Epoxyeicosatrienoic acid administration or soluble epoxide hydrolase inhibition attenuates renal fibrogenesis in obstructive nephropathy.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites with biological effects, including antiapoptotic, anti-inflammatory, and antifibrotic functions. Soluble epoxide hydrolase (sEH)-mediated hydrolysis of EETs to dihydroxyeicosatrienoic acids (DHETs) attenuates these effects. Recent studies have demonstrated that inhibition of sEH prevents renal tubulointerstitial fibrosis and inflammation in the chronic kidney disease model. Given the pathophysiological role of the EET pathway in chronic kidney disease, we investigated if administration of EET regioisomers and/or sEH inhibition will promote antifibrotic and renoprotective effects in renal fibrosis following unilateral ureteral obstruction (UUO). EETs administration abolished tubulointerstitial fibrogenesis, as demonstrated by reduced fibroblast activation and collagen deposition after UUO. The inflammatory response was prevented as demonstrated by decreased neutrophil and macrophage infiltration and expression of cytokines in EET-administered UUO kidneys. EET administration and/or sEH inhibition significantly reduced M1 macrophage markers, whereas M2 macrophage markers were highly upregulated. Furthermore, UUO-induced oxidative stress, tubular injury, and apoptosis were all downregulated following EET administration. Combined EET administration and sEH inhibition, however, had no additive effect in attenuating inflammation and renal interstitial fibrogenesis after UUO. Taken together, our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest EET treatment as a potential therapeutic strategy to treat fibrotic diseases.NEW & NOTEWORTHY Epoxyeicosatrienoic acids (EETs) are cytochrome P-450-dependent antihypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered renoprotective. We found that EET administration and/or soluble epoxide hydrolase inhibition significantly attenuates oxidative stress, renal cell death, inflammation, macrophage differentiation, and fibrogenesis following unilateral ureteral obstruction. Our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest that EET treatment may be a potential therapeutic strategy to treat fibrotic diseases.

Human milk oligosaccharides reduce necrotizing enterocolitis-induced neuroinflammation and cognitive impairment in mice.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in premature infants. One of the most devastating complications of NEC is the development of NEC-induced brain injury, which manifests as impaired cognition that persists beyond infancy and which represents a proinflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL) and 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we hypothesized that oral administration of these HMOs would reduce NEC-induced brain injury and sought to determine the mechanisms involved. We now show that the administration of either 2'-FL or 6'-SL significantly attenuated NEC-induced brain injury, reversed myelin loss in the corpus callosum and midbrain of newborn mice, and prevented the impaired cognition observed in mice with NEC-induced brain injury. In seeking to define the mechanisms involved, 2'-FL or 6'-SL administration resulted in a restoration of the blood-brain barrier in newborn mice and also had a direct anti-inflammatory effect on the brain as revealed through the study of brain organoids. Metabolites of 2'-FL were detected in the infant mouse brain by nuclear magnetic resonance (NMR), whereas intact 2'-FL was not. Strikingly, the beneficial effects of 2'-FL or 6'-SL against NEC-induced brain injury required the release of the neurotrophic factor brain-derived neurotrophic factor (BDNF), as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. Taken in aggregate, these findings reveal that the HMOs 2'-FL and 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury.NEW & NOTEWORTHY This study reveals that the administration of human milk oligosaccharides, which are present in human breast milk, can interfere with the proinflammatory gut-brain axis and prevent neuroinflammation in the setting of necrotizing enterocolitis, a major intestinal disorder seen in premature infants.

Hepatic and proximal tubule angiotensinogen play distinct roles in kidney dysfunction, glomerular and tubular injury, and fibrosis progression.

