Ischemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in the aging population. A reduction of hydrogen sulfide (H2S) production in the old kidney and renal IRI contribute to renal pathology and injury. Recent studies suggest that microRNAs (miRs) play an important role in the pathophysiology of AKI and a significant crosstalk exists between H2S and miRs. Among the miRs, miR-21 is highly expressed in AKI and is reported to have both pathological and protective role. In the present study, we sought to determine the effects of age-induced reduction in H2S and mir-21 antagonism in AKI. Wild type (WT, C57BL/6J) mice aged 12–14 weeks and 75–78 weeks underwent bilateral renal ischemia (27 min) and reperfusion for 7 days and were treated with H2S donor, GYY4137 (GYY, 0.25 mg/kg/day, ip) or locked nucleic acid anti-miR-21 (20 mg/kg b.w., ip) for 7 days. Following IRI, old kidney showed increased macrophage polarization toward M1 inflammatory phenotype, cytokine upregulation, endothelial–mesenchymal transition, and fibrosis compared to young kidney. Treatment with GYY or anti-miR-21 reversed the changes and improved renal vascular density, blood flow, and renal function in the old kidney. Anti-miR-21 treatment in mouse glomerular endothelial cells showed upregulation of H2S-producing enzymes, cystathionine β-synthase (CBS), and cystathionineγ-lyase (CSE), and reduction of matrix metalloproteinase-9 and collagen IV expression. In conclusion, exogenous H2S and inhibition of miR-21 rescued the old kidney dysfunction due to IRI by increasing H2S levels, reduction of macrophage-mediated injury, and promoting reparative process suggesting a viable approach for aged patients sustaining AKI.

e24162 Background: Platinum-based drug use in cancer treatment is restricted by dose-limiting side effects, including nausea/emesis, anorexia and weight loss that reduce patient quality of life and limit treatment adherence. Cisplatin increases GDF-15, a cytokine that induces aversion, anorexia and weight loss in preclinical models. GDF-15 signals through the hindbrain receptor glial cell-derived neurotrophic factor receptor alpha-like (GFRAL) and cisplatin-induced weight loss was attenuated in a GFRAL knockout mouse. Methods: In the current study, using mouse and/or nonhuman primate models, we examined whether GDF-15 inhibition via a potent and selective monoclonal antibody (mAB1) prevents platinum-induced emesis, anorexia, weight loss, with increased survival. Results: Circulating GDF-15 levels in NSCLC and colorectal cancer were higher (~1.5 fold) in patients on platinum therapy compared to non-platinum-based therapy. Higher levels of circulating GDF-15 were also associated with greater weight loss in colorectal cancer patients prior to receiving FOLFOX as part of cancer treatment. In wildtype mice, cisplatin, oxaliplatin and carboplatin each increased circulating GDF-15 (≥ 5-fold) and induced anorexia, skeletal muscle wasting, and weight loss. These effects were prevented in GDF-15 knockout mice, however only a partial blockade of carboplatin was observed. The GDF-15 neutralizing efficacy of mAB1 was confirmed by reversing AAV-GDF-15-induced weight loss in wildtype mice. In nonhuman primates, cisplatin treatment for 5 days (96% of the daily recommended clinical dose) also increased circulating GDF-15 ( > 5-fold), and induced anorexia and emesis. Treatment with mAB1 resulted in no detectable circulating levels of free GDF-15 and attenuated both cisplatin-induced anorexia and emesis. In a mouse cachectic tumor model (subcutaneous; NSCLC patient derived xenograft), cisplatin inhibited tumor growth; however, GDF-15 levels remained elevated and additional weight loss occurred compared to control. When mAB1 was given in combination with cisplatin, weight loss was reversed and tumor growth inhibition was maintained, resulting in greater survival compared to cisplatin alone. Conclusions: Taken together, these data support that GDF-15 inhibition with mAB1 holds the potential as an effective therapeutic approach to alleviate GDF-15 mediated emesis, anorexia and weight loss, with the aim to enable optimal cancer treatment as well as to improve patient quality of life and potentially survival.

