Tubular Fibrosis Research Articles

IFN Regulatory Factor 4 Controls Post-ischemic Inflammation and Prevents Chronic Kidney Disease.

Ischemia reperfusion injury (IRI) of the kidney results in interferon regulatory factor 4 (IRF4)–mediated counter-regulation of the acute inflammatory response. Beyond that, IRF4 exerts important functions in controlling the cytokine milieu, T-cell differentiation, and macrophage polarization. The latter has been implicated in tissue remodeling. It therefore remains elusive what the role of IRF4 is in terms of long-term outcome following IRI. We hypothesized that an inability to resolve chronic inflammation in Irf4−/− mice would promote chronic kidney disease (CKD) progression. To evaluate the effects of IRF4 in chronic upon acute injury in vivo, a mouse model of chronic injury following acute IRI was employed. The expression of Irf4 increased within 10 days after IRI in renal tissue. Both mRNA and protein levels remained high up to 5 weeks upon IRI, suggesting a regulatory function in the chronic phase. Mice deficient in IRF4 display increased tubular cell loss and defective clearance of infiltrating macrophages. These phenomena were associated with increased expression of pro-inflammatory macrophage markers together with reduced expression of alternatively activated macrophage markers. In addition, IRF4-deficient mice showed defective development of alternatively activated macrophages. Hints of a residual M1 macrophage signature were further observed in human biopsy specimens of patients with hypertensive nephropathy vs. living donor specimens. Thus, IRF4 restricts CKD progression and kidney fibrosis following IRI, potentially by enabling M2 macrophage polarization and restricting a Th1 cytokine response. Deteriorated alternative macrophage subpopulations in Irf4−/− mice provoke chronic intrarenal inflammation, tubular epithelial cell loss, and renal fibrosis in the long course after IRI in mice. The clinical significance of these finding for human CKD remains uncertain at present and warrants further studies.

Munc18-1-interacting protein 3 mitigates renal fibrosis through protection of tubular epithelial cells from apoptosis.

Tubulointerstitial fibrosis is a hallmark of chronic kidney disease (CKD), and is initiated by tubular epithelial cell (TEC) injury. Hypoxia promotes tubular cell death, fibrosis and CKD progression. Munc18-1-interacting protein 3 (Mint3) is a molecule that activates hypoxia-inducible factors (HIFs) by binding and suppressing factor inhibiting HIF-1 (FIH). However, the role of Mint3 in tubulointerstitial fibrosis remains unknown. We induced fibrosis of the kidney after unilateral ischemia-reperfusion injury (uIRI) in Mint3-knockout and littermate wild-type mice. The duration of ischemia was 23min and the kidneys were harvested at 24h and 7days after ischemia-reperfusion. The function of Mint3 was further investigated by using mouse cortical tubular (MCT) cells, which were treated with Mint3 and/or FIH small interfering RNA and exposed to normoxia or hypoxia. Knockout of Mint3 did not affect the acute injury induced by uIRI, but exacerbated the tubulointerstitial fibrosis, accompanied by an increase in TEC apoptosis. Consistently, hypoxia-induced apoptosis of MCT cells was aggravated by Mint3 knockdown. Unexpectedly, the additional knockdown of FIH did not suppress the increase in apoptosis by Mint3 knockdown, demonstrating the irrelevance of the FIH/HIF pathway. Therefore, we next focused on nuclear factor (NF)-κB, which has an anti-apoptotic role. Indeed, not only the expression of the inhibitory NF-κB p50 but also the DNA-binding activity of p50/p50 homodimer was increased by knockdown of Mint3 in the TECs, along with the decreased expressions of the NF-κB-targeted anti-apoptotic genes. An increase in NF-κB p50 was also confirmed in Mint3-knockout kidneys. Mint3 in epithelial cells protects the cells from apoptosis by up-regulating anti-apoptotic effects of NF-κB, leading to fibrosis suppression. This new pathophysiology of tubulointerstitial fibrosis could be a target of future therapy for CKD.

Rhein alleviates renal interstitial fibrosis by inhibiting tubular cell apoptosis in rats

BackgroundUreteral obstruction causes injury of the renal tissues and can irreversibly progress to renal fibrosis, with atrophy and apoptosis of tubular cells. The goal of the current study was to examine the effects of rhein on the apoptosis o renal tubular cells as well as renal fibrosis using a rodent model of unilateral ureteral obstruction (UUO).MethodsUUO was induced through ureteral ligation, then animals received treatments with rhein or vehicle. The control rats only received sham operation. The renal tissue was harvested 1 week after surgery for assessment of kidney fibrosis.ResultsThe expressions of collagen I and α-smooth muscle actin (α-SMA), as well as the severity of renal tubular apoptosis and fibrosis were time-dependently increased following UUO. Treatments with rhein partially inhibited such responses. Renal interstitial fibrosis was associated with STAT3 (signal transducer and activator of transcription 3) phosphorylation as well as altered expressions of Bax and Bcl2, both apoptosis-related proteins. Treatment with rhein also partly blocked these responses.ConclusionThese findings demonstrated that rhein mitigated apoptosis of renal tubular cell as well as renal fibrosis in a UUO rodent model. This curative effect is likely mediated via suppression of STAT3 phosphorylation.

