Mycophenolic acid directly protects podocytes by preserving the actin cytoskeleton and increasing cell survival.

Mycophenolate Mofetil (MMF) has an established role as a therapeutic agent in childhood nephrotic syndrome. While other immunosuppressants have been shown to positively affect podocytes, direct effects of MMF on podocytes remain largely unknown. The present study examines the effects of MMF's active component Mycophenolic Acid (MPA) on the transcriptome of podocytes and investigates its biological significance. We performed transcriptomics in cultured murine podocytes exposed to MPA to generate hypotheses on podocyte-specific effects of MPA. Accordingly, we further analyzed biological MPA effects on actin cytoskeleton morphology after treatment with bovine serum albumin (BSA) by immunofluorescence staining, as well as on cell survival following exposure to TNF-α and cycloheximide by neutral red assay. MPA treatment significantly (adjusted p < 0.05) affected expression of 351 genes in podocytes. Gene Ontology term enrichment analysis particularly clustered terms related to actin and inflammation-related cell death. Indeed, quantification of the actin cytoskeleton of BSA treated podocytes revealed a significant increase of thickness and number of actin filaments after treatment with MPA. Further, MPA significantly reduced TNFα and cycloheximide induced cell death. MPA has a substantial effect on the transcriptome of podocytes in vitro, particularly including functional clusters related to non-immune cell dependent mechanisms. This may provide a molecular basis for direct beneficial effects of MPA on the structural integrity and survival of podocytes under pro-inflammatory conditions.

Elevated n-3/n-6 PUFA ratio in early life diet reverses adverse intrauterine kidney programming in female rats.

Intrauterine growth restriction (IUGR) predisposes to chronic kidney disease via activation of proinflammatory pathways, and omega-3 PUFAs (n-3 PUFAs) have anti-inflammatory properties. In female rats, we investigated 1) how an elevated dietary n-3/n-6 PUFA ratio (1:1) during postnatal kidney development modifies kidney phospholipid (PL) and arachidonic acid (AA) metabolite content and 2) whether the diet counteracts adverse molecular protein signatures expected in IUGR kidneys. IUGR was induced by bilateral uterine vessel ligation or intrauterine stress through sham operation 3.5 days before term. Control (C) offspring were born after uncompromised pregnancy. On postnatal (P) days P2-P39, rats were fed control (n-3/n-6 PUFA ratio 1:20) or n-3 PUFA intervention diet (N3PUFA; ratio 1:1). Plasma parameters (P33), kidney cortex lipidomics and proteomics, as well as histology (P39) were studied. We found that the intervention diet tripled PL-DHA content (PC 40:6; P < 0.01) and lowered both PL-AA content (PC 38:4 and lyso-phosphatidylcholine 20:4; P < 0.05) and AA metabolites (HETEs, dihydroxyeicosatrienoic acids, and epoxyeicosatrienoic acids) to 25% in all offspring groups. After ligation, our network analysis of differentially expressed proteins identified an adverse molecular signature indicating inflammation and hypercoagulability. N3PUFA diet reversed 61 protein alterations (P < 0.05), thus mitigating adverse IUGR signatures. In conclusion, an elevated n-3/n-6 PUFA ratio in early diet strongly reduces proinflammatory PLs and mediators while increasing DHA-containing PLs regardless of prior intrauterine conditions. Counteracting a proinflammatory hypercoagulable protein signature in young adult IUGR individuals through early diet intervention may be a feasible strategy to prevent developmentally programmed kidney damage in later life.

Hippocampal mTOR Dysregulation and Morphological Changes in Male Rats after Fetal Growth Restriction.