e15633 Background: Almost half of cancer deaths are attributed to cancers most frequently associated with cachexia. Cachexia is a complex metabolic disease characterized by anorexia and unintentional weight loss. Skeletal muscle depletion has been recognized as a key feature of the disease, however muscle anabolic therapies have not been successful, suggesting that treatments that target multiple aspects of the disease will be most effective. Growth differentiation factor 15 (GDF-15) is a cytokine that induces anorexia and weight loss and is associated with cachexia in cancer patients. In preclinical cancer cachexia models, GDF-15 inhibition is sufficient to normalize food intake and body weight, including skeletal muscle mass. However, it remains to be determined whether the increased skeletal muscle mass also results in restoration of muscle function. Therefore, we examined the effect of GDF-15 inhibition on muscle mass and function in mouse models of cancer cachexia in comparison with myostatin inhibition, an established muscle anabolic pathway. Methods: Cachectic mouse tumor models were established with subcutaneous implantation of tumor cell lines reported to be GDF-15-dependent; mouse renal cell carcinoma (RENCA) and human ovarian cancer (TOV-21G) cell lines. Mice were treated with anti-GDF-15 (mAB2) or anti-myostatin (RK35) monoclonal antibodies and skeletal muscle function was assessed in vivo via maximum force, maximum rate of contraction and half relax time. In the RENCA tumor model, GDF-15 inhibition fully restored body weight and skeletal muscle mass whereas myostatin inhibition showed only a modest effect. Results: Consistent with the muscle mass improvement, GDF-15 inhibition dramatically increased functional muscle endpoints compared to the partial effect of myostatin inhibition. Interestingly, in the TOV-21G tumor model GDF-15 inhibition only partially restored body weight, however skeletal muscle mass and muscle function were completely normalized. Consistent with the functional assessment, GDF-15 inhibition in the RENCA tumor model decreased the expression of several catabolic genes (i.e. Trim63, Fbxo32, Myh7 and Myh2). The GDF-15 effect is likely to be secondary to the reversal of anorexia since wildtype mice pair-fed to Fc-GDF-15-treated mice demonstrated equivalent muscle mass loss. Conclusions: Taken together these data suggest that GDF-15 inhibition holds potential as an effective therapeutic approach to alleviate multiple aspects of cachexia.

Abstract Platinum-based chemotherapy is associated with nausea/emesis, anorexia and weight loss which reduce patient quality of life and limit treatment adherence potentially leading to poor treatment outcomes. Cisplatin increases circulating growth differentiation factor 15 (GDF-15), a cytokine that induces conditioned taste aversion, anorexia and weight loss in preclinical models. GDF-15 signals through the hindbrain receptor glial cell-derived neurotrophic factor receptor alpha-like (GFRAL). Cisplatin-induced anorexia/weight loss was attenuated in a GFRAL knockout mouse; therefore, we examined whether GDF-15 inhibition can prevent platinum-based chemotherapy-induced emesis, anorexia and weight loss, and also increase survival using mouse and/or nonhuman primate models. In cancer patients, platinum treatment increased circulating GDF-15 in non small cell lung carcinoma, colorectal, and ovarian cancer (~1.5 fold) compared to those receiving a non-platinum-based therapy. Furthermore, cisplatin, oxaliplatin and carboplatin administered individually all increased circulating GDF-15 in wildtype mice (≥ 5 fold) and induced anorexia, skeletal muscle wasting, and weight loss. GDF-15 mRNA was increased in kidney, liver, brain and white adipose tissue. These effects were prevented in GDF-15 knockout mice, however only a partial blockade was observed in the carboplatin group. In nonhuman primates, cisplatin treatment for 5 days (96% of the daily recommended clinical dose) also increased circulating GDF-15 (> 5 fold), and induced anorexia and emesis. Treatment with the anti-GDF-15 monoclonal antibody (mAB1) resulted in no detectable circulating levels of free GDF-15, and attenuated both cisplatin-induced anorexia and emesis. Furthermore, in a mouse cachectic tumor model (subcutaneous implantation of tumor tissue derived from human non-small cell lung carcinoma adenocarcinoma), cisplatin treatment inhibited tumor growth; however, GDF-15 levels were still elevated and additional weight loss occurred compared to vehicle control. When mAB1 was given in combination with cisplatin, weight loss was reversed and tumor growth inhibition was maintained, resulting in greater survival compared to cisplatin alone. Taken together, these data support the notion that GDF-15 inhibition with mAB1 holds the potential as an effective therapeutic approach to alleviate GDF-15 mediated emesis, anorexia and weight loss with the aim to enable optimal cancer treatment as well as to improve patient quality of life and potentially survival. Citation Format: Danna M. Breen, Kevin Beaumont, Donald Bennett, Julia Brosnan, Roberto Calle, Jeffrey R. Chabot, Susie Collins, Teresa Cunio, Ryan M. Esquejo, Stephanie Joaquim, Alison Joyce, Hanna Kim, Laura Lin, Betty Pettersen, Shuxi Qiao, Michelle Rossulek, Brendan Tierney, Karen M. Walters, Gregory Weber, Zhidan Wu, Bei B. Zhang, Morris J. Birnbaum. Growth differentiation factor 15 (GDF-15) inhibition attenuates platinum-based chemotherapy-induced emesis, anorexia and weight loss and increases survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3056.

