies on cesarean-delivered newborn pigs (n = 24) without (control) or with inoculation of enterotoxigenic Escherichia coli F18 (7.5 × 10/day for 8 days) fed either no (F18) or 10 g/L 2'-FL (2FL-F18). RESULTS: In vitro studies revealed decreased pathogen adhesion to intestinal epithelial cells with 2'-FL (5 g/L; P < 0.001). F18 pigs showed more diarrhea than control pigs (P < 0.01). Administration of 2'-FL to F18 pigs failed to prevent diarrhea, although the relative weight loss tended to be reduced (-19 vs -124 g/kg, P = 0.12), higher villi were observed in the distal small intestine (P < 0.05), and a trend toward increased proportion of mucosa and activities of some brush border enzymes in the proximal small intestine. In situ abundance of α-1,2-fucose and E coli was similar between groups, whereas sequencing showed higher abundance of Enterobacteriaceae in F18, Enterococcus in control and Lachnospiraceae in 2FL-F18 pigs. CONCLUSIONS: 2'-FL inhibited in vitro adhesion of E coli F18 to epithelial cells, but had limited effects on diarrhea and mucosal health in newborn pigs challenged with E coli F18.

Bovine colostrum improves neonatal growth, digestive function, and gut immunity relative to donor human milk and infant formula in preterm pigs.

Mother's own milk is the optimal first diet for preterm infants, but donor human milk (DM) or infant formula (IF) is used when supply is limited. We hypothesized that a gradual introduction of bovine colostrum (BC) or DM improves gut maturation, relative to IF during the first 11 days after preterm birth. Preterm pigs were fed gradually advancing doses of BC, DM, or IF (3-15 ml·kg(-1)·3 h(-1), n = 14-18) before measurements of gut structure, function, microbiology, and immunology. The BC pigs showed higher body growth, intestinal hexose uptake, and transit time and reduced diarrhea and gut permeability, relative to DM and IF pigs (P < 0.05). Relative to IF pigs, BC pigs also had lower density of mucosa-associated bacteria and of some putative pathogens in colon, together with higher intestinal villi, mucosal mass, brush-border enzyme activities, colonic short chain fatty acid levels, and bacterial diversity and an altered expression of immune-related genes (higher TNFα, IL17; lower IL8, TLR2, TFF, MUC1, MUC2) (all P < 0.05). Values in DM pigs were intermediate. Severe necrotizing enterocolitis (NEC) was observed in >50% of IF pigs, while only subclinical intestinal lesions were evident from DM and BC pigs. BC, and to some degree DM, are superior to preterm IF in stimulating gut maturation and body growth, using a gradual advancement of enteral feeding volume over the first 11 days after preterm birth in piglets. Whether the same is true in preterm infants remains to be tested.

Minimal short-term effect of dietary 2'-fucosyllactose on bacterial colonisation, intestinal function and necrotising enterocolitis in preterm pigs.

Human milk decreases the risk of necrotising enterocolitis (NEC), a severe gastrointestinal disease that occurs in 5-10 % of preterm infants. The prebiotic and immune-modulatory effects of milk oligosaccharides may contribute to this protection. Preterm pigs were used to test whether infant formula enriched with α1,2-fucosyllactose (2'-FL, the most abundant oligosaccharide in human milk) would benefit gut microbial colonisation and NEC resistance after preterm birth. Caesarean-delivered preterm pigs were fed formula (Controls, n 17) or formula with 5 g/l 2'-FL (2'-FL, n 16) for 5 d; eight 2'-FL pigs (50 %) and twelve Controls (71 %) developed NEC, with no difference in lesion scores (P=0·35); 2'-FL pigs tended to have less anaerobic bacteria in caecal contents (P=0·22), but no difference in gut microbiota between groups were observed by fluorescence in situ hybridisation and 454 pyrosequencing. Abundant α1,2-fucose was detected in the intestine with no difference between groups, and intestinal structure (villus height, permeability) and digestive function (hexose absorption, brush border enzyme activities) were not affected by 2'-FL. Formula enrichment with 2'-FL does not affect gut microbiology, digestive function or NEC sensitivity in pigs within the first few days after preterm birth. Milk 2'-FL may not be critical in the immediate postnatal period of preterm neonates when gut colonisation and intestinal immunity are still immature.

