Hypertension is one of the leading causes of chronic kidney disease (CKD) and affects approximate... more Hypertension is one of the leading causes of chronic kidney disease (CKD) and affects approximately one‐third of the United States population. Toll‐like receptor 4 (TLR4) plays a role in the innate immune system through induction of NF‐κB‐mediated inflammatory response, which if unresolved, can lead to tissue damage. Activation of TLR4 is through recognition of foreign antigens, such as lipopolysaccharide (LPS), which is a product of gram‐negative bacteria. Several recent studies have revealed the gut microbiome can influence multiple physiological processes, such as metabolism, immunity, and blood pressure (BP) regulation. Doxycycline is routinely prescribed for a variety of illnesses and infections, however, the exact effects on gut microbiome composition and blood pressure regulation and kidney function have not been evaluated. In this study, we sought to determine the link between TLR4, gut microbiota and the kidney during hypertension and whether the broad‐spectrum antibiotic d...
NHERF‐1 regulates several ion transporters in kidney proximal tubules including NHE3, NpT2a, NBC,... more NHERF‐1 regulates several ion transporters in kidney proximal tubules including NHE3, NpT2a, NBC, CFTR, and Na‐K ATPase (NKA) through modulation of protein complex formation and through modulation of hormone receptor signaling, in particular, PTH and dopamine. Regulation of ion balance changes with aging; however, the expression of NHERF‐1 in aging has not been studied. We hypothesize that loss of NHERF‐1 regulation of NHE3 and NKA with aging contributes to alterations in maintenance of sodium balance. To address this hypothesis, we compared expression of NHERF‐1, NHE3, and NKA in kidneys from young (4 m old) and old (24 m old) female F344 rats by western blot in isolated apical and basolateral membranes and by immunohistochemistry. Western blot showed complete loss of NHERF‐1 in both apical and basolateral membranes from kidneys of 24 m old rats. NKA expression significantly decreased in 24 m old rats as compared to 4 m old rats, but NHE3 expression did not change with aging. Simil...
Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) lea... more Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) leading to renal fibrosis, progressive deterioration of renal function, and eventually to end stage renal disease. Matrix metalloproteinases (MMPs) are known to regulate synthesis and degradation of the ECM. Earlier, we demonstrated that imbalanced MMPs promote adverse ECM remodeling leading to renal fibrosis in type-1 diabetes. Moreover, elevated macrophage infiltration, pro-inflammatory cytokines and epithelial‒mesenchymal transition (EMT) are known to contribute to the renal fibrosis. Various bioactive compounds derived from the medicinal plant, Azadirachta indica (neem) are shown to regulate inflammation and ECM proteins in different diseases. Nimbidiol is a neem-derived diterpenoid that is considered as a potential anti-diabetic compound due to its glucosidase inhibitory properties. We investigated whether Nimbidiol mitigates adverse ECM accumulation and renal fibrosis to improve kidne...
Previous studies from our laboratory revealed that the gaseous molecule; hydrogen sulfide (H2S), ... more Previous studies from our laboratory revealed that the gaseous molecule; hydrogen sulfide (H2S), a metabolic product of epigenetics which involves trans-sulfuration pathway for ensuring the metabolism and clearance of homocysteine (Hcy) from the body, helped mitigate the skeletal muscle’s pathological remodeling. Although the master circadian clock regulator that is known as the b rain and m uscle a ryl hydrocarbon receptor nuclear translocator l ike protein 1 (i.e., BMAL1) is intimately associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock system remains unexplored. We hypothesize that potential alterations in the functioning of circadian clock during sleep and wake cycle affect the skeletal muscle’s biology. To test this hypothesis, we measured serum matrix metalloproteinases (MMPs) activities using gelatin gels for analyzing the MMP-2 and MMP-9 activities. Further, employing the casein gels we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type (WT) and cystathionine β synthase deficient (CBS-/+) mice strains were treated with H2S and subjected for measurement of trans-sulfuration factors from their skeletal muscle tissues. The results suggested highly robust activation of MMPs activities in the wake mice versus sleep mice that appears somewhat akin to that of the “ 1-carbon metabolic dysregulation” which takes place during remodeling of extracellular matrix (ECM) in muscular dystrophy affected tissues. Interestingly, the levels of trans-sulphuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in the CBS-/+ mice. However, treatment with H2S mitigated the attenuation occurring in the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock system in trans-sulfuration pathway that affect the skeletal muscle remodeling and mitochondrial regeneration. HL-139047, DK116591 and AR-71789 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Hypertension is one of the leading causes of chronic kidney disease. Recent studies have highligh... more Hypertension is one of the leading causes of chronic kidney disease. Recent studies have highlighted the importance of gut microbiota on physiological processes, such as metabolism and immunity, th...
