OTUD6A in tubular epithelial cells mediates angiotensin II-induced kidney injury by targeting STAT3.

Kidney fibrosis is a prominent pathological feature of hypertensive kidney diseases (HKD). Recent studies have highlighted the role of ubiquitinating/deubiquitinating protein modification in kidney pathophysiology. Ovarian tumor domain-containing protein 6 A (OTUD6A) is a deubiquitinating enzyme involved in tumor progression. However, its role in kidney pathophysiology remains elusive. We aimed to investigate the role and underlying mechanism of OTUD6A during kidney fibrosis in HKD. The results revealed higher OTUD6A expression in kidney tissues of nephropathy patients and mice with chronic angiotensin II (Ang II) administration than that from the control ones. OTUD6A was mainly located in tubular epithelial cells. Moreover, OTUD6A deficiency significantly protected mice against Ang II-induced kidney dysfunction and fibrosis. Also, knocking OTUD6A down suppressed Ang II-induced fibrosis in cultured tubular epithelial cells, whereas overexpression of OTUD6A enhanced fibrogenic responses. Mechanistically, OTUD6A bounded to signal transducer and activator of transcription 3 (STAT3) and removed K63-linked-ubiquitin chains to promote STAT3 phosphorylation at tyrosine 705 position and nuclear translocation, which then induced profibrotic gene transcription in epithelial cells. These studies identified STAT3 as a direct substrate of OTUD6A and highlighted the pivotal role of OTUD6A in Ang II-induced kidney injury, indicating OTUD6A as a potential therapeutic target for HKD.NEW & NOTEWORTHY Ovarian tumor domain-containing protein 6 A (OTUD6A) knockout mice are protected against angiotensin II-induced kidney dysfunction and fibrosis. OTUD6A promotes pathological kidney remodeling and dysfunction by deubiquitinating signal transducer and activator of transcription 3 (STAT3). OTUD6A binds to and removes K63-linked-ubiquitin chains of STAT3 to promote its phosphorylation and activation, and subsequently enhances kidney fibrosis.

Dapagliflozin-entresto protected kidney from renal hypertension via downregulating cell-stress signaling and upregulating SIRT1/PGC-1α/Mfn2-medicated mitochondrial homeostasis.

This study tested whether combined dapagliflozin and entresto would be superior to mere one therapy on protecting the residual renal function and integrity of kidney parenchyma in hypertensive kidney disease (HKD) rat. In vitro results showed that the protein expressions of oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/oxidized-protein/cytosolic-cytochrome-C)/apoptotic (mitochondrial-Bax/cleaved caspeases 3, 9)/cell-stress (p-ERK/p-JNK/p-p38) biomarkers were significantly increased in H2O2-treated NRK-52E cells than those of controls that were reversed by dapagliflozin or entresto treatment. Adult-male SD rats (n = 50) were equally categorized into group 1 (sham-operated-control), group 2 (HKD by 5/6 nephrectomy + DOCA-salt/25 mg/kg/subcutaneous injection/twice weekly), group 3 (HKD + dapagliflozin/orally, 20 mg/kg/day for 4 weeks since day 7 after HKD induction), group 4 (HKD + entresto/orally, 100 mg/kg/day for 4 weeks since day 7 after HKD induction), and group 5 (HKD + dapagliflozin + entresto/the procedure and treatment strategy were identical to groups 2/3/4). By day 35, circulatory levels of blood-urine-nitrogen (BUN)/creatinine and urine protein/creatinine ratio were lowest in group 1, highest in group 2, and significantly lower in group 5 than in groups 3/4, but no difference between groups 3/4. Histopathological findings showed the kidney injury score/fibrotic area/cellular expressions of oxidative-stress/kidney-injury-molecule (8-OHdG+/KIM-1+) exhibited an identical trend, whereas the cellular expressions of podocyte components (synaptopodin/ZO-1/E-cadherin) exhibited an opposite pattern of BUN level among the groups. The protein expressions of oxidative stress/mitochondrial-damaged (NOX-1/NOX-2/oxidized protein/cytosolic-cytochrome-C/cyclophilin-D)/apoptotic (mitochondrial-Bax/cleaved-caspase 3)/mitochondrial-fission (PINK1/Parkin/p-DRP1)/autophagic (LC3BII/LC3BI ratio, Atg5/beclin-1)/MAPK-family (p-ERK/p-JNK/p-p38) biomarkers displayed a similar pattern, whereas the protein expression of mitochondria-biogenesis signaling (SIRT1/PGC-1α-Mfn2/complex I-V) displayed an opposite pattern of BUN among the groups. In conclusion, combined dapagliflozin-entresto therapy offered additional benefits on protecting the residual kidney function and architectural integrity in HKD rat.