Components of the renin-angiotensin system, including angiotensinogen (AGT), are critical contributors to chronic kidney disease (CKD) development and progression. However, the specific role of tissue-derived AGTs in CKD has not been fully understood. To define the contribution of liver versus kidney AGT in the CKD development, we performed 5/6 nephrectomy (Nx), an established CKD model, in wild-type (WT), proximal tubule (PT)- or liver-specific AGT knockout (KO) mice. Nx significantly elevated intrarenal AGT expression and elevated blood pressure (BP) in WT mice. The increase of intrarenal AGT protein was completely blocked in liver-specific AGT KO mice with BP reduction, suggesting a crucial role for liver AGT in BP regulation during CKD. Nx-induced glomerular and kidney injury and dysfunction, as well as fibrosis, were all attenuated to a greater extent in liver-specific AGT KO mice compared with PT-specific AGT KO and WT mice. However, the suppression of interstitial fibrosis in PT- and liver-specific AGT KO mouse kidneys was comparable. Our findings demonstrate that liver AGT acts as a critical contributor in driving glomerular and tubular injury, renal dysfunction, and fibrosis progression, whereas the role of PT AGT was limited to interstitial fibrosis progression in chronic renal insufficiency. Our results provide new insights for the development of tissue-targeted renin-angiotensin system intervention in the treatment of CKD.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a major unmet medical need with no effective treatment. Current findings demonstrate that hepatic and proximal tubule angiotensinogen have distinct roles in tubular and glomerular injury, fibrogenesis, and renal dysfunction during CKD development. As renin-angiotensin system components, including angiotensinogen, are important targets for treating CKD in the clinic, the results from our study may be applied to developing better tissue-targeted treatment strategies for CKD and other fibroproliferative diseases.

Proximal tubule cyclophilin D mediates kidney fibrogenesis in obstructive nephropathy.

The proximal tubule (PT) is highly vulnerable to acute injury, including ischemic insult and nephrotoxins, and chronic kidney injury. It has been established that PT injury is a primary cause of the development of chronic kidney disease, but the underlying molecular mechanism remains to be defined. Here, we tested whether PT cyclophilin D (CypD), a mitochondrial matrix protein, is a critical factor to cause kidney fibrosis progression. To define the role of CypD in kidney fibrosis, we used an established mouse model for kidney fibrosis: the unilateral ureteral obstruction (UUO) model in global and PT-specific CypD knockout (KO). Global CypD KO blunted kidney fibrosis progression with inhibition of myofibroblast activation and fibrosis. UUO-induced tubular atrophy was suppressed in kidneys of global CypD KO but not tubular dilation or apoptotic cell death. PT cell cycle arrest was highly increased in wild-type UUO kidneys but was markedly attenuated in global CypD KO UUO kidneys. The number of macrophages and neutrophils was less in UUO kidneys of global CypD KO than those of wild-type kidneys. Proinflammatory and profibrotic factors were all inhibited in global CypD KO. In line with those of global CypD KO, PT-specific CypD KO also blunted kidney fibrosis progression, along with less tubular atrophy, renal parenchymal loss, cell cycle arrest in PT, and inflammation, indicating a critical role for PT CypD in fibrogenesis. Collectively, our data demonstrate that CypD in the PT is a critical factor contributing to kidney fibrosis in UUO, providing a new paradigm for mitochondria-targeted therapeutics of fibrotic diseases.NEW & NOTEWORTHY It has been established that renal proximal tubule (PT) injury is a primary cause of the development of chronic kidney disease, but the underlying molecular mechanism remains to be defined. Here, we show that cyclophilin D, a mitochondrial matrix protein, in the PT causes kidney fibrogenesis in obstructive nephropathy. Our data suggest that targeting PT cyclophilin D could be beneficial to prevent fibrosis progression.

Nephron Progenitor Maintenance Is Controlled through Fibroblast Growth Factors and Sprouty1 Interaction.