The prevalence of hypertension increases with age. At the cellular level, oxidative stress is a m... more The prevalence of hypertension increases with age. At the cellular level, oxidative stress is a major contributing factor to the pathogenesis of hypertension-induced kidney damage. The nicotinamide adenine dinucleotide phosphate (NADPH) family is one of the major sources of reactive oxygen species generation in the body. Although the isoform, NADPH oxidase 4 (Nox4) is highly expressed in the kidney, its role in kidney diseases remains controversial as both pathogenic and protective effects have been described. In addition, its role in hypertension-induced kidney damage in aging remains unexplored. Current evidence supports the involvement of epigenetics in oxidative stress. The aim of the present study was to investigate the role of Nox4 in Ang-II induced kidney damage in aged mice and the potential role for epigenetic regulation. Wild type (WT, C57BL/6J) mice aged 12-14 wk and 75-78 wk were used in this study and treated with Ang-II (1000 ng/kg/min) for 4 weeks. Control mice received saline infusion. Blood pressure (BP) was measured twice weekly. Aged mice exhibited higher mean BP than young mice after Ang-II treatment (135.76±4.8 vs. 118.7±12.28 mmHg) and decreased renal vascular density on barium angiography. Dihydroethidium staining revealed increased oxidative stress in hypertensive kidney of aged mice. In the aged kidney, protein expression of mitochondrial antioxidant enzymes, manganese superoxide dismutase and catalase was decreased by > 2-fold compared to young, and decreased further after Ang-II infusion. The renal sensors of energy and redox state, SIRT1 and SIRT3, showed similar decrease in aged kidney compared to young kidney in response to Ang-II. Nox4 and p22 phox protein expression was upregulated in the hypertensive kidneys of aged mice. The epigenetic mechanism involving DNA methylation showed increased DNA methyltransferases, DNMT1, 3a and 3b in the aged kidney. Following Ang-II, the aged kidney showed further increase of DNMT1 and 3a and a decrease in DNMT3b. Conclusion: Endogenous expression of Nox4 is upregulated in hypertensive kidney and is associated with alteration of DNA methyltransferases suggesting epigenetic regulation of oxidative stress in aged mice.
Cellular autophagy is a protective mechanism where cells degrade damaged organelles to maintain i... more Cellular autophagy is a protective mechanism where cells degrade damaged organelles to maintain intracellular homeostasis. Apoptosis, on the other hand, is considered as programmed cell death. Interestingly, autophagy inhibits apoptosis by degrading apoptosis regulators. In hypertension, an imbalance of autophagy and apoptosis regulators can lead to renal injury and dysfunction. Previously, we have reported that toll-like receptor 4 (TLR4) mutant mice are protective against renal damage, in part, due to reduced oxidative stress and inflammation. However, the detailed mechanism remained elusive. In this study, we tested the hypothesis of whether TLR4 mutation reduces Ang-II-induced renal injury by inciting autophagy and suppressing apoptosis in the hypertensive kidney. Male mice with normal TLR4 expression (TLR4N, C3H/HeOuJ) and mutant TLR4 (TLR4M, C3H/HeJLps-d) aged 10–12 weeks were infused with Ang-II (1000 ng/kg/d) for 4 weeks to create hypertension. Saline infused appropriate control were used. Blood pressure was increased along with increased TLR4 expression in TLR4N mice receiving Ang-II compared to TLR4N control. Autophagy was downregulated, and apoptosis was upregulated in TLR4N mice treated with Ang-II. Also, kidney injury markers plasma lipocalin-2 (LCN2) and kidney injury molecule 1 (KIM-1) were upregulated in TLR4N mice treated with Ang-II. Besides, increased nuclear translocation and activity of NF-κB were measured in Ang-II-treated TLR4N mice. TLR4M mice remained protected against all these insults in hypertension. Together, these results suggest that Ang-II-induced TLR4 activation suppresses autophagy, induces apoptosis and kidney injury through in part by activating NF-κB signaling, and TLR4 mutation protects the kidney from Ang-II-induced hypertensive injury.