Maternal undernutrition aggravates renal tubular necrosis and interstitial fibrosis after unilateral ureteral obstruction in male rat offspring

Maternal undernutrition is known to reduce glomerular number but it may also affect tubulointerstitium, capillary density, and response to oxidative stress. To investigate whether the latter elements are affected, we examined the response to unilateral ureteral obstruction (UUO), an established model of renal tubulointerstitial fibrosis, in the kidney of offspring from control and nutrient restricted rats. Six-week old male offspring from rats given food ad libitum (CON) and those subjected to 50% food restriction throughout pregnancy (NR) were subjected to UUO for 7 days. Body weight was significantly lower in NR. Systolic blood pressure and blood urea nitrogen increased similarly in CON and NR after UUO. Tubular necrosis in the obstructed kidney, on the other hand, was more extensive in NR. Also, the collagen area, a marker of fibrosis, of the obstructed kidney was significantly increased compared with the contralateral kidney only in NR. Capillary density was decreased similarly in the obstructed kidney of CON and NR compared with the contralateral kidney. Urine nitrate/nitrite, a marker of nitric oxide production, from the obstructed kidney was significantly increased in NR compared with CON. Nitrotyrosine, a marker of nitric oxide-mediated free radical injury, was increased in the obstructed kidney compared with the contralateral kidney in both CON and NR, but the extent was significantly greater in NR.In conclusion, more severe tubular necrosis and fibrosis after UUO was observed in NR, which was thought to be due to increased nitrosative stress.

Abstract P2032: Differential Response To Angiotensin II Depends On Microbial Colonization

The gut microbiome is suspected to play a role in hypertension and target organ damage. In the present study, we used the germ-free mice (GF), which are devoid of bacteria, to investigate if bacterial colonization modulates the response to a hypertensive stimulus. GF C57BL/6 littermates were separated at 4 weeks and randomized to remain GF or receive bacterial colonization (COL). Angiotensin (Ang) II minipumps were implanted at 12 weeks (+AngII) to induce hypertension, sham-infused mice served as non-hypertensive controls. At 14 weeks, all mice were sacrificed, and inflammation and target organ damage were assessed. AngII induced a comparable cardiac hypertrophic response in both groups, as measured by left ventricular mass/tibia length. However, accounting for the low basal heart weight in non-hypertensive GF, the hypertrophic response was greater in GF than COL (in mg/cm; GF: 47.2, GF+AngII: 70.17, fold change: 1.5 and COL: 57.9, COL+AngII: 76.09, fold change: 1.3). Similarly, the increase in cardiac perivascular fibrosis in response to AngII was greater in GF than COL (fibrotic/medial area; GF: 3.8, GF+AngII: 5.8 and COL: 3.9, COL+AngII: 5). Inflammation of the cardiac tissue, as evidenced by relative mRNA expression of MCP-1 and TNFa, increased significantly in GF and not in COL. Using flow cytometry, we found that innate anti-inflammatory immune subsets (myeloid derived suppressor cells) are notably missing in GF, and the adaptive immune response to AngII (e.g. Th17 (in % of CD4+: GF: 0.26; GF+AngII: 0.5 and COL: 0.28; COL+AngII: 0.39)) was exacerbated. Kidney damage was also evident in AngII-infused GF and COL. However, genes indicative of tubular damage (KIM-1: GF: 0.3, GF+AngII: 3.3, fold change: 11.0 and COL: 0.2, COL+AngII: 1.5, fold change: 7.5 ), inflammation (TNFa, MCP-1, ICAM), and fibrosis (Col3a1, Col1a2) increased significantly in GF and were only mildly implicated in COL. Bacterial colonization sizably impacts the response to AngII and hypertensive target organ damage. The magnitude of the pathogenic response to AngII in GF compared to COL mice demonstrates the importance of the gut microbiota in modulating target-organ damage.

Metal-induced nephrotoxicity to diabetic and non-diabetic Wistar rats.