Renal Epithelial Mitochondria: Implications for Hypertensive Kidney Disease.

According to the Centers for Disease Control and Prevention, 1 in 2 U.S. adults have hypertension, and more than 1 in 7 chronic kidney disease. In fact, hypertension is the second leading cause of kidney failure in the United States; it is a complex disease characterized by, leading to, and caused by renal dysfunction. It is well-established that hypertensive renal damage is accompanied by mitochondrial damage and oxidative stress, which are differentially regulated and manifested along the nephron due to the diverse structure and functions of renal cells. This article provides a summary of the relevant knowledge of mitochondrial bioenergetics and metabolism, focuses on renal mitochondrial function, and discusses the evidence that has been accumulated regarding the role of epithelial mitochondrial bioenergetics in the development of renal tissue dysfunction in hypertension. © 2024 American Physiological Society. Compr Physiol 14:5225-5242, 2024.

Inhibition of USP1 ameliorates hypertensive nephropathy through regulating oxidative stress and ferroptosis: A precise treatment via SJB3-019A nanodelivery.

Hypertensive nephropathy is second only to diabetes for the causation of chronic kidney disease worldwide. As the mortality and morbidity of hypertensive nephropathy keep increasing, it is important to elucidate its pathogenesis and develop new treatment strategies. In this study, an angiotensin II (Ang II)-induced renal cell system was established, and the expression of ubiquitin specific peptidase 1 (USP1) in human kidney (HK-2) cells was found to be regulated by Ang II treatment through quantitative RT-PCR and Western blot assay. The detection of glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and lipid reactive oxygen species (ROS) levels revealed that interference with USP1 reversed Ang II-induced oxidative stress and ferroptosis, which was enhanced by overexpression of USP1. Subsequently, USP1 inhibitor SJB3-019A loaded in MIL-100 and PEGTK was modified to fabricate SJB3-019A@MIL-PEGTK nanoparticles, which was confirmed to exhibit excellent alleviation of hypertension-induced oxidative stress and ferroptosis in renal cells both in vitro and in vivo. Our study identified an important pathogenesis of hypertensive nephropathy and SJB3-019A@MIL-PEGTK nanoparticle was used to develop an effective clinical treatment for hypertensive nephropathy.

Non-Haemodynamic Mechanisms Underlying Hypertension-Associated Damage in Target Kidney Components.

Arterial hypertension (AH) is a global challenge that greatly impacts cardiovascular morbidity and mortality worldwide. AH is a major risk factor for the development and progression of kidney disease. Several antihypertensive treatment options are already available to counteract the progression of kidney disease. Despite the implementation of the clinical use of renin-angiotensin aldosterone system (RAAS) inhibitors, gliflozins, endothelin receptor antagonists, and their combination, the kidney damage associated with AH is far from being resolved. Fortunately, recent studies on the molecular mechanisms of AH-induced kidney damage have identified novel potential therapeutic targets. Several pathophysiologic pathways have been shown to play a key role in AH-induced kidney damage, including inappropriate tissue activation of the RAAS and immunity system, leading to oxidative stress and inflammation. Moreover, the intracellular effects of increased uric acid and cell phenotype transition showed their link with changes in kidney structure in the early phase of AH. Emerging therapies targeting novel disease mechanisms could provide powerful approaches for hypertensive nephropathy management in the future. In this review, we would like to focus on the interactions of pathways linking the molecular consequences of AH to kidney damage, suggesting how old and new therapies could aim to protect the kidney.

Macrophage Dectin-1 mediates Ang II renal injury through neutrophil migration and TGF-ß1 secretion.