Fetal growth restriction (FGR) has been linked to long-term neurocognitive impairment, especially in males. To determine possible underlying mechanisms, we examined hippocampal cellular composition and mTOR signaling of male rat FGR offspring during main brain growth and development (postnatal days (PND) 1 and 12). FGR was either induced by a low-protein diet throughout pregnancy, experimental placental insufficiency by bilateral uterine vessel ligation or intrauterine stress by "sham" operation. Offspring after unimpaired gestation served as common controls. Low-protein diet led to a reduced cell density in the molecular dentate gyrus subregion, while intrauterine surgical stress was associated with increased cell density in the cellular CA2 subregion. Experimental placental insufficiency caused increased mTOR activation on PND 1, whereas intrauterine stress led to mTOR activation on PND 1 and 12. To determine long-term effects, we additionally examined mTOR signaling and Tau phosphorylation, which is altered in neurodegenerative diseases, on PND 180, but did not find any changes among the experimental groups. Our findings suggest that hippocampal cellular proliferation and mTOR signaling are dysregulated in different ways depending on the cause of FGR. While a low-protein diet induced a decreased cell density, prenatal surgical stress caused hyperproliferation, possibly via increased mTOR signaling.

The effect of dietary lipid quality in early life on serum LysoPC(18:2) levels and their association with adult blood glucose levels in intrauterine growth restricted rats.

Being born small-for-gestational-age, especially with subsequent catch-up growth, is associated with impaired metabolic health in later-life. We previously showed that a postnatal diet with an adapted lipid droplet structure can ameliorate some of the adverse metabolic consequences in intrauterine growth-restricted (IUGR) rats. The aim of the present work was to explore possible underlying mechanism(s) and potential biomarkers. To this end, serum metabolomics was performed in postnatal day (PN) 42 and PN96 samples of the above-mentioned rat offspring, born after uterine vasculature ligation. Blood samples were collected at PN42, directly after a postnatal dietary intervention with either complex lipid matrix (CLM) or control (CTRL) diet, and at PN96 after a subsequent western-style diet (WSD). Offspring of Non-operated (NOP) dams fed CTRL in early life were included as control group. In the PN42 metabolomics data, 11 co-abundance modules of metabolites were identified, of which four were significantly correlated to adult blood glucose levels at PN96. Further analyses showed that Lysophosphatidylcholine(18:2) (LysoPC(18:2)) levels were reduced by ligation (p < 0.01) and restored in CLM fed animals (p < 0.05). LysoPC(18:2) levels at PN42 correlated inversely with adult blood glucose levels. These data indicate that early-life LysoPC(18:2) blood levels may predict adult blood glucose levels and are affected by a postnatal diet with an adapted lipid droplet structure in IUGR offspring.

Reduced neutrophil elastase inhibitor elafin and elevated transforming growth factor-ß1 are linked to inflammatory response in sputum of cystic fibrosis patients with Pseudomonas aeruginosa.

RESEARCH QUESTION: Pulmonary disease progression in patients with cystic fibrosis (CF) is characterised by inflammation and fibrosis and aggravated by Pseudomonas aeruginosa (Pa). We investigated the impact of Pa specifically on: 1) protease/antiprotease balance; 2) inflammation; and 3) the link of both parameters to clinical parameters of CF patients. METHODS: Transforming growth factor-ß1 (TGF-ß1), interleukin (IL)-1ß, IL-8, neutrophil elastase (NE) and elastase inhibitor elafin were measured (ELISA assays), and gene expression of the NF-κB pathway was assessed (reverse transcriptase PCR) in the sputum of 60 CF patients with a minimum age of 5 years. Spirometry was assessed according to American Thoracic Society guidelines. RESULTS: Our results demonstrated the following: 1) NE was markedly increased in Pa-positive sputum, whereas elafin was significantly decreased; 2) increased IL-1ß/IL-8 levels were associated with both Pa infection and reduced forced expiratory volume in 1 s, and sputum TGF-ß1 was elevated in Pa-infected CF patients and linked to an impaired lung function; and 3) gene expression of NF-κB signalling components was increased in sputum of Pa-infected patients, and these findings were positively correlated with IL-8. CONCLUSION: Our study links Pa infection to an imbalance of NE and NE inhibitor elafin and increased inflammatory mediators. Moreover, our data demonstrate an association between high TGF-ß1 sputum levels and a progress in chronic lung inflammation and pulmonary fibrosis in CF. Controlling the excessive airway inflammation by inhibition of NE and TGF-ß1 might be promising therapeutic strategies in future CF therapy and a possible complement to cystic fibrosis transmembrane conductance regulator (CFTR) modulators.