The present study was conducted to examine the nephrotoxic effects of heavy metals including lead (Pb), manganese (Mn), arsenic (As), and cadmium (Cd) in diabetic and non-diabetic Wistar rats. Animals were exposed to heavy metals for 30 days, Pb was injected as lead acetate (C4H6O4Pb), Mn was injected as manganese chloride (MnCl2), Cd was injected as cadmium chloride (CdCl2), and As was administered orally to rats in the form of sodium arsenite (AsO2Na). Results showed that metal deposition trends in tissues were Pb > As > Cd > Mn and the urinary metal levels were Pb > Cd > As > Mn. Diabetic metal alone, as well as metal mixture-treated groups, showed decreased urinary metal levels as compared with non-diabetic metal alone and metal mixture-treated groups. Both diabetic- and non-diabetic metal mixture-treated groups revealed an increasing trend of blood urea nitrogen (BUN) and serum creatinine. In addition, heavy metal treatments resulted in elevated malondialdehyde (MDA) levels in the kidney tissue while decreased levels of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GHS) were observed in the kidney tissue in comparison with the control group. The histological analysis of the kidney tissues showed tubular degeneration, fibrosis, and vacuolation as a result of heavy metal exposure. The present study revealed that co-exposure of heavy metals (Pb, Cd, Mn, As) induced more nephrotoxicity as compared with the metal alone treatment. Moreover, diabetic Wistar rats are more prone to kidney damage as a result of heavy metal exposure.

Localization and Regulation of Polymeric Ig Receptor in Healthy and Diseased Human Kidney

The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR invitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-β1 caused decreased expression. Furthermore, interferon-γ increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.

Protease-activated receptor 2 does not contribute to renal inflammation or fibrosis in the obstructed kidney.

Protease-activated receptor 2 (PAR2) has been implicated in the development of renal inflammation and fibrosis. In particular, activation of PAR2 in cultured tubular epithelial cells induces extracellular signal-regulated kinase signalling and secretion of fibronectin, C-C Motif Chemokine Ligand 2 (CCL2) and transforming growth factor-β1 (TGF-β1), suggesting a role in tubulointerstitial inflammation and fibrosis. We tested this hypothesis in unilateral ureteric obstruction (UUO) in which ongoing tubular epithelial cell damage drives tubulointerstitial inflammation and fibrosis. Unilateral ureteric obstruction surgery was performed in groups (n = 9/10) of Par2-/- and wild type (WT) littermate mice which were killed 7 days later. Non-experimental mice were controls. Wild type mice exhibited a 5-fold increase in Par2 messenger RNA (mRNA) levels in the UUO kidney. In situ hybridization localized Par2 mRNA expression to tubular epithelial cells in normal kidney, with a marked increase in Par2 mRNA expression by tubular cells, including damaged tubular cells, in WT UUO kidney. Tubular damage (tubular dilation, increased KIM-1 and decreased α-Klotho expression) and tubular signalling (extracellular signal-regulated kinase phosphorylation) seen in WT UUO were not altered in Par2-/- UUO. In addition, macrophage infiltration, up-regulation of M1 (NOS2) and M2 (CD206) macrophage markers, and up-regulation of pro-inflammatory molecules (tumour necrosis factor, CCL2, interleukin-36α) in WT UUO kidney were unchanged in Par2-/- UUO. Finally, the accumulation of α-SMA+ myofibroblasts, deposition of collagen IV and expression of pro-fibrotic factors (CTGF, TGF-β1) were not different between WT and Par2-/- UUO mice. Protease-activated receptor 2 expression is substantially up-regulated in tubular epithelial cells in the obstructed kidney, but this does not contribute to the development of tubular damage, renal inflammation or fibrosis.

Wnt/β-catenin/RAS signaling mediates age-related renal fibrosis and is associated with mitochondrial dysfunction.

Renal fibrosis is the common pathological feature in a variety of chronic kidney diseases. Aging is highly associated with the progression of renal fibrosis. Among several determinants, mitochondrial dysfunction plays an important role in aging. However, the underlying mechanisms of mitochondrial dysfunction in age‐related renal fibrosis are not elucidated. Herein, we found that Wnt/β‐catenin signaling and renin–angiotensin system (RAS) activity were upregulated in aging kidneys. Concomitantly, mitochondrial mass and functions were impaired with aging. Ectopic expression of Klotho, an antagonist of endogenous Wnt/β‐catenin activity, abolished renal fibrosis in d‐galactose (d‐gal)‐induced accelerated aging mouse model and significantly protected renal mitochondrial functions by preserving mass and diminishing the production of reactive oxygen species. In an established aging mouse model, dickkopf 1, a more specific Wnt inhibitor, and the mitochondria‐targeted antioxidant mitoquinone restored mitochondrial mass and attenuated tubular senescence and renal fibrosis. In a human proximal tubular cell line (HKC‐8), ectopic expression of Wnt1 decreased biogenesis and induced dysfunction of mitochondria, and triggered cellular senescence. Moreover, d‐gal triggered the transduction of Wnt/β‐catenin signaling, which further activated angiotensin type 1 receptor (AT1), and then decreased the mitochondrial mass and increased cellular senescence in HKC‐8 cells and primary cultured renal tubular cells. These effects were inhibited by AT1 blocker of losartan. These results suggest inhibition of Wnt/β‐catenin signaling and the RAS could slow the onset of age‐related mitochondrial dysfunction and renal fibrosis. Taken together, our results indicate that Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction.