Macrophage activation has been shown to play an essential role in renal fibrosis and dysfunction in hypertensive chronic kidney disease. Dectin-1 is a pattern recognition receptor that is also involved in chronic noninfectious diseases through immune activation. However, the role of Dectin-1 in Ang II-induced renal failure is still unknown. In this study, we found that Dectin-1 expression on CD68 + macrophages was significantly elevated in the kidney after Ang II infusion. We assessed the effect of Dectin-1 on hypertensive renal injury using Dectin-1-deficient mice infused by Angiotensin II (Ang II) at 1000 ng/kg/min for 4 weeks. Ang II-induced renal dysfunction, interstitial fibrosis, and immune activation were significantly attenuated in Dectin-1-deficient mice. A Dectin-1 neutralizing antibody and Syk inhibitor (R406) were used to examine the effect and mechanism of Dectin-1/Syk signaling axle on cytokine secretion and renal fibrosis in culturing cells. Blocking Dectin-1 or inhibiting Syk significantly reduced the expression and secretion of chemokines in RAW264.7 macrophages. The in vitro data showed that the increase in TGF-ß1 in macrophages enhanced the binding of P65 and its target promotor via the Ang II-induced Dectin-1/Syk pathway. Secreted TGF-ß1 caused renal fibrosis in kidney cells through Smad3 activation. Thus, macrophage Dectin-1 may be involved in the activation of neutrophil migration and TGF-ß1 secretion, thereby promoting kidney fibrosis and dysfunction.

High-Salt Diet Aggravates Endothelial-to-Mesenchymal Transition in Glomerular Fibrosis in Dahl Salt-Sensitive Rats.

BACKGROUND: Both diabetic and hypertensive nephropathy eventually progress to glomerulosclerosis. Previous studies revealed a potential role of endothelial-to-mesenchymal transition (EndMT) in the pathophysiology of glomerulosclerosis in diabetic rats. Therefore, we hypothesized that EndMT was also involved in the development of glomerulosclerosis in salt-sensitive hypertension. We aimed to explore the effects of high-salt diet on endothelial-to-mesenchymal transition (EndMT) in glomerulosclerosis in Dahl salt-sensitive (Dahl-SS) rats. METHODS: Eight-week-old male rats were fed high-salt (8%NaCl; DSH group) or normal salt (0.3%NaCl; DSN group) for eight weeks, with systolic blood pressure (SBP), serum creatinine, urea, 24-hour urinary protein/sodium, renal interlobar artery blood flow, and pathological examination measured. We also examined endothelial-(CD31) and fibrosis-related protein(α-SMA) expressions in glomeruli. RESULTS: High-salt diet increased SBP (DSH vs. DSN, 205.2 ±â€…8.9 vs. 135.4 ±â€…7.9 mm Hg, P < 0.01), 24-hour urinary protein (132.55 ±â€…11.75 vs. 23.52 ±â€…5.94 mg/day, P < 0.05), urine sodium excretions (14.09 ±â€…1.49 vs. 0.47 ±â€…0.06 mmol/day, P < 0.05), and renal interlobar artery resistance. Glomerulosclerosis increased (26.1 ±â€…4.6 vs. 7.3 ±â€…1.6%, P < 0.05), glomerular CD31 expressions decreased while α-SMA expression increased in DSH group. Immunofluorescence staining showed that CD31 and α-SMA co-expressed in glomeruli of the DSH group. The degree of glomerulosclerosis negatively correlated with CD31 expressions (r = -0.823, P < 0.01) but positively correlated with α-SMA expressions (r = 0.936, P < 0.01). CONCLUSIONS: We demonstrated that a high-salt diet led to glomerulosclerosis involving the EndMT process, which played an essential role in glomerulosclerosis in hypertensive Dahl-SS rats.

Complement component C3 as a new target to lower albuminuria in hypertensive kidney disease.

BACKGROUND AND PURPOSE: Complement activation may drive hypertension through its effects on immunity and tissue integrity. EXPERIMENTAL APPROACH: We examined expression of C3, the central protein of the complement cascade, in hypertension. KEY RESULTS: Increased C3 expression was found in kidney biopsies and micro-dissected glomeruli of patients with hypertensive nephropathy. Renal single cell RNA sequence data from normotensive and hypertensive patients confirmed expression of C3 in different cellular compartments of the kidney. In angiotensin II (Ang II) induced hypertension renal C3 expression was up-regulated. C3-/- mice revealed a significant lower albuminuria in the early phase of hypertension. However, no difference was found for blood pressure, renal injury (histology, glomerular filtration rate, inflammation) and cardiac injury (fibrosis, weight, gene expression) between C3-/- and wildtype mice after Ang II infusion. Also, in deoxycorticosterone acetate (DOCA) salt hypertension, a significantly lower albuminuria was found in the first weeks of hypertension in C3 deficient mice but no significant difference in renal and cardiac injury. Down-regulation of C3 by C3 targeting GalNAc (n-acetylgalactosamine) small interfering RNA (siRNA) conjugate decreased C3 in the liver by 96% and lowered albuminuria in the early phase but showed no effect on blood pressure and end-organ damage. Inhibition of complement C5 by siRNA showed no effect on albuminuria. CONCLUSION AND IMPLICATIONS: Increased C3 expression is found in the kidneys of hypertensive mice and men. Genetic and therapeutic knockdown of C3 improved albuminuria in the early phase of hypertension but did not ameliorate arterial blood pressure nor renal and cardiac injury.