Platinum-based cancer therapy is restricted by dose-limiting side effects and is associated with elevation of growth differentiation factor 15 (GDF-15). But whether this elevation contributes to such side effects has been unclear. Here, we explored the effects of GDF-15 blockade on platinum-based chemotherapy-induced emesis, anorexia, and weight loss in mice and/or nonhuman primate models. We found that circulating GDF-15 is higher in subjects with cancer receiving platinum-based chemotherapy and is positively associated with weight loss in colorectal cancer (NCT00609622). Further, chemotherapy agents associated with high clinical emetic score induce circulating GDF-15 and weight loss in mice. Platinum-based treatment-induced anorexia and weight loss are attenuated in GDF-15 knockout mice, while GDF-15 neutralization with the monoclonal antibody mAB1 improves survival. In nonhuman primates, mAB1 treatment attenuates anorexia and emesis. These results suggest that GDF-15 neutralization is a potential therapeutic approach to alleviate chemotherapy-induced side effects and improve the quality of life.

Hypertension is the second leading cause of chronic kidney disease in the world. Angiotensin II (Ang II) induced Toll-like receptor 4, TLR4, activation contributes to hypertension-induced renal inflammation and fibrosis. Recently, we showed that mice with dysfunctional TLR4 are protected from renal injury by reducing inflammation and oxidative stress. Autophagy is a protective mechanism that degrades protein aggregates and damaged organelles to maintain intracellular homeostasis and cell integrity. Consequently, defective autophagy in the kidney can lead to cellular injury. A growing body of evidence implicates altered autophagy to various kidney diseases including hypertension. TLRs are known to play a significant role in autophagy, however, the role of TLR4 mediated autophagy in hypertension induced renal injury remains unclear. We hypothesized that TLR4 deficiency reduces Ang II induced renal injury by suppressing autophagy in hypertensive kidney. C3H/Heouj mice with normal TLR4 and C3H/Hej LPS-d with mutant TLR4 (TLR4 deficiency) aged 10-12 weeks were treated without or with Ang II (1000 ng/kg/d) for 4 weeks. In response to Ang II, TLR4 deficient mice showed increased glomerular filtration rate (GFR) (892.43 ± 56.72 vs. 712.63 ± 46.82 μL/min./100g b.w.) and increased renal vascular density and reduced renal cortical resistive index compared to mice with normal TLR4. Ang II treated mice with normal TLR 4 showed increased reactive oxygen species (ROS) generation and upregulation of IκB and NF-kβ compared to TLR4 deficient mice. Ang II increased the expression of autophagy marker proteins, LC3 I/II, p62, Atg5, Beclin1 in the kidney of mice with normal TLR4 compared to mice with TLR4 deficiency. Our data suggests that Ang II induced TLR4 activation upregulates autophagy whereas in C3H/Hej LPS-d mice with TLR4 deficiency, autophagy is unaffected and protects the kidney from hypertension injury.