BACKGROUND: Nephron progenitor cells (NPCs) give rise to all segments of functional nephrons and are of great interest due to their potential as a source for novel treatment strategies for kidney disease. Fibroblast growth factor (FGF) signaling plays pivotal roles in generating and maintaining NPCs during kidney development, but little is known about the molecule(s) regulating FGF signaling during nephron development. Sprouty 1 (SPRY1) is an antagonist of receptor tyrosine kinases. Although SPRY1 antagonizes Ret-GDNF signaling, which modulates renal branching, its role in NPCs is not known. METHODS: Spry1, Fgf9, and Fgf20 compound mutant animals were used to evaluate kidney phenotypes in mice to understand whether SPRY1 modulates FGF signaling in NPCs and whether FGF8 functions with FGF9 and FGF20 in maintaining NPCs. RESULTS: Loss of one copy of Spry1 counters effects of the loss of Fgf9 and Fgf20, rescuing bilateral renal agenesis premature NPC differentiation, NPC proliferation, and cell death defects. In the absence of SPRY1, FGF9, and FGF20, another FGF ligand, FGF8, promotes nephrogenesis. Deleting both Fgf8 and Fgf20 results in kidney agenesis, defects in NPC proliferation, and cell death. Deleting one copy of Fgf8 reversed the effect of deleting one copy of Spry1, which rescued the renal agenesis due to loss of Fgf9 and Fgf20. CONCLUSIONS: SPRY1 expressed in NPCs modulates the activity of FGF signaling and regulates NPC stemness. These findings indicate the importance of the balance between positive and negative signals during NPC maintenance.

IDH2 gene deficiency accelerates unilateral ureteral obstruction-induced kidney inflammation through oxidative stress and activation of macrophages.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) produces NADPH, which is known to inhibit mitochondrial oxidative stress. Ureteral obstruction induces kidney inflammation and fibrosis via oxidative stress. Here, we investigated the role and underlying mechanism of IDH2 in unilateral ureteral obstruction (UUO)-induced kidney inflammation using IDH2 gene deleted mice (IDH2-/-). Eight- to 10-week-old female IDH2-/- mice and wild type (IDH2+/+) littermates were subjected to UUO and kidneys were harvested 5 days after UUO. IDH2 was not detected in the kidneys of IDH2-/- mice, while UUO decreased IDH2 in IDH2+/+ mice. UUO increased the expressions of markers of oxidative stress in both IDH2+/+ and IDH2-/- mice, and these changes were greater in IDH2-/- mice compared to IDH2+/+ mice. Bone marrow-derived macrophages of IDH2-/- mice showed a more migrating phenotype with greater ruffle formation and Rac1 distribution than that of IDH2+/+ mice. Correspondently, UUO-induced infiltration of monocytes/macrophages was greater in IDH2-/- mice compared to IDH2+/+ mice. Taken together, these data demonstrate that IDH2 plays a protective role against UUO-induced inflammation through inhibition of oxidative stress and macrophage infiltration.

Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury.

Regardless of the etiology, acute kidney injury involves aspects of mitochondrial dysfunction and ATP depletion. Fatty acid oxidation is the preferred energy source of the kidney and is inhibited during acute kidney injury. A pivotal role for the mitochondrial matrix protein, cyclophilin D in regulating overall cell metabolism is being unraveled. We hypothesize that mitochondrial interaction of proximal tubule cyclophilin D and the transcription factor PPARα modulate fatty acid beta-oxidation in cisplatin-induced acute kidney injury. Cisplatin injury resulted in histological and functional damage in the kidney with downregulation of fatty acid oxidation genes and increase of intrarenal lipid accumulation. However, proximal tubule-specific deletion of cyclophilin D protected the kidneys from the aforementioned effects. Mitochondrial translocation of PPARα, its binding to cyclophilin D, and sequestration led to inhibition of its nuclear translocation and transcription of PPARα-regulated fatty acid oxidation genes during cisplatin-induced acute kidney injury. Genetic or pharmacological inhibition of cyclophilin D preserved nuclear expression and transcriptional activity of PPARα and prevented the impairment of fatty acid oxidation and intracellular lipid accumulation. Docking analysis identified potential binding sites between PPARα and cyclophilin D. Thus, our results indicate that proximal tubule cyclophilin D elicits impaired mitochondrial fatty acid oxidation via mitochondrial interaction between cyclophilin D and PPARα. Hence, targeting their interaction may be a potential therapeutic strategy to prevent energy depletion, lipotoxicity and cell death in cisplatin-induced acute kidney injury.