HIV Protease Inhibitor Ritonavir Induces Renal Fibrosis and Dysfunction: Role of Platelet-Derived TGF-β1 and Intervention via Anti-Oxidant Pathways

Background: Chronic kidney disease (CKD) characterized by tubular injury and fibrosis occurs in HIV infection treated with certain protease inhibitor (PI)-based antiretroviral therapies (ART). The pathophysiology is unclear. Methods: We hypothesized that fibrosis, mediated by platelet-derived transforming growth factor (TGF)-β1 and macrophage polarization to an inflammatory subtype underlies PI-associated CKD. We induced this in mice exposed to the PI ritonavir (RTV) in the absence of HIV, and intervened with low-dose inhaled carbon monoxide (CO), to activate erythroid 2- related factor (Nrf2)-associated anti-oxidant pathways. C57BL/6 mice, wild-type and deficient in platelet TGF-β1, were given RTV (10mg/kg) or vehicle daily for 8 weeks. Select groups were exposed to CO (250ppm) for 4 hours after RTV or vehicle injection. Renal pathology, fibrosis, and TGF-β1- and Nrf2-based signaling were examined by histology, immunofluorescence, and flow cytometry. Renal damage and dysfunction were assessed by KIM-1 and cystatin C ELISAs. Clinical correlations were sought among HIV-infected individuals. Findings: RTV induced glomerular and tubular injury, elevating urinary KIM-1. It enhanced TGF-β1-related signaling, accompanied by kidney fibrosis, macrophage polarization to an inflammatory phenotype, and renal dysfunction with cystatin C elevation. Mice with platelet TGF-β1 deletion were partially protected from these abnormalities. CO inhibited RTVinduced fibrosis and macrophage polarization in association with upregulation of Nrf2 and heme oxygenase-1 (HO-1). RTV use correlated with elevated cystatin C and HO-1 levels in HIV-infected individuals vs. controls and HIV-infected subjects receiving no therapy or non-PI-based regimens. Interpretation: Platelet TGF-β1 contributes to RTV-induced kidney fibrosis and dysfunction, which may be amenable to anti-oxidant-based interventions. Funding Statement: This work was supported by NIH grants R21 HL125044 (J.L. and J.A.), R01 HL123605 (J.A.), RO1 HL133801 and RO1 HL060234 (M.E.C.), COBRE grant GM114731, and funding from PHF (J.A.) and the Angelo Donghia Foundation (J.L.). Declaration of Interests: The spouse of M.E.C is a co-founder, shareholder and serves on the Scientific Advisory Board of Proterris, Inc. The remaining authors have no conflicts. Ethics Approval Statement: Animal studies were approved by the Institutional Animal Care and Use Committee of OMRF and WCMC. Patient samples were obtained through study approval by the Institutional Review Board of WCMC.

TREM1/3 Deficiency Impairs Tissue Repair After Acute Kidney Injury and Mitochondrial Metabolic Flexibility in Tubular Epithelial Cells.

Long-term sequelae of acute kidney injury (AKI) are associated with incomplete recovery of renal function and the development of chronic kidney disease (CKD), which can be mediated by aberrant innate immune activation, mitochondrial pathology, and accumulation of senescent tubular epithelial cells (TECs). Herein, we show that the innate immune receptor Triggering receptor expressed on myeloid cells-1 (TREM-1) links mitochondrial metabolism to tubular epithelial senescence. TREM-1 is expressed by inflammatory and epithelial cells, both players in renal repair after ischemia/reperfusion (IR)-induced AKI. Hence, we subjected WT and TREM1/3 KO mice to different models of renal IR. TREM1/3 KO mice displayed no major differences during the acute phase of injury, but increased mortality was observed in the recovery phase. This detrimental effect was associated with maladaptive repair, characterized by persistent tubular damage, inflammation, fibrosis, and TEC senescence. In vitro, we observed an altered mitochondrial homeostasis and cellular metabolism in TREM1/3 KO primary TECs. This was associated with G2/M arrest and increased ROS accumulation. Further exposure of cells to ROS-generating triggers drove the cells into a stress-induced senescent state, resulting in decreased wound healing capacity. Treatment with a mitochondria anti-oxidant partly prevented the senescent phenotype, suggesting a role for mitochondria herein. In summary, we have unraveled a novel (metabolic) mechanism by which TREM1/3 deficiency drives senescence in TECs. This involves redox imbalance, mitochondrial dysfunction and a decline in cellular metabolic activities. These finding suggest a novel role for TREM-1 in maintaining tubular homeostasis through regulation of mitochondrial metabolic flexibility.