Maternal Exercise Mediates Hepatic Metabolic Programming via Activation of AMPK-PGC1α Axis in the Offspring of Obese Mothers.

Maternal obesity is associated with an increased risk of hepatic metabolic dysfunction for both mother and offspring and targeted interventions to address this growing metabolic disease burden are urgently needed. This study investigates whether maternal exercise (ME) could reverse the detrimental effects of hepatic metabolic dysfunction in obese dams and their offspring while focusing on the AMP-activated protein kinase (AMPK), representing a key regulator of hepatic metabolism. In a mouse model of maternal western-style-diet (WSD)-induced obesity, we established an exercise intervention of voluntary wheel-running before and during pregnancy and analyzed its effects on hepatic energy metabolism during developmental organ programming. ME prevented WSD-induced hepatic steatosis in obese dams by alterations of key hepatic metabolic processes, including activation of hepatic ß-oxidation and inhibition of lipogenesis following increased AMPK and peroxisome-proliferator-activated-receptor-γ-coactivator-1α (PGC-1α)-signaling. Offspring of exercised dams exhibited a comparable hepatic metabolic signature to their mothers with increased AMPK-PGC1α-activity and beneficial changes in hepatic lipid metabolism and were protected from WSD-induced adipose tissue accumulation and hepatic steatosis in later life. In conclusion, this study demonstrates that ME provides a promising strategy to improve the metabolic health of both obese mothers and their offspring and highlights AMPK as a potential metabolic target for therapeutic interventions.

Vascular tone regulation in renal interlobar arteries of male rats is dysfunctional after intrauterine growth restriction.

Intrauterine growth restriction (IUGR) due to an adverse intrauterine environment predisposes to arterial hypertension and loss of kidney function. Here, we investigated whether vascular dysregulation in renal interlobar arteries (RIAs) may contribute to hypertensive glomerular damage after IUGR. In rats, IUGR was induced by bilateral uterine vessel ligation. Offspring of nonoperated rats served as controls. From postnatal day 49, blood pressure was telemetrically recorded. On postnatal day 70, we evaluated contractile function in RIAs and mesenteric arteries. In addition, blood, urine, and glomerular parameters as well as renal collagen deposition were analyzed. IUGR RIAs not only showed loss of stretch activation in 9 of 11 arteries and reduced stretch-induced myogenic tone but also showed a shift of the concentration-response relation of acetylcholine-induced relaxation toward lower concentrations. However, IUGR RIAs also exhibited augmented contractions through phenylephrine. Systemic mean arterial pressure [mean difference: 4.8 mmHg (daytime) and 5.7 mmHg (night)], mean glomerular area (IUGR: 9,754 ± 338 µm2 and control: 8,395 ± 227 µm2), and urinary protein-to-creatinine ratio (IUGR: 1.67 ± 0.13 g/g and control: 1.26 ± 0.10 g/g) were elevated after IUGR. We conclude that male IUGR rat offspring may have increased vulnerability toward hypertensive glomerular damage due to loss of myogenic tone and augmented endothelium-dependent relaxation in RIAs.NEW & NOTEWORTHY For the first time, our study presents wire myography data from renal interlobar arteries (RIAs) and mesenteric arteries of young adult rat offspring after intrauterine growth restriction (IUGR). Our data indicate that myogenic tone in RIAs is dysfunctional after IUGR. Furthermore, IUGR offspring suffer from mild arterial hypertension, glomerular hypertrophy, and increased urinary protein-to-creatinine ratio. Dysregulation of vascular tone in RIAs could be an important variable that impacts upon vulnerability toward glomerular injury after IUGR.

Impact of early-life diet on long-term renal health.