Approximately 10% of the United States population suffers from diabetes causing diabetic nephropathy (DN) and eventually leading to renal failure. Fatty acids (FAs) are byproducts of gut microbiota and can regulate physiological process such as metabolism and inflammation as well as influenced through dietary supplements. Certain FA receptors have been shown to exert anti‐inflammatory effects in diabetes and other diseases; however, the role of these receptors in the kidney is not well defined. Diallyl trisulfide (DATS) is a major organosulfur component of garlic and is metabolized into hydrogen sulfide, an important gasotransmitter with noted cardiovascular and renal benefits. Whether DATS regulates FAs and their receptors in the kidney is unclear. In this study, we sought to determine if dietary supplementation with DATS improves kidney function by reducing inflammation through FA receptors in a Type‐II diabetic mouse model. Initial results showed improved kidney function of diabetic mice supplemented with DATS, as evidenced by an increase in glomerular filtration rate (GFR) as well as a lower resistive index (RI) of the renal artery compared to diabetic mice on a normal diet. The expression of short chain fatty acid receptor Gpr43 and long chain fatty acid receptor Gpr120 was altered in kidney of mice given DATS compared to untreated animals. In addition, Ptgs2, an enzyme implicated in the inflammatory response and can be inhibited by some naturally occurring FAs, was reduced in the kidney of diabetic mice supplemented with DATS. 16S sequencing showed DATS supplementation affected host gut microbiome composition, showing an enrichment in Verrucomicrobia and increased alpha diversity in diabetic mice on DATS diet. In conclusion, our data suggests FA receptors may play a role in the diabetic kidney by reducing inflammation with DATS supplementation, influencing the expression of these receptors, and improving kidney function. Further, a shift in the gut microbiome of diabetic mice toward that of non‐diabetic mice suggests a role for gut microbiota in DN and DATS supplementation may help delay or prevent the onset of this disease.Support or Funding InformationThis study was supported in part by U.S. National Institutes of Health (NIH) Grant DK104653 (U.S.) and American Heart Association Scientist Development Grant 15SDG25840013 (S.P.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Accumulating evidence suggests that epigenetic mechanisms contribute to aging and target organ injury during hypertension. Aberrant DNA methylation of mitochondrial genes can affect mitochondrial copy numbers, replication and metabolism. Recently, the presence of mitochondrial DNA (mtDNA) methylation was reported; however, its functional significance in aging and hypertension-induced chronic kidney disease is unknown. We hypothesized that alteration in mtDNA methylation aggravates hypertension injury in aged kidney by disrupting mitochondrial homeostasis leading to reduced mitochondrial biogenesis and dysfunction. Hydrogen sulfide (H 2 S) was shown to modulate chromatin recently, we therefore investigated whether H 2 S can act as an epigenetic modifying agent to protect renal mitochondria and improve renal function. Aged (72-75 weeks) and young (10-12 weeks) wild type (WT, C57BL/6J) mice were treated without or with angiotensin-II (Ang II, 1000 ng/Kg/d) and H 2 S donor, GYY4137 (0.5 mg/Kg/d, i.p.), for 28 d. Ang-II increased mean blood pressure in aged mice compared to young (136.54±3.55 vs. 108.68±2.86 mmHg) and reduced glomerular filtration rate (688.3±24.83 vs. 1029.3±30.8 μL/min/100 g b.w.). H 2 S treatment reduced mean BP (122.34±3.47) and increased GFR (962.7±21.5) in aged mice receiving Ang II. In aged mice, querying renal mitochondrial genes for methylation status revealed increased methylation of mt-ND1 and reduction of mt-CO1 and mt-CO2 and H 2 S treatment reversed the changes. Methylation of mt-Cytb was undetected and mt-Dloop was low in Ang II treated aged mice and increased with H 2 S treatment. Ang II treated aged mice exhibited decreased PGC-1α and nuclear transcription factors, NRFI, NRF2, ERRα and TFAM, indicating reduced mitochondrial biogenesis compared to young mice and H 2 S restored their levels. ATP synthase activity, ATP generation was reduced in aged mice receiving Ang II compared to young and increased with H 2 S treatment. Together, our results suggest that alteration in mtDNA methylation and impaired nuclear DNA genes is associated with reduced mitochondrial replication and disrupted metabolism and H 2 S therapy protects the aged kidney from hypertension-induced damage by restoring mitochondrial homeostasis.