Early gradual feeding with bovine colostrum improves gut function and NEC resistance relative to infant formula in preterm pigs.

It is unclear when and how to start enteral feeding for preterm infants when mother's milk is not available. We hypothesized that early and slow advancement with either formula or bovine colostrum stimulates gut maturation and prevents necrotizing enterocolitis (NEC) in preterm pigs, used as models for preterm infants. Pigs were given either total parenteral nutrition (TPN, n = 14) or slowly advancing volumes (16-64 ml·kg(-1)·day(-1)) of preterm infant formula (IF, n = 15) or bovine colostrum (BC, n = 13), both given as adjunct to parenteral nutrition. On day 5, both enteral diets increased intestinal mass (27 ± 1 vs. 22 ± 1 g/kg) and glucagon-like peptide 2 release, relative to TPN (P < 0.05). The incidence of mild NEC lesions was higher in IF than BC and TPN pigs (60 vs. 0 and 15%, respectively, P < 0.05). Only the IF pigs showed reduced gastric emptying and gastric inhibitory polypeptide release, and increased tissue proinflammatory cytokine levels (IL-1ß and IL-8, P < 0.05) and expression of immune-related genes (AOAH, LBP, CXCL10, TLR2), relative to TPN. The IF pigs also showed reduced intestinal villus-to-crypt ratio, lactose digestion, and some plasma amino acids (Arg, Cit, Gln, Tyr, Val), and higher intestinal permeability, compared with BC pigs (all P < 0.05). Colonic microbiota analyses showed limited differences among groups. Early feeding with formula induces intestinal dysfunction whereas bovine colostrum supports gut maturation when mother's milk is absent during the first week after preterm birth. A diet-dependent feeding guideline may be required for newborn preterm infants.

Preterm Birth Reduces Nutrient Absorption With Limited Effect on Immune Gene Expression and Gut Colonization in Pigs.

OBJECTIVES: The primary risk factors for necrotizing enterocolitis (NEC) are preterm birth, enteral feeding, and gut colonization. It is unclear whether feeding and colonization induce excessive expression of immune genes that lead to NEC. Using a pig model, we hypothesized that reduced gestational age would upregulate immune-related genes and cause bacterial imbalance after birth. METHODS: Preterm (85%-92% gestation, n = 53) and near-term (95%-99% gestation, n = 69) pigs were delivered by cesarean section and euthanized at birth or after 2 days of infant formula or bovine colostrum feeding. RESULTS: At birth, preterm delivery reduced 5 of 30 intestinal genes related to nutrient absorption and innate immunity, relative to near-term pigs, whereas 2 genes were upregulated. Preterm birth also reduced ex vivo intestinal glucose and leucine uptake (40%-50%), but failed to increase cytokine secretions from intestinal explants relative to near-term birth. After 2 days of formula feeding, NEC incidence was increased in preterm versus near-term pigs (47% vs 0%-13%). A total of 6 of the 30 genes related to immunity (TLR2, IL1B, and IL8), permeability (CLDN3, and OCLN), and absorption (SGLT) decreased in preterm pigs without affecting Gram-negative bacteria-related responses (TLR4, IKBA, NFkB1, TNFAIP3, and PAFA). Bacterial abundance tended to be higher in preterm versus near-term pigs (P = 0.09), whereas the composition was unaffected. CONCLUSIONS: Preterm birth predisposes to NEC and reduces nutrient absorption but does not induce upregulation of immune-related genes or cause bacterial dyscolonization in the neonatal period. Excessive inflammation and bacterial overgrowth may occur relatively late in NEC progression in preterm neonates.

Transforming growth factor-ß2 and endotoxin interact to regulate homeostasis via interleukin-8 levels in the immature intestine.