In the last years, great advances have been made in the effort to understand how nutritional influences can affect long-term renal health. Evidence has accumulated that maternal nutrition before and during pregnancy and lactation as well as early postnatal nutrition is of special significance. In this review, we summarize epidemiologic and experimental data on the renal effects of perinatal exposure to energy restriction, low-protein diet, high-fat diet, high-fructose diet, and high- and low-salt diet as well as micronutrient deficiencies. Interestingly, different modifications during early-life diet may end up with similar sequelae for the offspring. On the other hand, molecular pathways can be influenced in opposite directions by different dietary interventions during early life. Importantly, postnatal nutrition significantly modifies the phenotype induced by maternal diet. Sequelae of altered macro- or micronutrient intakes include altered nephron count, blood pressure dysregulation, altered sodium handling, endothelial dysfunction, inflammation, mitochondrial dysfunction, and oxidative stress. In addition, renal prostaglandin metabolism as well as renal AMPK, mTOR, and PPAR signaling can be affected and the renin-angiotensin-aldosterone system may be dysregulated. Lately, the influence of early-life diet on gut microbiota leading to altered short chain fatty acid profiles has been discussed in the etiology of arterial hypertension. Against this background, the preventive and therapeutic potential of perinatal nutritional interventions regarding kidney disease is an emerging field of research. Especially individuals at risk (e.g., newborns from mothers who suffered from malnutrition during gestation) could disproportionately benefit from well-targeted dietary interventions.

Maternal exercise conveys protection against NAFLD in the offspring via hepatic metabolic programming.

Maternal exercise (ME) during pregnancy has been shown to improve metabolic health in offspring and confers protection against the development of non-alcoholic fatty liver disease (NAFLD). However, its underlying mechanism are still poorly understood, and it remains unclear whether protective effects on hepatic metabolism are already seen in the offspring early life. This study aimed at determining the effects of ME during pregnancy on offspring body composition and development of NAFLD while focusing on proteomic-based analysis of the hepatic energy metabolism during developmental organ programming in early life. Under an obesogenic high-fat diet (HFD), male offspring of exercised C57BL/6J-mouse dams were protected from body weight gain and NAFLD in adulthood (postnatal day (P) 112). This was associated with a significant activation of hepatic AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor alpha (PPARα) and PPAR coactivator-1 alpha (PGC1α) signaling with reduced hepatic lipogenesis and increased hepatic ß-oxidation at organ programming peak in early life (P21). Concomitant proteomic analysis revealed a characteristic hepatic expression pattern in offspring as a result of ME with the most prominent impact on Cholesterol 7 alpha-hydroxylase (CYP7A1). Thus, ME may offer protection against offspring HFD-induced NAFLD by shaping hepatic proteomics signature and metabolism in early life. The results highlight the potential of exercise during pregnancy for preventing the early origins of NAFLD.

Altered molecular signatures during kidney development after intrauterine growth restriction of different origins.

This study was performed to identify transcriptional alterations in male intrauterine growth restricted (IUGR) rats during and at the end of nephrogenesis in order to generate hypotheses which molecular mechanisms contribute to adverse kidney programming. IUGR was induced by low protein (LP) diet throughout pregnancy, bilateral uterine vessel ligation (LIG), or intrauterine stress (IUS) by sham operation. Offspring of unimpaired dams served as controls. Significant acute kidney damage was ruled out by negative results for proteins indicative of ER-stress, autophagy, apoptosis, or infiltration with macrophages. Renal gene expression was examined by transcriptome microarrays, demonstrating 53 (LP, n = 12; LIG, n = 32; IUS, n = 9) and 134 (LP, n = 10; LIG, n = 41; IUS, n = 83) differentially expressed transcripts on postnatal days (PND) 1 and 7, respectively. Reduced Pilra (all IUGR groups, PND 7), Nupr1 (LP and LIG, PND 7), and Kap (LIG, PND 1) as well as increased Ccl20, S100a8/a9 (LIG, PND 1), Ifna4, and Ltb4r2 (IUS, PND 7) indicated that inflammation-related molecular dysregulation could be a "common" feature after IUGR of different origins. Network analyses of transcripts and predicted upstream regulators hinted at proinflammatory adaptions mainly in LIG (arachidonic acid-binding, neutrophil aggregation, toll-like-receptor, NF-kappa B, and TNF signaling) and dysregulation of AMPK and PPAR signaling in LP pups. The latter may increase susceptibility towards obesity-associated kidney damage. Western blots of the most prominent predicted upstream regulators confirmed significant dysregulation of RICTOR in LP (PND 7) and LIG pups (PND 1), suggesting that mTOR-related processes could further modulate kidney programming in these groups of IUGR pups. KEY MESSAGES: Inflammation-related transcripts are dysregulated in neonatal IUGR rat kidneys. Upstream analyses indicate renal metabolic dysregulation after low protein diet. RICTOR is dysregulated after low protein diet and uterine vessel ligation.