Ischemia‐reperfusion (IR) is one of the most common causes of acute kidney injury (AKI) in the aging population. MicroRNAs (miRs) are endogenous non‐coding RNA which have the ability to target the expression of multiple genes involved in the inflammatory and repair processes, thus influencing the course of disease. Although elevation of miR‐21 following renal IR has been observed in several studies involving young animals including humans, its role in the aged kidney remains unknown. The purpose of this study was to determine whether miR‐21 contributes directly to persistent inflammation in the aged kidney following IR injury and whether its inhibition attenuates inflammation to promote repair. Because profibrotic cues originating from inflammatory cells are known to favor endothelial‐mesenchymal transition (endoMT) by activation of myofibroblasts, we also investigated whether miR‐21 inhibition reduces endoMT. Wild type (WT, C57BL/6J) mice aged 12–14 wk and 75–78 wk were used in this study. After treatment without or with Locked Nucleic Acid Anti‐miR‐21 (20 mg/kg b.w.), mice underwent bilateral renal ischemia (27 min.) followed by reperfusion for 7 days. Aged mice exhibited severe renal IR injury and anti‐miR‐21 treatment improved glomerular filtration rate, renal blood flow and decreased renal resistive index. Anti‐miR‐21 treated mice demonstrated predominance of M2 macrophages associated with increased expression of arginase‐1 and IL‐4 and reduction of inflammatory cytokines. Further, anti‐miR‐21 reduced TGF‐β, matrix metalloproteinase‐9 and collagen expression in the aged kidney. Double labeling for endothelial marker, Tie2, and fibroblast markers, FSP‐1/α‐SMA showed significant reduction in their coexpression suggesting reduction in endoMT. Our findings suggest that miR‐21 inhibition reduces macrophage mediated inflammation and endoMT to promote reparative process following IR injury in the aged kidney.Support or Funding InformationAHA grant to SP: 15SDG25840013NIH grant to US: DK104653This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 doxycycline could reverse the effects to mitigate renal dysfunction. C3H/HeJ (TLR4 deficiency) and C3H/HeOuJ (normal TLR4) mice were treated without or with Ang‐II (1000ng/kg −1 /min −1 ) via mini osmotic pump for 4 weeks. Initial results indicated a blunted response in TLR4 deficient mice treated with Ang‐II as observed by a lower mean blood pressure compared to mice with functional TLR4. In addition, kidney injury marker (KIM‐1) and lactate dehydrogenase (LDH) were reduced in the tubules and glomeruli of the kidney, respectively, in TLR4‐deficient mice. Moreover, TLR4‐deficient mice were protected from inflammation‐mediated damage as evidenced by a decrease in IL‐6 and IL‐1β expression. Kidney function of mice lacking TLR4 treated with Ang‐II was improved as observed by a higher glomerular filtration rate and a lower resistive index of cortex and renal artery compared to controls. Hypertensive mice with normal TLR4 function were also found to have a decrease in plasma hydrogen sulfide (H 2 S) levels as well as hypermethylation of the H 2 S converting enzyme 3‐mercaptopyruvate sulfurtransferase compared to deficient TLR4 mice. Expression of zonulin, claudin 1, and occluding, tight‐junction proteins involved in the maintenance and integrity of the gut‐epithelial barrier, were found to be similar in Ang‐II TLR4‐deficient mice compared to untreated mice. In contrast, normal TLR4 mice showed lack of tight junction protein expression, indicating a compromised gut‐epithelial barrier. This result was supported by increased levels of plasma FITC‐dextran that was administered to mice at the end of the treatment period to assess gut leakage, as well as an increase in 16S bacterial expression in the kidney. In conclusion, our data suggests TLR4‐deficient mice have improved renal function and an intact gut‐epithelial barrier as well as normalized H 2 S levels. Current efforts are aimed at profiling the microbiome of these mice as well as assess the effects of doxycycline on gut microbiota composition and whether this antibiotic affects blood pressure regulation. Support or Funding Information NIH Grant DK104653 to Utpal Sen AHA Grant 15SDG25840013 to Sathnur Pushpakumar This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Heart failure is a functional