Renal Sympathetic Nerve-Derived Signaling in Acute and Chronic kidney Diseases.

The kidney is innervated by afferent sensory and efferent sympathetic nerve fibers. Norepinephrine (NE) is the primary neurotransmitter for post-ganglionic sympathetic adrenergic nerves, and its signaling, regulated through adrenergic receptors (AR), modulates renal function and pathophysiology under disease conditions. Renal sympathetic overactivity and increased NE level are commonly seen in chronic kidney disease (CKD) and are critical factors in the progression of renal disease. Blockade of sympathetic nerve-derived signaling by renal denervation or AR blockade in clinical and experimental studies demonstrates that renal nerves and its downstream signaling contribute to progression of acute kidney injury (AKI) to CKD and fibrogenesis. This review summarizes our current knowledge of the role of renal sympathetic nerve and adrenergic receptors in AKI, AKI to CKD transition and CKDand provides new insights into the therapeutic potential of intervening in its signaling pathways.

Downregulation of exocyst Sec10 accelerates kidney tubule cell recovery through enhanced cell migration.

Migration of surviving kidney tubule cells after sub-lethal injury, for example ischemia/reperfusion (I/R), plays a critical role in recovery. Exocytosis is known to be involved in cell migration, and a key component in exocytosis is the highly-conserved eight-protein exocyst complex. We investigated the expression of a central exocyst complex member, Sec10, in kidneys following I/R injury, as well as the role of Sec10 in wound healing following scratch injury of cultured Madin-Darby canine kidney (MDCK) cells. Sec10 overexpression and knockdown (KD) in MDCK cells were used to investigate the speed of wound healing and the mechanisms underlying recovery. In mice, Sec10 decreased after I/R injury, and increased during the recovery period. In cell culture, Sec10 OE inhibited ruffle formation and wound healing, while Sec10 KD accelerated it. Sec10 OE cells had higher amounts of diacylglycerol kinase (DGK) gamma at the leading edge than did control cells. A DGK inhibitor reversed the inhibition of wound healing and ruffle formation in Sec10 OE cells. Conclusively, downregulation of Sec10 following I/R injury appears to accelerate recovery of kidney tubule cells through activated ruffle formation and enhanced cell migration.

Mitochondrial NADP+-Dependent Isocitrate Dehydrogenase Deficiency Exacerbates Mitochondrial and Cell Damage after Kidney Ischemia-Reperfusion Injury.

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, synthesizing NADPH, which is essential for mitochondrial redox balance. Ischemia-reperfusion (I/R) is one of most common causes of AKI. I/R disrupts the mitochondrial redox balance, resulting in oxidative damage to mitochondria and cells. Here, we investigated the role of IDH2 in I/R-induced AKI. I/R injury in mice led to the inactivation of IDH2 in kidney tubule cells. Idh2 gene deletion exacerbated the I/R-induced increase in plasma creatinine and BUN levels and the histologic evidence of tubule injury, and augmented the reduction of NADPH levels and the increase in oxidative stress observed in the kidney after I/R. Furthermore, Idh2 gene deletion exacerbated I/R-induced mitochondrial dysfunction and morphologic fragmentation, resulting in severe apoptosis in kidney tubule cells. In cultured mouse kidney proximal tubule cells, Idh2 gene downregulation enhanced the mitochondrial damage and apoptosis induced by treatment with hydrogen peroxide. This study demonstrates that Idh2 gene deletion exacerbates mitochondrial damage and tubular cell death via increased oxidative stress, suggesting that IDH2 is an important mitochondrial antioxidant enzyme that protects cells from I/R insult.

Hepatic ischemia/reperfusion injury disrupts the homeostasis of kidney primary cilia via oxidative stress.