Interferon regulatory factor 4 deletion protects against kidney inflammation and fibrosis in deoxycorticosterone acetate/salt hypertension.

BACKGROUND: Inflammation and renal interstitial fibrosis are the main pathological features of hypertensive nephropathy. Interferon regulatory factor 4 (IRF-4) has an important role in the pathogenesis of inflammatory and fibrotic diseases. However, its role in hypertension-induced renal inflammation and fibrosis remains unexplored. METHOD AND RESULTS: We showed that deoxycorticosterone acetate (DOCA)-salt resulted in an elevation of blood pressure and that there was no difference between wild-type and IRF-4 knockout mice. IRF-4 -/- mice presented less severe renal dysfunction, albuminuria, and fibrotic response after DOCA-salt stress compared with wild-type mice. Loss of IRF-4 inhibited extracellular matrix protein deposition and suppressed fibroblasts activation in the kidneys of mice subjected to DOCA-salt treatment. IRF-4 disruption impaired bone marrow-derived fibroblasts activation and macrophages to myofibroblasts transition in the kidneys in response to DOCA-salt treatment. IRF-4 deletion impeded the infiltration of inflammatory cells and decreased the production of proinflammatory molecules in injured kidneys. IRF-4 deficiency activated phosphatase and tensin homolog and weakened phosphoinositide-3 kinase/AKT signaling pathway in vivo or in vitro . In cultured monocytes, TGFß1 also induced expression of fibronectin and α-smooth muscle actin and stimulated the transition of macrophages to myofibroblasts, which was blocked in the absence of IRF-4. Finally, macrophages depletion blunted macrophages to myofibroblasts transition, inhibited myofibroblasts accumulation, and ameliorated kidney injury and fibrosis. CONCLUSION: Collectively, IRF-4 plays a critical role in the pathogenesis of kidney inflammation and fibrosis in DOCA-salt hypertension.

GPR97 deficiency ameliorates renal interstitial fibrosis in mouse hypertensive nephropathy.

Hypertensive nephropathy (HTN) ranks as the second-leading cause of end-stage renal disease (ESRD). Accumulating evidence suggests that persistent hypertension injures tubular cells, leading to tubulointerstitial fibrosis (TIF), which is involved in the pathogenesis of HTN. G protein-coupled receptors (GPCRs) are implicated in many important pathological and physiological processes and act as important drug targets. In this study, we explored the intrarenal mechanisms underlying hypertension-associated TIF, and particularly, the potential role of GPR97, a member of the adhesion GPCR subfamily, in TIF. A deoxycorticosterone acetate (DOCA)/salt-induced hypertensive mouse model was used. We revealed a significantly upregulated expression of GPR97 in the kidneys, especially in renal tubules, of the hypertensive mice and 10 patients with biopsy-proven hypertensive kidney injury. GPR97-/- mice showed markedly elevated blood pressure, which was comparable to that of wild-type mice following DOCA/salt treatment, but dramatically ameliorated renal injury and TIF. In NRK-52E cells, we demonstrated that knockdown of GPR97 suppressed the activation of TGF-ß signaling by disturbing small GTPase RhoA-mediated cytoskeletal reorganization, thus inhibiting clathrin-mediated endocytosis of TGF-ß receptors and subsequent Smad activation. Collectively, this study demonstrates that GPR97 contributes to hypertension-associated TIF at least in part by facilitating TGF-ß signaling, suggesting that GPR97 is a pivotal intrarenal factor for TIF progression under hypertensive conditions, and therapeutic strategies targeting GPR97 may improve the outcomes of patients with HTN.

Urinary extracellular vesicles as a source of protein-based biomarkers in feline chronic kidney disease and hypertension.