lack of myocardial performance due to a loss of molecular control over increases in calcium and ROS, resulting in proteolytic degradative advances and cardiac remodeling. Mitochondria are the molecular powerhouse of cells, shifting the sphere of cardiomyocyte stability and performance. Functional mitochondria rely on the molecular abilities of safety factors such as TFAM to maintain physiological parameters. Mitochondrial transcription factor A (TFAM) creates a mitochondrial nucleoid structure around mtDNA, protecting it from mutation, inhibiting NFAT (ROS activator/hypertrophic stimulator), and transcriptionally activating Serca2a to decrease calcium mishandling. Current literature depicts major decreases in TFAM as HF progresses. We aim to assess TFAM function against Calpain 1 and MMP 9 proteolytic activity and its role in cardiac remodeling. To this date, no publication has surfaced describing the effects of aortic banding (AB) as a surgical heart failure model in TFAM‐TG mice. HF models were created via AB in TFAM Transgenic (TFAM‐TG) and C57BLJ‐6 (control) mice. Eight weeks post‐AB, functional and histological analysis revealed a successful banding procedure, resulting in cardiac hypertrophy as observed via echocardiography. Pulse wave and color doppler show increased aortic flow rates as well as turbulent flow at the banding site. Preliminary results of cardiac tissue immuno‐histochemistry of HF‐control mice show decreased TFAM and compensatory increases in Serca2a fluorescent expression, along with increased calpain 1 and MMP9 expression. Protein, RNA, and IHC analysis will further assess TFAM‐TG results post‐banding. Echocardiography show more cardiac stability and functionality in HF induced TFAM‐TG mice than the control counterpart. These preliminary findings, along with our invitro results, suggest that TFAM has molecular therapeutic potential to reduce protease activity. Support or Funding Information The study is supported by NIH Grants HL‐74185 and HL‐108621 to SCT and NIH F31 Grant 1F31HL132527‐01 to GHK

Hypertension affects nearly one third of the adult US population and is a significant risk factor for chronic kidney disease (CKD). An expanding body of recent studies indicates that gut microbiome has crucial roles in regulating physiological processes through, among other mechanisms, one mode of short chain fatty acids (SCFA) and their target receptors. In addition, these SCFA receptors are potential targets of regulation by host miRNAs, however, the mechanisms through which this occurs is not clearly defined. Hydrogen sulfide (H 2 S) is an important gasotransmitter involved in multiple physiological processes and is known to alleviate adverse effects of hypertension such as reducing inflammation in the kidney. To determine the role of host microRNAs in regulating short chain fatty acid receptors in the kidney as well as the gut, C57BL/6J wild-type mice were treated with or without Ang-II (1000 ng/kg -1 /min -1 ) and H 2 S donor GYY4137 (GYY) (133 μM/kg -1 /d -1 ) for 4 weeks to assess whether GYY would normalize adverse effects observed in hypertensive mice and whether this was in part due to altered gut microbiome composition. We observed several changes of SCFA receptors and predicted microRNA regulators in the kidney among the different treatments. Haptoglobin, a marker for intestinal epithelial barrier integrity, was increased in hypertensive mice (97 ng/mL) compared to control (48 ng/mL), GYY (64 ng/mL) and Ang-II+GYY (71 ng/mL). The glomerular filtration rate (GFR) was improved in mice treated with Ang-II+GYY (963 μl/min -1 /100 g body wt -1 ) compared with Ang-II (480 μl/min -1 /100 g body wt -1 ) indicating improved kidney function. GYY supplemented mice had increased function (1219 μl/min -1 /100 g body wt -1 ) compared to controls (981 μl/min -1 /100 g body wt -1 ). The Erysipelotrichia class of bacteria, linked with altered SCFA production, was enriched in hypertensive animals but reduced with GYY supplementation. The TM7-3 phyla of bacteria are speculated to have anti-inflammatory properties and were found enriched in GYY only mice, supporting an anti-inflammation effect of H 2 S. These data point towards a role for miRNA regulation in hypertension and are beneficially influenced by H 2 S in both the kidney and potentially through changes in gut microflora.