Acute kidney injury (AKI) is a major complication of hepatic surgeries. The primary cilium protrudes to the lumen of kidney tubules and plays an important role in renal functions. Disruption of primary cilia homeostasis is highly associated with human diseases including AKI. Here, we investigated whether transient hepatic ischemia induces length change and deciliation of kidney primary cilia, and if so, whether reactive oxygen species (ROS)/oxidative stress regulates those. HIR induced damages to the liver and kidney with increases in ROS/oxidative stress. HIR shortened the cilia of kidney epithelial cells and caused them to shed into the urine. This shortening and shedding of cilia was prevented by Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, an antioxidant). The urine of patient undergone liver resection contained ciliary proteins. These findings indicate that HIR induces shortening and deciliation of kidney primary cilia into the urine via ROS/oxidative stress, suggesting that primary cilia is associated with HIR-induced AKI and that the presence of ciliary proteins in the urine could be a potential indication of kidney injury.

Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis.

The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.

Simultaneous deletion of Bax and Bak is required to prevent apoptosis and interstitial fibrosis in obstructive nephropathy.

Proximal tubular injury and apoptosis are key mediators of the development of kidney fibrosis, a hallmark of chronic kidney disease. However, the molecular mechanism by which tubular apoptotic cell death leads to kidney fibrosis is poorly understood. In the present study, we tested the roles of Bcl-2-associated X (Bax) and Bcl-2 antagonist/killer (Bak), two crucial proteins involved in intrinsic apoptotic cell death, in the progression of kidney fibrosis. Mice with proximal tubule-specific Bax deletion, systemic deletion of Bak, and dual deletion of Bax and Bak were subjected to unilateral ureteral obstruction (UUO). Dual deficiency of Bax and Bak inhibited tubular apoptosis and atrophy. Consistent with decreased tubular injury, dual ablation of Bax and Bak suppressed UUO-induced inflammation and kidney fibrosis with decreased tubular cell cycle arrest, expression of fibrogenic and inflammatory cytokines, and oxidative stress in the kidney. Bax or Bak deficiency was insufficient to prevent apoptosis and all other aforementioned malevolent effects, suggesting compensatory mediation by each other in the respective signaling pathways. These data suggest that dual ablation of Bax and Bak in the kidney is required to prevent UUO-induced tubular apoptosis and the consequent kidney inflammation and fibrosis.

Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site.

Increase of interstitial cell population, resulting in the expansion of interstitium, excessive production of extracellular matrix, and reduction of functioning tubules, is critical in fibrotic progression in the kidney of patients suffering from chronic renal diseases. Here, we investigated the contribution of bone marrow-derived cells (BMDC) in kidney fibrosis caused by ureteral obstruction (UO) using eGFP bone marrow-reconstituted chimeric mice. UO caused dramatic increases in the numbers of interstitial cells and expansion of the interstitium. Most kidney interstitial cells expressed GFP. Twenty nine percent of interstitial cells were cells that had proliferated and approximately 89% among them were BMDCs. Proliferation of fibroblasts differentiated from BMDCs significantly occurred in the interstitium of UO-kidney. Removal of BMDCs by whole body irradiation after UO resulted in reduction of kidney fibrosis, while injection of RAW264.7 cells, monocytes/macrophages, into irradiated mice induced a reversal of this reduction. Treatment with apocynin, an inhibitor of NADPH oxidase, reduced infiltration of BMDCs into the UO-kidney, leading to reduction of kidney fibrosis. In addition, only a few slow-cycling cells were observed in the interstitium of normal kidney. Even after UO, no change in the number of those cells was observed. Our findings demonstrate that BMDCs are a major source for interstitial expansion during kidney fibrosis via infiltration into damaged sites, differentiation to fibroblasts, and subsequent proliferation, contributing kidney fibrosis. These data provide a clear therapeutic target for treatment of chronic kidney disease.

Activation of ERK accelerates repair of renal tubular epithelial cells, whereas it inhibits progression of fibrosis following ischemia/reperfusion injury.

Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-ß1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.

Defect in Runx2 gene accelerates ureteral obstruction-induced kidney fibrosis via increased TGF-ß signaling pathway.