512-P: Sexual Differences in Progression of Diabetic Nephropathy in over 26 Type 1 Diabetic Mice

As a transgenic type 1 diabetic model, OVE26 mice develop diabetic nephropathy (DN) with the feature close to advanced DN in human, which makes it a valuable rodent model to study DN. Nevertheless, sexual differences in renal lesions, especially dynamic progression with different ages, remain undefined in OVE26 mice. To explore these issues, male and female OVE26 and wild type nondiabetic FVB mice at 4-, 12-, 24 - and 36-weeks old were used (N=6). Urine albumin-to-creatinine ratio (UACR) was detected to evaluate renal function. Renal histopathology and immunochemical and Western blotting protein expression of inflammation, tubular epithelial-myofibroblast transdifferentiation and fibrosis markers were examined. In FVB mice, blood glucose, triglyceride, UACR and podocytes numbers were constant during aging and comparable in male and female. Age-dependent slightly increase of glomerular mesangial matrix, inflammatory cells infiltration and renal fibrosis were observed in FVB mice. Compared with the age- and sex- matched FVB mice, male OVE26 mice developed hyperglycemia, mesangial matrix accumulation and increased trend of triglyceride at 4-week-old and albuminuria, glomerular enlargement, and upregulations of TGF-β1, α-SMA at 12 weeks old. Significant loss of podocytes, tubular dilation, collagen accumulation, increased trend of macrophage and T lymphocytes infiltrations, and increased protein levels of TNF-α, ICAM and fibronectin in kidney were observed in 24-week old male OVE26 mice. Compared with age-matched male OVE26 mice, female OVE26 mice had significant hypertriglyceridemia, worse renal function and kidney lesions at older age, including higher UACR and sustained loss of podocytes at age of 24 weeks; more inflammatory cells infiltration and renal fibrosis at age of 36 weeks. Our findings suggest the sexual difference in the progression of DN in OVE26 mice to provide evidence for researchers to properly select the age and sex of OVE26 mice in the future type 1 DN study. Disclosure W. Wang: None. L. Cai: None.

492-P: A Novel Renoprotective Target of SGLT2 Inhibitor: HIF-1α Inhibition and AMPK Activation in Renal Proximal Tubular Epithelial Cells

The renoprotective mechanism of SGLT2 inhibitors has not been fully elucidated. Tubular hypoxia is major driving force for proximal tubulopathy in diabetic kidney. In addition, diabetes causes dysregulation of AMP-activated protein kinase (AMPK) in kidney cortex. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on hypoxia inducible factor-1α (HIF-1α) expression and AMPK phosphorylation in cultured human renal proximal tubular epithelial cells (HRPTECs) and on tubulointerstitial pathological changes in diabetic mice. Luseogliflozin inhibited hypoxia (1%O2, 24h)-induced HIF-1α protein expression in HRPTECs in a dose-dependent manner (1-100µM). In addition, luseogliflozin increased AMPKα protein phosphorylation (Th172) in normoxic and hypoxic condition. Intriguingly, luseogliflozin suppressed oxygen consumption rate (OCR) measured by oxygen quenching phosphorescent probe by 69% in HRPTECs from normoxic condition. Hypoxia decreased OCR by 52% and luseogliflozin further decreased OCR by 33% from hypoxic condition. Moreover, luseogliflozin rescued hypoxic state in HRPTECs even under hypoxic conditions assessed by a hypoxia-sensitive dye, pimonidazole. These data indicate luseogliflozin inhibits HIF-1α expression through suppression of mitochondrial OCR, which leads to restore intracellular hypoxia. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin ameliorated tubular injury compared to non-treated db/db mice. Furthermore, immunostaining of HIF-1α and fibronectin in cortical tubules revealed that luseogliflozin decreased HIF-1α and fibronectin expression in db/db mice. In conclusion, luseogliflozin decreases mitochondrial OCR and ameliorates tubular fibrosis at least partly by suppressing HIF-1α expression and activating AMPK in renal proximal tubules of diabetic mice. These results may provide a novel renoprotective target of SGLT2 inhibitor. Disclosure R. Bessho: None. Y. Takiyama: None. T. Takiyama: None. T. Ota: None.

Finasteride-Induced Inhibition of 5α-Reductase Type 2 Could Lead to Kidney Damage-Animal, Experimental Study.