OBJECTIVES: To validate a methodology for isolating feline urinary extracellular vesicles and characterise the urinary extracellular vesicle population and proteome in cats with normal renal function and cats with normotensive or hypertensive chronic kidney disease. METHODS: Feline urinary extracellular vesicles were isolated using three different methods (precipitation alone, precipitation followed by size exclusion chromatography and ultrafiltration followed by size exclusion chromatography, which were compared via transmission electron microscopy and nanoparticle tracking analysis. Cats with normal renal function (n=9), normotensive chronic kidney disease (n=10) and hypertensive chronic kidney disease (n=9) were identified and urinary extracellular vesicles isolated from patient urine samples via ultrafiltration followed by size exclusion chromatography. Extracellular vesicle size and concentration were determined using nanoparticle tracking analysis, and subsequently underwent proteomic analysis using liquid chromatography with tandem mass spectrometry to identify differences in protein expression between categories. RESULTS: Urinary extracellular vesicle preparations contained particles of the expected size and morphology, and those obtained by ultrafiltration + size exclusion chromatography had a significantly higher purity (highest particle: protein ratio). The urinary extracellular vesicle proteomes contained extracellular vesicle markers and proteins originating from all nephron segments. Urinary extracellular vesicle concentration and size were unaffected by renal disease or hypertension. There were no differentially expressed proteins detected when comparing urinary extracellular vesicles derived from cats in the healthy category with the combined chronic kidney disease category, but five differentially expressed proteins were identified between the normotensive chronic kidney disease and hypertensive chronic kidney disease categories. CLINICAL SIGNIFICANCE: Feline urinary extracellular vesicles can be successfully isolated from stored urine samples. Differentially expressed urinary extracellular vesicle proteins were discovered in cats with hypertensive chronic kidney disease, and warrant further investigation into their utility as biomarkers or therapeutic targets.

Glomerular proteomic profiling of kidney biopsies with hypertensive nephropathy reveals a signature of disease progression.

Hypertensive nephropathy (HN) requires a kidney biopsy as diagnostic gold-standard but histological findings are unspecific and specific prognostic markers are missing. We aimed at identifying candidate prognostic markers based on glomerular protein signatures. We studied adult patients (n = 17) with eGFR >30 ml/min/1.73m2 and proteinuria <3 g/d from the Norwegian Kidney Biopsy Registry, including subjects non progressing (NP, n = 9), or progressing (P, n = 8) to end-stage renal disease (ESRD) within an average follow-up of 22 years. Glomerular cross-sections from archival kidney biopsy sections were microdissected and processed for protein extraction. Proteomic analyses were performed using Q-exactive HF mass spectrometer and relative glomerular protein abundances were compared between P and NP patients. Immunohistochemistry (IHC) was used to validate selected data. Amongst 1870 quality filtered proteins, 58 were differentially expressed in P and NP patients' glomeruli, with absolute fold changes (FC) ≥1.5, p ≤ 0.05. Supervised classifier analysis (K nearest neighbor) identified a set of five proteins, including Gamma-butyrobetaine dioxygenase (BBOX1, O75936) and Cadherin 16 (CDH16, O75309), overexpressed in P, and Eosinophil peroxidase (EPX, P11678), DnaJ homolog subfamily B member 1 (DNAJB1, P25685) and Alpha-1-syntrophin (SNTA1, Q13424), overexpressed in NP glomeruli, correctly classifying 16/17 kidney biopsy samples. Geneset Enrichment Analysis (GSEA), showed that metabolic pathways were generally enriched in P, and structural cell pathways in NP. Pathway analysis identified Epithelial Adherens Junction Signaling as most affected canonical pathway. IHC analysis confirmed overexpression of BBOX1 and Cadherin 16 in glomeruli from P patients. In conclusion, glomerular proteomic profiling can be used to discriminate P from NP HN patients.

A Single Nucleotide Polymorphism in SH2B3/LNK Promotes Hypertension Development and Renal Damage.

BACKGROUND: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. METHODS: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. RESULTS: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8+ T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8+ T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8+ T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. CONCLUSIONS: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Inhibition of MyD88 attenuates angiotensin II-induced hypertensive kidney disease via regulating renal inflammation.