A balance between pro- and anti-inflammatory signals from milk and microbiota controls intestinal homeostasis just after birth, and an optimal balance is particularly important for preterm neonates that are sensitive to necrotizing enterocolitis (NEC). We suggest that the intestinal cytokine IL-8 plays an important role and hypothesize that transforming growth factor-ß2 (TGF-ß2) acts in synergy with bacterial lipopolysaccharide (LPS) to control IL-8 levels, thereby supporting intestinal homeostasis. Preterm pigs were fed colostrum (containing TGF-ß2) or infant formula (IF) with or without antibiotics (COLOS, n = 27; ANTI, n = 11; IF, n = 40). Intestinal IL-8 levels and NEC incidence were much higher in IF than in COLOS and ANTI pigs (P < 0.001), but IL-8 levels did not correlate with NEC severity. Intestinal TGF-ß2 levels were high in COLOS but low in IF and ANTI pigs. Based on these observations, the interplay among IL-8, TGF-ß2, and LPS was investigated in a porcine intestinal epithelial cell line. TGF-ß2 attenuated LPS-induced IL-6, IL-1ß, and TNF-α release by reducing early ERK activation, whereas IL-8 secretion was synergistically induced by LPS and TGF-ß2 via NF-κB. The TGF-ß2/LPS-induced IL-8 levels stimulated cell proliferation and migration following epithelial injury, without continuous NF-κB activation and cyclooxygenase-2 expression. We suggest that a combined TGF-ß2-LPS induction of IL-8 stimulates epithelial repair just after birth when the intestine is first exposed to colonizing bacteria and TGF-ß2-containing milk. Moderate IL-8 levels may act to control intestinal inflammation, whereas excessive IL-8 production may enhance the damaging proinflammatory cascade leading to NEC.

Postnatal amniotic fluid intake reduces gut inflammatory responses and necrotizing enterocolitis in preterm neonates.

Preterm neonates are susceptible to gastrointestinal disorders such as necrotizing enterocolitis (NEC). Maternal milk and colostrum protects against NEC via growth promoting, immunomodulatory, and antimicrobial factors. The fetal enteral diet amniotic fluid (AF), contains similar components, and we hypothesized that postnatal AF administration reduces inflammatory responses and NEC in preterm neonates. Preterm pigs (92% gestation) were delivered by caesarean section and fed parental nutrition (2 days) followed by enteral (2 days) porcine colostrum (COLOS, n = 7), infant formula (FORM, n = 13), or AF supplied before and after introduction of formula (AF, n = 10) in experiment 1, and supplied only during the enteral feeding period in experiment 2 (FORM, n = 16; AF, n = 14). The NEC score was reduced in both AF and COLOS pigs, relative to FORM, when AF was provided prior to full enteral feeding (9.9 and 7.7 compared with 17.3, P < 0.05). There was no effect of AF when provided only during enteral feeding. AF pigs showed decreased bacterial abundance in colon and intestinal inflammation-related genes (e.g., TNF-α, IL-1α, IL-6, NOS) were downregulated, relative to FORM pigs with NEC. Anti-inflammatory properties of AF were supported by delayed maturation and decreased TNF-α production in murine dendritic cells, as well as increased proliferation and migration, and downregulation of IL-6 expression in intestinal cells (IEC-6, IPEC-J2). Like colostrum, AF may reduce NEC development in preterm neonates by suppressing the proinflammatory responses to enteral formula feeding and gut colonization when provided before the onset of NEC.

Whey protein processing influences formula-induced gut maturation in preterm pigs.

Immaturity of the gut predisposes preterm infants to nutritional challenges potentially leading to clinical complications such as necrotizing enterocolitis. Feeding milk formulas is associated with greater risk than fresh colostrum or milk, probably due to loss of bioactive proteins (e.g., immunoglobulins, lactoferrin, insulin-like growth factor, transforming growth factor-ß) during industrial processing (e.g., pasteurization, filtration, spray-drying). We hypothesized that the processing method for whey protein concentrate (WPC) would affect gut maturation in formula-fed preterm pigs used as a model for preterm infants. Fifty-five caesarean-delivered preterm pigs were distributed into 4 groups given 1 of 4 isoenergetic diets: formula containing conventional WPC (filtration, multi-pasteurization, standard spray-drying) (CF); formula containing gently treated WPC (reduced filtration and pasteurization, gentle spray-drying) (GF); formula containing minimally treated WPC (rennet precipitation, reduced filtration, heat treatment <40°C, freeze-drying) (MF); and bovine colostrum (used as a positive reference group) (BC). Relative to CF, GF, and MF pigs, BC pigs had greater villus heights, lactose digestion, and absorption and lower gut permeability (P < 0.05). MF and BC pigs had greater plasma citrulline concentrations than CF and GF pigs and intestinal interleukin-8 was lower in BC pigs than in the other groups (P < 0.05). MF pigs had lower concentrations of intestinal claudin-4, cleaved caspase-3, and phosphorylated c-Jun than CF pigs (P < 0.05). The conventional and gently treated WPCs had similar efficacy in stimulating proliferation of porcine intestinal epithelial cells. We conclude that processing of WPC affects intestinal structure, function, and integrity when included in formulas for preterm pigs. Optimization of WPC processing technology may be important to preserve the bioactivity and nutritional value of formulas for sensitive newborns.