The relationship between hydrogen sulfide (H 2 S), microRNAs (miRs), matrix metalloproteinases (MMPs) and poly-ADP-ribose-polymerase-1 (PARP-1) in diabetic kidney remodeling remains mostly obscured. We aimed at investigating whether alteration of miR-194-dependent MMPs and PARP-1 causes renal fibrosis in diabetes kidney, and whether H 2 S ameliorates fibrosis. Wild type, diabetic Akita mice as well as mouse glomerular endothelial cells (MGECs) were used as experimental models, and GYY4137 as H 2 S donor. In diabetic mice, plasma H 2 S levels were decreased while ROS and expression of its modulator (ROMO1) were increased. In addition, alteration of MMPs-9, −13 and −14 expression, PARP-1, HIF1α, and increased collagen biosynthesis as well as collagen cross-linking protein, P4HA1 and PLOD2 were observed along with diminished vascular density in diabetic kidney. These changes were ameliorated by GYY4137. Further, downregulated miRNA-194 was normalized by GYY4137 in diabetic kidney. Similar results were obtained in in vitro condition. Interestingly, miR-194 mimic also diminished ROS production, and normalized ROMO1, MMPs-9, −13 and −14, and PARP-1 along with collagen biosynthesis and cross-linking protein in HG condition. We conclude that decrease H 2 S diminishes miR-194, induces collagen deposition and realignment leading to fibrosis and renovascular constriction in diabetes. GYY4137 mitigates renal fibrosis in diabetes through miR-194-dependent pathway.

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, through production of short chain fatty acids (SCFA), which are linked with blood pressure (BP) regulation. Moreover, host epigenetic regulators, such as microRNAs, can directly affect the microbiome, and therefore blood pressure. However, the precise epigenetic regulation of BP is poorly understood. The gaseous molecule, hydrogen sulfide (H2S) is involved in regulating several physiological processes, however, its role and influence on the host gut microbiome and on renal function remains to be elucidated. In this study, we sought to determine the roles of host microRNAs in regulating short chain fatty acid receptors in the hypertensive kidney and whether H2S donor, GYY4137 (GYY), could reverse the effects to mitigate renal dysfunction. C57BL6/J wild‐type mice were treated without or with Ang‐II (1000ng/kg−1/min−1) and GYY (133μM/kg−1/d−1) for 4 weeks. Quantitative PCR, Western blot, immunofluorescence assays, metagenomic sequencing, and glomerular filtration rate were measured. Results indicated that increased expression of miR‐329 and miR‐132 in hypertensive mice were reduced by GYY treatment. SCFA receptors Gpr41 and Gpr43 are expressed in the kidney and are also predicted targets of miR‐329 and miR‐132, respectively. The mRNA and protein expression of the above receptors was reduced in Ang‐II treated mice while animals treated with Ang‐II and GYY showed normalized expression. miR‐129 was induced by GYY supplementation and the predicted SCFA receptor target Olfr78 was decreased in the normal kidney with the opposite effect in hypertensive kidney. Immunofluorescence analysis showed decreased signal of Gpr41 and Gpr43 in the blood vessel and tubules in Ang‐II mice and were normalized in mice treated with GYY. Metagenomic sequencing showed H2S supplementation affected the host gut microbiome composition and improved renal function. In conclusion, our data suggests that microRNAs can regulate SCFA receptors in the kidney and hydrogen sulfide supplementation has a positive effect on renal function through the gut microbiome and SCFA receptors.Support or Funding InformationDK‐104653 and HL‐104103 to U.S.,15SDG25840013 to S.P.