Hypertensive nephropathy is associated with progressive alteration of extracellular matrix (ECM) ... more Hypertensive nephropathy is associated with progressive alteration of extracellular matrix (ECM) components. Both mesangial and glomerular endothelial cells have the ability to synthesize and degrade ECM proteins such as collagens by changes in the activity of matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Endo180 is an extracellular fibronectin type II domain involved in lysosomal degradation of collagen which has been shown to mitigate renal fibrosis. More recently, hydrogen sulfide (H2S) has also been shown to mitigate hypertensive renal remodeling, however, its mechanism remains unclear. In this study, our aim was to investigate whether Angiotensin-II (Ang II) treatment alters the expression of Endo180, MMPs and TIMPs leading to dysregulation of collagen metabolism and whether GYY4137 (H2S donor) restores their levels to achieve homeostasis. Mesangial and mouse glomerular endothelial cells (MCs and MGECs respectively) were treated without or with Ang II (200 nM) and GYY4137 (250 μM) for 48hrs. Cell lysates were analyzed for MMP-13, -14, TIMP-1, Endo180, and collagen IV by Western blot, RT-PCR, and immunohistochemistry. In MGECs, Ang II treatment compared to its control decreased MMP-13/TIMP-1 ratio (0.75±0.44 vs. 2.48 ±0.73), and upregulated MMP-14/TIMP-1 ratio (0.64±2.10 vs. 0.96±1.47), and collagen IV (0.77±0.07 vs. 0.58±0.06). GYY4137 treatment mitigated these changes. In contrast, Ang II treatment in MCs decreased Endo180 compared to control (0.72±0.04 vs. 1.07±0.23), but did not alter the expression of MMP-13/TIMP-1, MMP-14/TIMP-1 ratios, and collagen IV level compared to control or MGECs. Similarly, immunostaining showed downregulation of MMP-13 and Endo180 in Ang II treated MGECs which was normalized following GYY4137 treatment. Endo180 was also normalized in MCs following GYY4137 treatment however, there was no change in MMP-14/TIMP-1 ratio or collagen IV level. We conclude that Ang II treatment causes adverse ECM remodeling in MGECs via downregulation of Endo180 and MMP-13 and upregulation of MMP-14 and TIMP-1 and in MCs by decreasing Endo180, and GYY4137 mitigates these changes in part, by modulating Endo180/MMP/TIMP pathway.
Hypertension is one of the leading causes of chronic kidney disease (CKD) and affects approximate... more Hypertension is one of the leading causes of chronic kidney disease (CKD) and affects approximately one‐third of the United States population. Toll‐like receptor 4 (TLR4) plays a role in the innate immune system through induction of NF‐κB‐mediated inflammatory response, which if unresolved, can lead to tissue damage. Activation of TLR4 is through recognition of foreign antigens, such as lipopolysaccharide (LPS), which is a product of gram‐negative bacteria. Several recent studies have revealed the gut microbiome can influence multiple physiological processes, such as metabolism, immunity, and blood pressure (BP) regulation. Doxycycline is routinely prescribed for a variety of illnesses and infections, however, the exact effects on gut microbiome composition and blood pressure regulation and kidney function have not been evaluated. In this study, we sought to determine the link between TLR4, gut microbiota and the kidney during hypertension and whether the broad‐spectrum antibiotic d...
NHERF‐1 regulates several ion transporters in kidney proximal tubules including NHE3, NpT2a, NBC,... more NHERF‐1 regulates several ion transporters in kidney proximal tubules including NHE3, NpT2a, NBC, CFTR, and Na‐K ATPase (NKA) through modulation of protein complex formation and through modulation of hormone receptor signaling, in particular, PTH and dopamine. Regulation of ion balance changes with aging; however, the expression of NHERF‐1 in aging has not been studied. We hypothesize that loss of NHERF‐1 regulation of NHE3 and NKA with aging contributes to alterations in maintenance of sodium balance. To address this hypothesis, we compared expression of NHERF‐1, NHE3, and NKA in kidneys from young (4 m old) and old (24 m old) female F344 rats by western blot in isolated apical and basolateral membranes and by immunohistochemistry. Western blot showed complete loss of NHERF‐1 in both apical and basolateral membranes from kidneys of 24 m old rats. NKA expression significantly decreased in 24 m old rats as compared to 4 m old rats, but NHE3 expression did not change with aging. Simil...
Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) lea... more Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) leading to renal fibrosis, progressive deterioration of renal function, and eventually to end stage renal disease. Matrix metalloproteinases (MMPs) are known to regulate synthesis and degradation of the ECM. Earlier, we demonstrated that imbalanced MMPs promote adverse ECM remodeling leading to renal fibrosis in type-1 diabetes. Moreover, elevated macrophage infiltration, pro-inflammatory cytokines and epithelial‒mesenchymal transition (EMT) are known to contribute to the renal fibrosis. Various bioactive compounds derived from the medicinal plant, Azadirachta indica (neem) are shown to regulate inflammation and ECM proteins in different diseases. Nimbidiol is a neem-derived diterpenoid that is considered as a potential anti-diabetic compound due to its glucosidase inhibitory properties. We investigated whether Nimbidiol mitigates adverse ECM accumulation and renal fibrosis to improve kidne...