In the pharmacological treatment of prostate cancer, benign prostatic hyperplasia and androgenetic alopecia finasteride is commonly used. This drug inhibits 5α-reductase type 2, which is why finasteride affects androgen homeostasis, since testosterone (T) cannot be reduced to dihydrotestosterone (DHT). As studies on sex-related renal injuries suggest a high probability of androgen-induced renal dysfunction, the aim of this study was to determine the potential harmful effects of finasteride on the kidneys of rats. The study was performed on sexually mature male Wistar rats given finasteride. Histological sections of the kidneys were used for immunohistochemical visualization of the androgen receptor (AR), junctional proteins (occluding (Occ); E-cad, N-cad, E-/N-cadherin; β-cat, β-catenin; connexin 43 (Cx43)), proliferating cell nuclear antigen (PCNA), IL-6, and lymphocyte markers (CD3 for T cell, CD19 for B cell). The TUNEL method was used for cell apoptosis identification, and picro sirius red staining was used to assess collagen fibers thickness. The levels of T, DHT and estradiol (E2) were determined in blood serum. It was shown that finasteride treatment affected steroid hormone homeostasis, altered the expression of AR and intracellular junction proteins, changed the ratio between cell apoptosis and proliferation, and caused lymphocyte infiltration and an increase of IL-6. The thickening of collagen fibers was observed as tubular fibrosis and glomerulosclerosis. Summarizing, finasteride-induced hormonal imbalance impaired the morphology (i.e., dysplastic glomeruli, swollen proximal convoluted tubules) and physiology (changed level of detected proteins/markers expression) of the kidneys. Therefore, it is suggested that patients with renal dysfunction or following renal transplantation, with androgen or antiandrogen supplementation, should be under special control and covered by extended diagnostics, because the adverse negative effect of DHT deficiency on the progression of kidney disease cannot be ignored.

Hirsutella sinensis Treatment Shows Protective Effects on Renal Injury and Metabolic Modulation in db/db Mice.

Hirsutella sinensis (HS) is the anamorph of the traditional Chinese medicine Cordyceps sinensis. Although the renal protective effect of HS has been reported, its effect on diabetic nephropathy (DN) remains unclear. In this study, db/db mice were used as the DN model, and the renal protective effect was evaluated after oral administration of HS for 6 and 12 weeks. Plasma, urine, and kidney samples were collected, and biochemical indicator measurements, pathological analysis, and metabolomics studies were performed. Biochemical assays showed that HS reduced the levels of fasting blood glucose (FBG), urinary albumin/creatinine ratio (ACR), and N-acetyl-beta-D-glucosaminidase (NAG) and increased the creatinine clearance (Ccr). HS alleviated glomerular and tubular glycogen accumulation and fibrosis and normalized the disordered ultrastructure of the glomerular filtration barrier. Metabolomics analysis of metabolites in the plasma, urine, and kidney indicated that HS modulated the perturbed glycolipid metabolism and amino acid turnover. HS reduced the elevated levels of metabolites involved in energy metabolism (TCA cycle, glycolysis, and pentose phosphate pathway) and nucleotide metabolism (pyrimidine metabolism and purine metabolism) in the kidneys of db/db mice. These results suggest that HS can protect against renal injury and that its efficacy involved metabolic modulation of the disturbed metabolome in db/db mice.

AKI to CKD Transition is driven by Alpha 2 Adrenergic Receptors

BackgroundEnhanced tissue level and activity of norepinephrine (NE) have been recognized as risk factors of development and progression of chronic kidney disease (CKD). Our reports showed renal denervation (DNx) can prevent renal fibrosis and inflammation in renal fibrogenesis models. However, the underlying mechanism by which renal DNx prevents the long‐term sequelae of acute kidney injury (AKI) remains to be elucidated.MethodsBased on our published data, we hypothesized that NE mediates its effect through activation of alpha 2 adrenergic receptors (α2‐ARs). In this study, we investigated whether genetic and/or pharmacological inhibition of α2‐ARs prevents ischemia/reperfusion injury (IRI)‐induced inflammation and CKD progression.ResultsRenal bilateral IRI resulted in severe kidney fibrosis in medullary region over 2 weeks with persistent tubular injury and massive macrophage infiltration. Renal DNx suppressed renal inflammation, tubular damage, and fibrosis progression, as well as increase in blood pressure, 2 weeks post‐IRI. Pharmacological inhibition of α2‐AR also inhibited fibrosis progression with reduced downstream targets of NE‐α2‐AR axis, including angiotensinogen and fibrogenic factors. Consistent with results in renal DNx and pharmacological inhibition of α2‐AR, inhibition of α2A‐ and α2C‐AR subtypes by pharmacological or genetic manipulation additively prevented macrophage accumulation with downregulation of M1 and M2 macrophage markers, resulting in suppression of persistent renal tubular injury and fibrosis progression.ConclusionTaken together, blockage of α2‐AR subtypes prevents IRI‐induced AKI to CKD transition through inhibition of macrophage accumulation, persistent renal tubular injury and fibrosis progression, suggesting that targeting α2‐ARs may be a potential therapeutic strategy to prevent long‐term sequelae of ischemic AKI.Support or Funding InformationThis work was supported by NIH DK‐083291 and DK‐090332 and UNMC College of Medicine Bridge fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Novel Dual Soluble Epoxide Hydrolase Inhibitor/Cyclooxygenase‐2 Inhibitor Treats Type 2 Diabetic Complications in Obese ZSF1 Rats