Runt-related transcription factor 2 (Runx2) plays an important role in bone formation and de novo synthesis of proteins, including type 1 collagen. Runx2 has a potent effect on signaling of transforming growth factor (TGF)-ß and vice versa, implicating its significant role in fibrosis. Chronic renal failure comprises fibrosis, characterized as an increase in TGF-ß signaling, and expression of α-smooth muscle actin (α-SMA), and extracellular matrix proteins. Here, we evaluated the role of Runx2 in ureteral obstruction (UO)-induced kidney fibrosis using mice whose Runx2 gene expression is genetically down-regulated. UO caused tubular atrophy and dilation, expansion of interstitium, and increased expression of collagens and α-SMA with a concomitant decrease in expression of Runx2. Deficiency of Runx2 gene (Runx2(+/-) mice) showed higher expression of collagens and α-SMA in the kidney following UO compared to wild type (Runx2(+/+)) mice. UO-induced activation of TGF-ß signaling was higher in the Runx2(+/-) kidney than Runx2(+/+) kidney, suggesting an inhibitory effect of Runx2 on TGF-ß signaling in kidney fibrosis. Besides, overexpression of the Runx2 gene using an adenoviral vector in kidney tubule cells resulted in attenuated TGF-ß-induced Smad3 phosphorylation and expressions of α-SMA and collagen I. Furthermore, Runx2 gene deficient mouse embryonic fibroblasts induced greater activation of Smad3 and expression of α-SMA in response to TGF-ß. Collectively, Runx2 plays a protective role in UO-induced kidney fibrosis by inhibition of TGF-ß signaling, suggesting Runx2 as a novel target for protection against fibrosis-related diseases such as chronic renal failure.

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction.

Hydrogen sulfide (H2S) produced by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H2S in kidney fibrosis remains to be defined. Here, we investigated the role of H2S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H2S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H2S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H2S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-ß1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H2S in UO-induced kidney fibrosis, suggesting that H2S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.

Recruitment and subsequent proliferation of bone marrow-derived cells in the postischemic kidney are important to the progression of fibrosis.

Acute kidney injury (AKI) is an independent risk factor of the development of chronic kidney disease. Kidney fibrosis is a typical feature of chronic kidney disease and is characterized as an expansion of the interstitium due to increases in extracellular matrix molecules and interstitial cells caused by accumulations of extrarenal cells and by the proliferation or differentiation of intrarenal cells. However, the role of bone marrow-derived cells (BMDCs) in AKI-induced kidney fibrosis remains to be defined. Here, we investigated the role of BMDCs in kidney fibrosis after ischemia-reperfusion injury (IRI)-induced AKI in green fluorescent protein (GFP)-expressing bone marrow chimeric mice. IRI resulted in severe fibrotic changes in kidney tissues and dramatically increased interstitial cell numbers. Furthermore, GFP-expressing BMDCs accounted for >80% of interstitial cells in fibrotic kidneys. Interstitial GFP-expressing cells expressed α-smooth muscle actin (a myofibroblast marker), fibroblast-specific protein-1 (a fibroblast marker), collagen type III, and F4/80 (a macrophage marker). Over 20% of interstitial cells were bromodeoxyuridine-incorporating (proliferating) cells, and of these, 80% cells were GFP-expressing BMDCs. Daily treatment of IRI mice with apocynin (a NADPH oxidase inhibitor that functions as an antioxidant) from the day after surgery until euthanization slightly inhibited these changes with a small reduction of fibrosis. Taken together, our findings show that BMDCs make a major contribution to IRI-induced fibrosis due to their infiltration, subsequent differentiation, and proliferation in injured kidneys, suggesting that BMDCs be considered an important target for the treatment of kidney fibrosis.