Hypertension is a major risk factor for chronic kidney disease and renal inflammation is an integral part in this pathology. In recent years, hydrogen sulfide (H 2 S) has emerged as an important regulator of blood pressure. Studies in spontaneously hypertensive rats have shown that endogenous hydrogen sulfide (H 2 S) production is impaired due to decreased CBS/CSE expression, and exogenous H 2 S supplementation decreased blood pressure and vascular remodeling. MicroRNAs are short, single‐stranded RNA genes that regulate post‐transcriptional gene expression by targeting messenger RNAs (mRNAs), and are associated with a broad spectrum of physiological processes. While there are several studies aimed at investigating the role of non‐coding RNA in hypertension, the roles and implications of these RNAs are not well defined. In this study, we sought to identify microRNAs that are dysregulated in response to angiotensin‐induced hypertension in the kidney and whether hydrogen sulfide donor, GYY 4137, could reverse the microRNA alteration. Wild‐type mice were treated without or with Ang‐II (1000ng/Kg/Min) and without or with GYY4137 for 4 weeks. Blood pressure, renal blood flow, renal resistive index (RI) measurements were performed. microRNA microarrays were conducted and subsequent target prediction revealed genes associated with proinflammatory response. Ang‐II treated mice showed a significant increase in blood pressure accompanied by a decrease in blood flow in the renal cortex and an increase in the resistive index. These effects were attenuated in mice treated with GYY4137. Microarrays of the kidneys revealed downregulation of miR‐129 and miR‐299b in Ang‐II treated mice and upregulation following GYY4137 treatment. In contrast, miR‐369 was upregulated in Ang‐II treatment and suppressed in Ang‐II+GYY4137 mice. Quantitation of genes involved in proinflammatory response revealed upregulation of Tnfα , Il1β , Mcp1 and Mip2 mRNA. Our data suggests that downregulation of miR‐129 and ‐299b plays a significant role in Ang‐II induced renal inflammation, and GYY4137 reduces inflammation and is associated with reversal of miR expression. Support or Funding Information NIH: HL104103, AHA: 15SDG25840013

MicroRNAs regulate several physiological processes and are implicated in various pathologies, including hypertension. Previous work indicates miR-132 targets Sirtuin 1 (Sirt1), a histone deacetylase and regulator of epigenetic gene silencing in various cellular processes. Sirt1 is expressed in the kidney; however, its role in hypertensive kidney and whether it is regulated by physiological gaseous molecules, such as hydrogen sulfide (H 2 S), is not known. In this study, we sought to determine the role of miR-132 in regulating Sirt1, Ace2 and At1 in hypertensive kidney and whether H 2 S donor, GYY4137 (GYY), could reverse these effects and mitigates renal dysfunction. Wild-type mice were treated without or with Ang-II (1000 ng/Kg/Min) and GYY (133 μM) for 4 weeks. Quantitative PCR, Western blot, and immunofluorescence assays were performed. Increased expression levels of miR-132 in hypertensive mice (3.79 fold vs control) were reduced in mice receiving GYY treatment (2.43 fold vs control). Sirt1 expression was reduced (-1.15 fold) in Ang-II mice but was upregulated in GYY (1.25 fold) and Ang-II+GYY (1.9 fold) groups. A similar effect was seen with Sirt1 protein where the expression was increased in animals treated with GYY and Ang-II+GYY (1.16, 1.03 respectively) compared to Ang-II (0.47). Ace2 in Ang-II+GYY (0.45) was increased compared to Ang-II (0.17), while At1 was reduced (0.46) compared to Ang-II (0.86). Immunofluorescence showed decreased signal of Sirt1 in the glomerulus in Ang-II mice and increased At1 in the blood vessels surrounding the glomerulus, leading to constriction of renal artery, decreased blood flow, and kidney dysfunction. These effects were alleviated in mice treated with GYY. Our data suggests that upregulation of miR-132 in hypertensive kidney decreases Sirt1 and Ace2 expression, leading to increased Ang-II signaling through the At1 receptor and GYY supplementation reverses these expression patterns, leading to increased blood flow and kidney function.

Prediction of human pharmacokinetics (PK) can be challenging for monoclonal antibodies (mAbs) exhibiting target-mediated drug disposition (TMDD). In this study, we performed a quantitative analysis of a diverse set of six mAbs exhibiting TMDD to explore translational rules that can be utilized to predict human PK. A TMDD model with rapid-binding approximation was utilized to fit PK and PD (i.e., free and/or total target levels) data, and average absolute fold error (AAFE) was calculated for each model parameter. Based on the comparative analysis, translational rules were developed and applied to a test antibody not included in the original analysis. AAFE of less than two-fold was observed between monkey and human for baseline target levels (R 0), body-weight (BW) normalized central elimination rate (K el/BW−0.25) and central volume (V c/BW1.0). AAFE of less than three-fold was estimated for the binding affinity constant (K D). The other four parameters, i.e., complex turnover rate (K int), target turnover rate (K deg), central to peripheral distribution rate constant (K pt) and peripheral to central rate constant (K tp) were poorly correlated between monkey and human. The projected human PK of test antibody based on the translation rules was in good agreement with the observed nonlinear PK. In conclusion, we recommend a TMDD model-based prediction approach that integrates in vitro human biomeasures and in vivo preclinical data using translation rules developed in this study.