Diabetes is a highly prevalent chronic disease worldwide that represents a global healthcare burden. A devastating type 2 diabetes complication is diabetic nephropathy. We developed a novel dual modulator, PTUPB that concurrently acts as a soluble epoxide hydrolase (sEH) inhibitor as well as a cyclooxyganse‐2 inhibitor (sEHi/COXi) to combat diabetic nephropathy. The objective for the current investigation is to determine if PTUPB can treat diabetic nephropathy in obese ZSF1 rats. PTUPB was compared to an angiotensin converting enzyme inhibitor that is the current standard of care for diabetic nephropathy. Obese ZSF‐1 rats at 16 weeks of age received PTUPB (10mg/kg/d i.p.) or enalapril (10mg/kg/d, p/o.) for 8 weeks. Type 2 diabetes was assessed in obese ZSF1 rats. Renal injury was determined using several biochemical, histological, and immunohistological techniques. Obese ZSF1 rats were diabetic with fasting blood glucose (19±0.4 vs. 4.8±0.1 mmol/l in lean ZSF1) . Neither PTUPB nor enalapril treated diabetes and did not reduce hyperglycemia or HbA1c in obese ZSF1 rats. Obese ZSF1 rats developed diabetic nephropathy with elevated albuminuria (49±6 vs. 1.3±0.2 mg/mg creatinine in lean ZSF1), tubular cast formation (cortex, 14±1 vs. 1.0±0.1%; medulla, 22.4±2 vs. 0.9±0.2%), renal fibrosis (cortex, 11±1 vs. 4.4±0.3%; medulla, 10±1.2 vs. 4.0±0.8%) and glomerular injury (3.4±0.6 vs. 0.6±0.08 a.u.) compared to lean ZSF1 lean (all P<0.05). PTUPB treated diabetic nephropathy by reducing albuminuria (by 50%), renal tubular cast formation (by 60% in cortex; 70% in medulla), renal fibrosis (by 50% in cortex; 36% in medulla), and glomerular injury (by 55%) compared to vehicle. Enalapril treatment reduced diabetic nephropathy in obese ZSF1 rats like PTUPB and reduced the renal injury parameters by 30 to 50% compared to vehicle. Diabetic renal injury in obese ZSF1 rats was accompanied by inflammation with elevated urinary MCP‐1 levels (29±2 vs. 5±0.6 ng/mg creatinine in lean ZSF1 rat, P<0.05) and renal infiltration of CD‐68 cells (95±10 vs. 16±1.2 cell/mm2, P<0.05). PTUPB or enalapril treated renal inflammation and reduced MCP‐1 excretion (PTUPB reduced by 50% and enalapril reduced by 20%) and renal CD‐68 positive cell infiltration (by 60% and 50%, respectively by PTUPB or enalapril). These findings demonstrate that PTUPB is not anti‐diabetic but markedly treat diabetic nephropathy. We also demonstrate that PTUPB has renal actions comparable to enalapril which is currently the standard of care to treat diabetic nephropathy.Support or Funding InformationNational Institute of Health (NIH) grant (DK103616) and Dr. Ralph and Marian Falk Medical Research Trust Bank of America, N.A., Trustee grant to JDI; NIEHS/R01 ES002710 (HAM1400) and NIEHS/Superfund Research Program P42 ES004699 (SUPR?30) to BDHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of Physiopathology of Acute Kidney Injury Associated or Not with Diabetes Mellitus