BACKGROUND: Kidney damage is a frequent event in the course of hypertension. Recent researches highlighted a critical role of non-hemodynamic activities of angiotensin II (Ang II) in hypertension-associated kidney fibrosis and inflammation. These activities are mediated through toll-like receptors (TLRs) but the mechanisms by which Ang II links TLRs to downstream inflammatory and fibrogenic responses is not fully known. In this study, we investigated the role of TLR adapter protein called myeloid differentiation primary-response protein-88 (MyD88) as the potential link. METHODS: C57BL/6 mice were administered Ang II by micro-osmotic pump infusion for 4 weeks to develop nephropathy. Mice were treated with small-molecule MyD88 inhibitor LM8. In vitro, MyD88 was blocked using siRNA or LM8 in Ang II-challenged renal tubular epithelial cells. RESULTS: We show that MyD88 is mainly located in tubular epithelial cells and Ang II increases the interaction between TLR4 and MyD88. This interaction activates MAPKs and nuclear factor-κB (NF-κB), leading to increased production of inflammatory and fibrogenic factors. Inhibition of MyD88 by siRNA or selective inhibitor LM8 supresses MyD88-TLR4 interaction, NF-κB activation, and elaboration of inflammatory cytokines and fibrosis-associated factors. These protective actions resulted in decreased renal pathological changes and preserved renal function in LM8-treated hypertensive mice, without affecting hypertension. CONCLUSION: These results demonstrate that Ang II induces inflammation and fibrosis in renal tubular epithelial cells through MyD88 and present MyD88 as a potential point of intervention for hypertension-associated kidney disease.

Ang II (Angiotensin II)-Induced FGFR1 (Fibroblast Growth Factor Receptor 1) Activation in Tubular Epithelial Cells Promotes Hypertensive Kidney Fibrosis and Injury.

BACKGROUND: Elevated Ang II (angiotensin II) level leads to a range of conditions, including hypertensive kidney disease. Recent evidences indicate that FGFR1 (fibroblast growth factor receptor 1) signaling may be involved in kidney injuries. In this study, we determined whether Ang II alters FGFR1 signaling to mediate renal dysfunction. METHODS: Human archival kidney samples from patients with or without hypertension were examined. Multiple genetic and pharmacological approaches were used to investigate FGFR1-mediated signaling in tubular epithelial NRK-52E cells in response to Ang II stimulation. C57BL/6 mice were infused with Ang II for 28 days to develop hypertensive kidney disease. Mice were treated with either adeno-associated virus expressing FGFR1 shRNA or FGFR1 inhibitor AZD4547. RESULTS: Kidney specimens from subjects with hypertension and mice challenged with Ang II have increased FGFR1 activity in renal epithelial cells. Renal epithelial cells in culture initiate extracellular matrix programming in response to Ang II, through the activation of FGFR1, which is independent of both AT1R (angiotensin II receptor type 1) and AT2R (angiotensin II receptor type 2). The RNA sequencing analysis indicated that disrupting FGFR1 suppresses Ang II-induced fibrogenic responses in epithelial cells. Mechanistically, Ang II-activated FGFR1 leads to STAT3 (signal transducer and activator of transcription 3) activation, which is responsible for fibrogenic factor expression in kidneys. In the mouse model of hypertensive kidney disease, genetic knockdown of FGFR1 or pharmacological inhibition of its activity protected kidneys from dysfunction and fibrosis upon Ang II challenge. CONCLUSIONS: Our studies uncover a novel mechanism causing renal fibrosis in hypertension and indicate FGFR1 as a potential target to preserve renal function and integrity.

Renoprotective Effect of KLF2 on Glomerular Endothelial Dysfunction in Hypertensive Nephropathy.

Kruppel-like factor 2 (KLF2) regulates endothelial cell metabolism; endothelial dysfunction is associated with hypertension and is a predictor of atherosclerosis development and cardiovascular events. Here, we investigated the role of KLF2 in hypertensive nephropathy by regulating KLF2 expression in human primary glomerular endothelial cells (hPGECs) and evaluating this expression in the kidney tissues of a 5/6 nephrectomy mouse model as well as patients with hypertension. Hypertension-mimicking devices and KLF2 siRNA were used to downregulate KLF2 expression, while the expression of KLF2 was upregulated by administering simvastatin. After 4 mmHg of pressure was applied on hPGECs for 48 h, KLF2 mRNA expression decreased, while alpha-smooth muscle actin (αSMA) mRNA expression increased. Apoptosis and fibrosis rates were increased under pressure, and these phenomena were aggravated following KLF2 knockdown, but were alleviated after simvastatin treatment; additionally, these changes were observed in angiotensin II, angiotensin type-1 receptor (AT1R) mRNA, and interleukin-18 (IL-18), but not in angiotensin type-2 receptor mRNA. Reduced expression of KLF2 in glomerular endothelial cells due to hypertension was found in both 5/6 nephrectomy mice and patients with hypertensive nephropathy. Thus, our study demonstrates that the pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, and are associated with IL-18.