Ccr2 Dependent Monocytes Exacerbate Intestinal Inflammation and Modulate Gut Serotonergic Signaling Following Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) leads to acute gastrointestinal dysfunction and mucosal damage, resulting in feeding intolerance. Ccr2+ monocytes are crucial immune cells that regulate the gut's inflammatory response via the brain-gut axis. Using CCR2KO mice, we investigated the intricate interplay between these cells to better elucidate the role of systemic inflammation after TBI. METHODS: A murine-controlled cortical impact model was utilized, and results were analyzed on post-injury days (PID) 1 and 3. The experimental groups included (1) Sham C57Bl/6 wild-type (WT), (2) TBI WT, (3) Sham CCR2KO and (4) TBI CCR2KO. Mice were euthanized on PID 1 and 3 to harvest the ileum and study intestinal dysfunction and serotonergic signaling using a combination of quantitative real-time PCR (qRT-PCR), immunohistochemistry, FITC-dextran motility assays, and flow cytometry. Student's t-test and one-way ANOVA were used for statistical analysis, with significance achieved when p < 0.05. RESULTS: TBI resulted in severe dysfunction and dysmotility of the small intestine in WT mice as established by significant upregulation of inflammatory cytokines iNOS, Lcn2, TNFα, and IL1ß and the innate immunity receptor toll-like receptor 4 (Tlr4). This was accompanied by disruption of genes related to serotonin synthesis and degradation. Notably, CCR2KO mice subjected to TBI showed substantial improvements in intestinal pathology. TBI CCR2KO groups demonstrated reduced expression of inflammatory mediators (iNOS, Lcn2, IL1ß, and Tlr4) and improvement in serotonin synthesis genes, including tryptophan hydroxylase 1 (Tph1) and dopa decarboxylase (Ddc). CONCLUSION: Our study reveals a critical role for Ccr2+ monocytes in modulating intestinal homeostasis after TBI. Ccr2+ monocytes aggravate intestinal inflammation and alter gut-derived serotonergic signaling. Therefore, targeting Ccr2+ monocyte-dependent responses could provide a better understanding of TBI-induced gut inflammation. Further studies are required to elucidate the impact of these changes on brain neuroinflammation and cognitive outcomes. STUDY TYPE: Original Article (Basic Science, level of evidence N/A).

Colitis-Induced Small Intestinal Hypomotility Is Dependent on Enteroendocrine Cell Loss in Mice.

BACKGROUND & AIMS: The abdominal discomfort experienced by patients with colitis may be attributable in part to the presence of small intestinal dysmotility, yet mechanisms linking colonic inflammation with small-bowel motility remain largely unexplored. We hypothesize that colitis results in small intestinal hypomotility owing to a loss of enteroendocrine cells (EECs) within the small intestine that can be rescued using serotonergic-modulating agents. METHODS: Male C57BL/6J mice, as well as mice that overexpress (EECOVER) or lack (EECDEL) NeuroD1+ enteroendocrine cells, were exposed to dextran sulfate sodium (DSS) colitis (2.5% or 5% for 7 days) and small intestinal motility was assessed by 70-kilodalton fluorescein isothiocyanate-dextran fluorescence transit. EEC number and differentiation were evaluated by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, and quantitative reverse-transcriptase polymerase chain reaction. Mice were treated with the 5-hydroxytryptamine receptor 4 agonist prucalopride (5 mg/kg orally, daily) to restore serotonin signaling. RESULTS: DSS-induced colitis was associated with a significant small-bowel hypomotility that developed in the absence of significant inflammation in the small intestine and was associated with a significant reduction in EEC density. EEC loss occurred in conjunction with alterations in the expression of key serotonin synthesis and transporter genes, including Tph1, Ddc, and Slc6a4. Importantly, mice overexpressing EECs revealed improved small intestinal motility, whereas mice lacking EECs had worse intestinal motility when exposed to DSS. Finally, treatment of DSS-exposed mice with the 5-hydroxytryptamine receptor 4 agonist prucalopride restored small intestinal motility and attenuated colitis. CONCLUSIONS: Experimental DSS colitis induces significant small-bowel dysmotility in mice owing to enteroendocrine loss that can be reversed by genetic modulation of EEC or administering serotonin analogs, suggesting novel therapeutic approaches for patients with symptomatic colitis.