Maternal High Fat Diet and in-Utero Metformin Exposure Significantly Impact upon the Fetal Renal Proteome of Male Mice.

There is accumulating evidence for fetal programming of later kidney disease by maternal obesity or associated conditions. We performed a hypothesis-generating study to identify potentially underlying mechanisms. Female mice were randomly split in two groups and fed either a standard diet (SD) or high fat diet (HFD) from weaning until mating and during pregnancy. Half of the dams from both groups were treated with metformin ((M), 380 mg/kg), resulting in four experimental groups (SD, SD-M, HFD, HFD-M). Caesarean section was performed on gestational day 18.5. Fetal kidney tissue was isolated from cryo-slices using laser microdissection methods and a proteomic screen was performed. For single proteins, a fold change ≥1.5 and q-value <0.05 were considered to be statistically significant. Interestingly, HFD versus SD had a larger effect on the proteome of fetal kidneys (56 proteins affected; interaction clusters shown for proteins concerning transcription/translation, mitochondrial processes, eicosanoid metabolism, H2S-synthesis and membrane remodeling) than metformin exposure in either SD (29 proteins affected; clusters shown for proteins involved in transcription/translation) or HFD (6 proteins affected; no cluster). By further analysis, ATP6V1G1, THY1, PRKCA and NDUFB3 were identified as the most promising candidates potentially mediating reprogramming effects of metformin in a maternal high fat diet.

IL-6/Smad2 signaling mediates acute kidney injury and regeneration in a murine model of neonatal hyperoxia.

Prematurity is linked to incomplete nephrogenesis and risk of chronic kidney diseases (CKDs). Oxygen is life-saving in that context but induces injury in numerous organs. Here, we studied the structural and functional impact of hyperoxia on renal injury and its IL-6 dependency. Newborn wild-type (WT) and IL-6 knockout (IL-6-/-) mice were exposed to 85% O2 for 28 d, followed by room air until postnatal d (P) 70. Controls were in room air throughout life. At P28, hyperoxia reduced estimated kidney cortex area (KCA) in WT; at P70, KCA was greater, number of glomeruli was fewer, fractional potassium excretion was higher, and glomerular filtration rate was slightly lower than in controls. IL-6-/- mice were protected from these changes after hyperoxia. Mechanistically, the acute renal injury phase (P28) showed in WT but not in IL-6-/- mice an activation of IL-6 (signal transducer and activator of transcription 3) and TGF-ß [mothers against decapentaplegic homolog (Smad)2] signaling, increased inflammatory markers, disrupted mitochondrial biogenesis, and reduced tubular proliferation. Regenerative phase at P70 was characterized by tubular proliferation in WT but not in IL-6-/- mice. These data demonstrate that hyperoxia increases the risk of CKD through a novel IL-6-Smad2 axis. The amenability of these pathways to pharmacological approaches may offer new avenues to protect premature infants from CKD.-Mohr, J., Voggel, J., Vohlen, C., Dinger, K., Dafinger, C., Fink, G., Göbel, H., Liebau, M. C., Dötsch, J., Alejandre Alcazar, M. A. IL-6/Smad2 signaling mediates acute kidney injury and regeneration in a murine model of neonatal hyperoxia.