Nephroprotective Effects of Semaglutide as Mono- and Combination Treatment with Lisinopril in a Mouse Model of Hypertension-Accelerated Diabetic Kidney Disease.

Background: Obesity, hyperglycemia and hypertension are critical risk factors for development of diabetic kidney disease (DKD). Emerging evidence suggests that glucagon-like peptide-1 receptor (GLP-1R) agonists improve cardiovascular and renal outcomes in type 2 diabetes patients. Here, we characterized the effect of the long-acting GLP-1R agonist semaglutide alone and in combination with an ACE inhibitor (lisinopril) in a model of hypertension-accelerated, advanced DKD facilitated by adeno-associated virus-mediated renin overexpression (ReninAAV) in uninephrectomized (UNx) female diabetic db/db mice. Methods: Female db/db mice received a single intravenous injection of ReninAAV 1 week prior to UNx. Six weeks post-nephrectomy, db/db UNx-ReninAAV mice were administered (q.d.) vehicle, semaglutide (30 nmol/kg, s.c.) or semaglutide (30 nmol/kg, s.c.) + lisinopril (30 mg/kg, p.o.) for 11 weeks. Endpoints included blood pressure, plasma/urine biochemistry, kidney histopathology and RNA sequencing. Results: Vehicle-dosed db/db UNx-ReninAAV mice developed hallmarks of DKD characterized by severe albuminuria and advanced glomerulosclerosis. Semaglutide robustly reduced hyperglycemia, hypertension and albuminuria concurrent with notable improvements in glomerulosclerosis severity, podocyte filtration slit density, urine/renal kidney injury molecule-1 (KIM-1) levels and gene expression markers of inflammation and fibrogenesis in db/db UNx-ReninAAV mice. Co-administration of lisinopril further ameliorated hypertension and glomerulosclerosis. Conclusions: Semaglutide improves disease hallmarks in the db/db UNx-ReninAAV mouse model of advanced DKD. Further benefits on renal outcomes were obtained by adjunctive antihypertensive standard of care. Collectively, our study supports the development of semaglutide for management of 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.

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.

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.

Rat pancreatectomy combined with isoprenaline or uninephrectomy as models of diabetic cardiomyopathy or nephropathy.

Cardiovascular and renal complications are the predominant causes of morbidity and mortality amongst patients with diabetes. Development of novel treatments have been hampered by the lack of available animal models recapitulating the human disease. We hypothesized that experimental diabetes in rats combined with a cardiac or renal stressor, would mimic diabetic cardiomyopathy and nephropathy, respectively. Diabetes was surgically induced in male Sprague Dawley rats by 90% pancreatectomy (Px). Isoprenaline (Iso, 1 mg/kg, sc., 10 days) was administered 5 weeks after Px with the aim of inducing cardiomyopathy, and cardiac function and remodeling was assessed by echocardiography 10 weeks after surgery. Left ventricular (LV) fibrosis was quantified by Picro Sirius Red and gene expression analysis. Nephropathy was induced by Px combined with uninephrectomy (Px-UNx). Kidney function was assessed by measurement of glomerular filtration rate (GFR) and urine albumin excretion, and kidney injury was evaluated by histopathology and gene expression analysis. Px resulted in stable hyperglycemia, hypoinsulinemia, decreased C-peptide, and increased glycated hemoglobin (HbA1c) compared with sham-operated controls. Moreover, Px increased heart and LV weights and dimensions and caused a shift from α-myosin heavy chain (MHC) to ß-MHC gene expression. Isoprenaline treatment, but not Px, decreased ejection fraction and induced LV fibrosis. There was no apparent interaction between Px and Iso treatment. The superimposition of Px and UNx increased GFR, indicating hyperfiltration. Compared with sham-operated controls, Px-UNx induced albuminuria and increased urine markers of kidney injury, including neutrophil gelatinase-associated lipocalin (NGAL) and podocalyxin, concomitant with upregulated renal gene expression of NGAL and kidney injury molecule 1 (KIM-1). Whereas Px and isoprenaline separately produced clinical endpoints related to diabetic cardiomyopathy, the combination of the two did not accentuate disease development. Conversely, Px in combination with UNx resulted in several clinical hallmarks of diabetic nephropathy indicative of early disease development.