IntroductionAcute kidney injury (AKI) and diabetes mellitus (DM) are the major causes of chronic kidney disease (CKD). However, the pathophysiological mechanisms of AKI associated or not with diabetes are unclear. Thus, the aim of this study is to identify the pathophysiology of AKI in an experimental model of renal ischemia and reperfusion (I/R) associated or not with streptozotocin (STZ)‐induced diabetes.MethodsThe protocols are in agreement with CEUA (nº 12/2017). C57BL/6J mice, aged 60 days, were randomly grouped into: Control (CTL) ‐ vehicle and sham surgery; renal ischemia and reperfusion (I/R) ‐ vehicle and ischemia and reperfusion surgery; streptozotocin (STZ) ‐ STZ injection (55 mg/kg/day) for five consecutive days followed by sham surgery; STZ + I/R ‐ STZ injection (55 mg/kg/day) for five consecutive days followed by ischemia and reperfusion surgery. On the fifth day after the last injection of STZ, hyperglycemic animals (glycemia ≥ 250 mg/dL, monitored by glycosimeter) were considered diabetic. On the 12th day after the last STZ injection, the animals were submitted to surgical procedures and after 48 hours were submitted to euthanasia. Renal function was assessed by plasma creatinine and urea concentration. Proteinuria was assessed by commercial kit. Renal tissue was used for evaluation of gene expression by qPCR and protein expression by Western Blotting. Statistical analysis was performed by one‐way ANOVA, followed by Bonferroni post‐test. Values of p<0.05 were considered statistically significant.ResultsRenal ischemia followed by reperfusion resulted in increased plasma creatinine levels (p<0.05 vs CTL) and plasma urea levels (p<0.001 vs CTL); albuminuria (p<0.05 vs CTL); decreased nephrin mRNA expression (p<0.01 vs CTL), indicating glomerular injury and increased Kim‐1 mRNA expression (p<0.001 vs CTL), indicating tubular injury. Renal ischemia followed by reperfusion also induced inflammation, confirmed by the expression of mRNA for TNF‐α (p<0.01 vs CTL) and interleukin 1β (p<0.001 vs CTL), as well as the pro‐fibrotic response indicated by increased expression of collagen I (p<0.001 vs CTL), collagen III (p<0.001 vs CTL) and collagen IV (p<0.001 vs CTL). In the STZ‐treated group, the parameters described above were not altered because it is a condition of early diabetes. However, the diabetic group submitted to renal ischemia and reperfusion presented exacerbated responses in all parameters evaluated. In protein expression studies, renal ischemia followed by reperfusion decreased the expression of phosphorylated adenosine monophosphate‐activated protein kinase (AMPK) (p<0.01 vs CTL) and did not alter the protein expression of LC3A/B I (microtubule‐associated protein 1 light chain 3), indicating a loss of autophagic response.ConclusionAKI induces glomerular and tubular injury, inflammation, fibrosis and loss of autophagic response. DM behaves as an underlying disease that aggravates post‐ischemic AKI, primarily by accentuating tubular injury and inflammatory response.Support or Funding InformationFAPESP (2016/12354‐0), CNPq (140182/2017‐9) and CAPES.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Canagliflozin Prevents Intrarenal Angiotensinogen Augmentation and Mitigates Kidney Injury and Hypertension in Mouse Model of Type 2 Diabetes Mellitus

Background: Hypertension and renal injury are common complications of type 2 diabetes mellitus (T2DM). Hyperglycemia stimulates renal proximal tubular angiotensinogen (AGT) expression via elevated oxidative stress contributing to the development of high blood pressure and diabetic nephropathy. The sodium glucose cotransporter 2 (SGLT2) in proximal tubules is responsible for the majority of glucose reabsorption by renal tubules. We tested the hypothesis that SGLT2 inhibition with canagliflozin (CANA) prevents intrarenal AGT augmentation and ameliorates kidney injury and hypertension in T2DM. Methods: We induced T2DM in New Zealand obese mice with a high fat diet (DM, 30% fat) with control mice receiving regular fat diet (ND, 4% fat). When DM mice exhibited > 350 mg/dL blood glucose levels, both DM- and ND-fed mice were treated with 10 mg/kg/day CANA or vehicle by oral gavage for 6 weeks. We evaluated intrarenal AGT, blood pressure, and the development of kidney injury. Results: Systolic blood pressure in DM mice (133.9 ± 2.0 mm Hg) was normalized by CANA (113.9 ± 4.0 mm Hg). CANA treatment ameliorated hyperglycemia-associated augmentation of renal AGT mRNA (148 ± 21 copies/ng RNA in DM, and 90 ± 16 copies/ng RNA in DM + CANA) and protein levels as well as elevation of urinary 8-isoprostane levels. Tubular fibrosis in DM mice (3.4 ± 0.9-fold, fibrotic score, ratio to ND) was suppressed by CANA (0.9 ± 0.3-fold). Furthermore, CANA attenuated DM associated increased macrophage infiltration and cell proliferation in kidneys of DM mice. Conclusions: CANA prevents intrarenal AGT upregulation and oxidative stress and which may mitigate high blood pressure, renal tubular fibrosis, and renal inflammation in T2DM.

DR region specific antibody ameliorated but ouabain worsened renal injury in nephrectomized rats through regulating Na,K-ATPase mediated signaling pathways.

Reduced Na+-K+-ATPase function is reported in various renal diseases. This implies that increase of Na+-K+-ATPase function may be a new target in treatment of renal injury. We previously reported that Na+-K+-ATPase was stabilized by DRm217, a specific antibody against DR region of Na+-K+-ATPase. In this study, we compared the protective effect of DRm217 and ouabain on kidney in a chronic kidney disease rat model and investigated the mechanism under it. We found that DRm217 improved renal function, alleviated glomerulus atrophy, inhibited renal tubular cells apoptosis, tubulointerstitial injury and renal fibrosis in 5/6 nephrectomized rats. Contrary to DRm217, ouabain worsened renal damage. Activated Na+-K+-ATPase /Src signaling pathway, increased oxidant stress and activated inflammasome were responsible for nephrectomized or ouabain-induced renal injury. DRm217 inhibited Na+-K+-ATPase /Src signaling pathway, retarded oxidant stress, suppressed inflammasome activation, and improved renal function, suggesting a novel approach to prevent renal damage.