Previous studies from our laboratory revealed that the gaseous molecule; hydrogen sulfide (H2S), ... more Previous studies from our laboratory revealed that the gaseous molecule; hydrogen sulfide (H2S), a metabolic product of epigenetics which involves trans-sulfuration pathway for ensuring the metabolism and clearance of homocysteine (Hcy) from the body, helped mitigate the skeletal muscle’s pathological remodeling. Although the master circadian clock regulator that is known as the b rain and m uscle a ryl hydrocarbon receptor nuclear translocator l ike protein 1 (i.e., BMAL1) is intimately associated with S-adenosylhomocysteine hydrolase (SAHH) and Hcy metabolism but how trans-sulfuration pathway is influenced by the circadian clock system remains unexplored. We hypothesize that potential alterations in the functioning of circadian clock during sleep and wake cycle affect the skeletal muscle’s biology. To test this hypothesis, we measured serum matrix metalloproteinases (MMPs) activities using gelatin gels for analyzing the MMP-2 and MMP-9 activities. Further, employing the casein gels we also studied MMP-13 that is known to be influenced by the growth arrest and DNA damage-45 (GADD45) protein during sleep and wake cycle. The wild type (WT) and cystathionine β synthase deficient (CBS-/+) mice strains were treated with H2S and subjected for measurement of trans-sulfuration factors from their skeletal muscle tissues. The results suggested highly robust activation of MMPs activities in the wake mice versus sleep mice that appears somewhat akin to that of the “ 1-carbon metabolic dysregulation” which takes place during remodeling of extracellular matrix (ECM) in muscular dystrophy affected tissues. Interestingly, the levels of trans-sulphuration factors such as CBS, cystathionine γ lyase (CSE), methyl tetrahydrofolate reductase (MTHFR), phosphatidylethanolamine N-methyltransferase (PEMT), and Hcy-protein bound paraoxonase 1 (PON1) were attenuated in the CBS-/+ mice. However, treatment with H2S mitigated the attenuation occurring in the trans-sulfuration pathway. In addition, levels of mitochondrial peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC 1-α) and mitofusin-2 (MFN-2) were significantly improved by H2S intervention. Our findings suggest participation of the circadian clock system in trans-sulfuration pathway that affect the skeletal muscle remodeling and mitochondrial regeneration. HL-139047, DK116591 and AR-71789 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Hypertension is one of the leading causes of chronic kidney disease. Recent studies have highligh... more Hypertension is one of the leading causes of chronic kidney disease. Recent studies have highlighted the importance of gut microbiota on physiological processes, such as metabolism and immunity, th...
The prevalence of hypertension increases with age. At the cellular level, oxidative stress is a m... more The prevalence of hypertension increases with age. At the cellular level, oxidative stress is a major contributing factor to the pathogenesis of hypertension-induced kidney damage. The nicotinamide adenine dinucleotide phosphate (NADPH) family is one of the major sources of reactive oxygen species generation in the body. Although the isoform, NADPH oxidase 4 (Nox4) is highly expressed in the kidney, its role in kidney diseases remains controversial as both pathogenic and protective effects have been described. In addition, its role in hypertension-induced kidney damage in aging remains unexplored. Current evidence supports the involvement of epigenetics in oxidative stress. The aim of the present study was to investigate the role of Nox4 in Ang-II induced kidney damage in aged mice and the potential role for epigenetic regulation. Wild type (WT, C57BL/6J) mice aged 12-14 wk and 75-78 wk were used in this study and treated with Ang-II (1000 ng/kg/min) for 4 weeks. Control mice received saline infusion. Blood pressure (BP) was measured twice weekly. Aged mice exhibited higher mean BP than young mice after Ang-II treatment (135.76±4.8 vs. 118.7±12.28 mmHg) and decreased renal vascular density on barium angiography. Dihydroethidium staining revealed increased oxidative stress in hypertensive kidney of aged mice. In the aged kidney, protein expression of mitochondrial antioxidant enzymes, manganese superoxide dismutase and catalase was decreased by > 2-fold compared to young, and decreased further after Ang-II infusion. The renal sensors of energy and redox state, SIRT1 and SIRT3, showed similar decrease in aged kidney compared to young kidney in response to Ang-II. Nox4 and p22 phox protein expression was upregulated in the hypertensive kidneys of aged mice. The epigenetic mechanism involving DNA methylation showed increased DNA methyltransferases, DNMT1, 3a and 3b in the aged kidney. Following Ang-II, the aged kidney showed further increase of DNMT1 and 3a and a decrease in DNMT3b. Conclusion: Endogenous expression of Nox4 is upregulated in hypertensive kidney and is associated with alteration of DNA methyltransferases suggesting epigenetic regulation of oxidative stress in aged mice.