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.

Human milk oligosaccharide effects on intestinal function and inflammation after preterm birth in pigs.

Human milk oligosaccharides (HMOs) may mediate prebiotic and anti-inflammatory effects in newborns. This is particularly important for preterm infants who are highly susceptible to intestinal dysfunction and necrotizing enterocolitis (NEC). We hypothesized that HMO supplementation of infant formula (IF) improves intestinal function, bacterial colonization and NEC resistance immediately after preterm birth, as tested in a preterm pig model. Mixtures of HMOs were investigated in intestinal epithelial cells and in preterm pigs (n=112) fed IF supplemented without (CON) or with a mixture of four HMOs (4-HMO) or >25 HMOs (25-HMO, 5-10 g/L given for 5 or 11 days). The 25-HMO blend decreased cell proliferation and both HMO blends decreased lipopolysaccharide-induced interleukin-8 secretion in IPEC-J2 cells, relative to control (P<.05). All HMOs were found in urine and feces of HMO-treated pigs, and short-chain fatty acids in the colon were higher in HMO vs. CON pigs (P<.05). After 5 days, NEC lesions were similar between HMO and CON pigs and 25-HMO increased colon weights (P<.01). After 11 days, the 4-HMO diet did not affect NEC (56 vs. 79%, P=.2) but increased dehydration and diarrhea (P<.05) and expression of immune-related genes (IL10, IL12, TGFß, TLR4; P<.05). Bacterial adherence and diversity was unchanged after HMO supplementation. CONCLUSION: Complex HMO-blends affect intestinal epithelial cells in vitro and gut gene expression and fermentation in preterm pigs. However, the HMOs had limited effects on NEC and diarrhea when supplemented to IF. Longer-term exposure to HMOs may be required to improve the immature intestinal function in formula-fed preterm neonates.

Modulation of intestinal inflammation by minimal enteral nutrition with amniotic fluid in preterm pigs.

BACKGROUND: Necrotizing enterocolitis (NEC) is a severe inflammatory disorder, associated with the difficult transition from parenteral to enteral feeding after preterm birth. We hypothesized that minimal enteral nutrition (MEN) with amniotic fluid (AF), prior to enteral formula feeding, would improve resistance to NEC in preterm pigs. METHODS: Experiment 1: IEC-6 cells were incubated with porcine (pAF) and human AF (hAF) to test AF-stimulated enterocyte proliferation and migration in vitro. Experiment 2: Cesarean-delivered, preterm pigs were fed parenteral nutrition and MEN with pAF, hAF, or control fluid (MEN-pAF, MEN-hAF, or MEN-CTRL; all n = 9) for 2 days before tissue collection. Experiment 3: Preterm pigs were fed MEN diets as in experiment 2, but followed by 2 days of enteral formula feeding, which predisposes to NEC (NEC-pAF, NEC-hAF, or NEC-CTRL; n = 10-12). RESULTS: Both pAF and hAF stimulated enterocyte proliferation and migration in vitro. In experiment 2, MEN-pAF and MEN-hAF pigs showed increased body weight gain and reduced intestinal interleukin (IL)-8 and colonic IL-6 levels, indicating reduced inflammatory response. In experiment 3, body weight gain was highest in the 2 groups fed AF as MEN, but NEC incidences were similar (NEC-pAF) or increased (NEC-hAF) compared with controls. CONCLUSIONS: Intake of pAF or hAF improved body growth and modulated intestinal inflammatory cytokines during a period of parenteral nutrition, but did not protect against later formula-induced NEC in preterm pigs. Further studies are required to show if MEN feeding with species-specific AF, combined with an optimal enteral diet (eg, human milk), will improve adaptation during the transition from parenteral to enteral feeding in preterm neonates.