Cellular autophagy is a protective mechanism where cells degrade damaged organelles to maintain i... more Cellular autophagy is a protective mechanism where cells degrade damaged organelles to maintain intracellular homeostasis. Apoptosis, on the other hand, is considered as programmed cell death. Interestingly, autophagy inhibits apoptosis by degrading apoptosis regulators. In hypertension, an imbalance of autophagy and apoptosis regulators can lead to renal injury and dysfunction. Previously, we have reported that toll-like receptor 4 (TLR4) mutant mice are protective against renal damage, in part, due to reduced oxidative stress and inflammation. However, the detailed mechanism remained elusive. In this study, we tested the hypothesis of whether TLR4 mutation reduces Ang-II-induced renal injury by inciting autophagy and suppressing apoptosis in the hypertensive kidney. Male mice with normal TLR4 expression (TLR4N, C3H/HeOuJ) and mutant TLR4 (TLR4M, C3H/HeJLps-d) aged 10–12 weeks were infused with Ang-II (1000 ng/kg/d) for 4 weeks to create hypertension. Saline infused appropriate control were used. Blood pressure was increased along with increased TLR4 expression in TLR4N mice receiving Ang-II compared to TLR4N control. Autophagy was downregulated, and apoptosis was upregulated in TLR4N mice treated with Ang-II. Also, kidney injury markers plasma lipocalin-2 (LCN2) and kidney injury molecule 1 (KIM-1) were upregulated in TLR4N mice treated with Ang-II. Besides, increased nuclear translocation and activity of NF-κB were measured in Ang-II-treated TLR4N mice. TLR4M mice remained protected against all these insults in hypertension. Together, these results suggest that Ang-II-induced TLR4 activation suppresses autophagy, induces apoptosis and kidney injury through in part by activating NF-κB signaling, and TLR4 mutation protects the kidney from Ang-II-induced hypertensive injury.
Hypertensive nephropathy is associated with progressive alteration of extracellular matrix (ECM) ... more Hypertensive nephropathy is associated with progressive alteration of extracellular matrix (ECM) components. Both mesangial and glomerular endothelial cells have the ability to synthesize and degrade ECM proteins such as collagens by changes in the activity of matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Endo180 is an extracellular fibronectin type II domain involved in lysosomal degradation of collagen which has been shown to mitigate renal fibrosis. More recently, hydrogen sulfide (H2S) has also been shown to mitigate hypertensive renal remodeling, however, its mechanism remains unclear. In this study, our aim was to investigate whether Angiotensin-II (Ang II) treatment alters the expression of Endo180, MMPs and TIMPs leading to dysregulation of collagen metabolism and whether GYY4137 (H2S donor) restores their levels to achieve homeostasis. Mesangial and mouse glomerular endothelial cells (MCs and MGECs respectively) were treated without or with Ang II (200 nM) and GYY4137 (250 μM) for 48hrs. Cell lysates were analyzed for MMP-13, -14, TIMP-1, Endo180, and collagen IV by Western blot, RT-PCR, and immunohistochemistry. In MGECs, Ang II treatment compared to its control decreased MMP-13/TIMP-1 ratio (0.75±0.44 vs. 2.48 ±0.73), and upregulated MMP-14/TIMP-1 ratio (0.64±2.10 vs. 0.96±1.47), and collagen IV (0.77±0.07 vs. 0.58±0.06). GYY4137 treatment mitigated these changes. In contrast, Ang II treatment in MCs decreased Endo180 compared to control (0.72±0.04 vs. 1.07±0.23), but did not alter the expression of MMP-13/TIMP-1, MMP-14/TIMP-1 ratios, and collagen IV level compared to control or MGECs. Similarly, immunostaining showed downregulation of MMP-13 and Endo180 in Ang II treated MGECs which was normalized following GYY4137 treatment. Endo180 was also normalized in MCs following GYY4137 treatment however, there was no change in MMP-14/TIMP-1 ratio or collagen IV level. We conclude that Ang II treatment causes adverse ECM remodeling in MGECs via downregulation of Endo180 and MMP-13 and upregulation of MMP-14 and TIMP-1 and in MCs by decreasing Endo180, and GYY4137 mitigates these changes in part, by modulating Endo180/MMP/TIMP pathway.
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