Comparative analysis of hypertensive nephrosclerosis in animal models of hypertension and its relevance to human pathology. Glomerulopathy.

Current research on hypertension utilizes more than fifty animal models that rely mainly on stable increases in systolic blood pressure. In experimental hypertension, grading or scoring of glomerulopathy in the majority of studies is based on a wide range of opinion-based histological changes that do not necessarily comply with lesional descriptors for glomerular injury that are well-established in clinical pathology. Here, we provide a critical appraisal of experimental hypertensive glomerulopathy with the same approach used to assess hypertensive glomerulopathy in humans. Four hypertensive models with varying pathogenesis were analyzed-chronic angiotensin II infused mice, mice expressing active human renin in the liver (TTRhRen), spontaneously hypertensive rats (SHR), and Goldblatt two-kidney one-clip rats (2K1C). Analysis of glomerulopathy utilized the same criteria applied in humans-hyalinosis, focal segmental glomerulosclerosis (FSGS), ischemic, hypertrophic and solidified glomeruli, or global glomerulosclerosis (GGS). Data from animal models were compared to human reference values. Kidneys in TTRhRen mice, SHR and the nonclipped kidneys in 2K1C rats had no sign of hyalinosis, FSGS or GGS. Glomerulopathy in these groups was limited to variations in mesangial and capillary compartment volumes, with mild increases in collagen deposition. Histopathology in angiotensin II infused mice corresponded to mesangioproliferative glomerulonephritis, but not hypertensive glomerulosclerosis. The number of nephrons was significantly reduced in TTRhRen mice and SHR, but did not correlate with severity of glomerulopathy. The most substantial human-like glomerulosclerotic lesions, including FSGS, ischemic obsolescent glomeruli and GGS, were found in the clipped kidneys of 2K1C rats. The comparison of affected kidneys to healthy control in animals produces lesion values that are numerically impressive but correspond to mild damage if compared to humans. Animal studies should be standardized by employing the criteria and classifications established in human pathology to make experimental and human data fully comparable for comprehensive analysis and model improvements.

Protective effects of endothelial progenitor cell microvesicles on Ang II­induced rat kidney cell injury.

Chronic hypertension can lead to kidney damage, known as hypertensive nephropathy or hypertensive nephrosclerosis. Further understanding of the molecular mechanisms via which hypertensive nephropathy develops is essential for effective diagnosis and treatment. The present study investigated the mechanisms by which endothelial progenitor cells (EPCs) repair primary rat kidney cells (PRKs). ELISA, Cell Counting Kit­8 and flow cytometry assays were used to analyze the effects of EPCs or EPC­MVs on the oxidative stress, inflammation, cell proliferation, apoptosis and cycle of PRKs induced by AngII. A PRK injury model was established using angiotensin II (Ang II). After Ang II induction, PRK proliferation was decreased, apoptosis was increased and the cell cycle was blocked at the G1 phase before entering the S phase. It was found that the levels of reactive oxygen species and malondialdehyde were increased, while the levels of glutathione peroxidase and superoxide dismutase were decreased. Moreover, the levels of the inflammatory cytokines IL­1ß, IL­6 and TNF­α were significantly increased. Thus, Ang II damaged PRKs by stimulating oxidative stress and promoting the inflammatory response. However, when PRKs were co­cultured with EPCs, the damage induced by Ang II was significantly reduced. The current study collected the microvesicles (MVs) secreted by EPCs and co­cultured them with Ang II­induced PRKs, and identified that EPC­MVs retained their protective effect on PRKs. In conclusion, EPCs protect PRKs from Ang II­induced damage via secreted MVs.

Fibroblast growth factor 21 attenuates salt-sensitive hypertension-induced nephropathy through anti-inflammation and anti-oxidation mechanism.

BACKGROUND: Patients with salt-sensitive hypertension are often accompanied with severe renal damage and accelerate to end-stage renal disease, which currently lacks effective treatment. Fibroblast growth factor 21 (FGF21) has been shown to suppress nephropathy in both type 1 and type 2 diabetes mice. Here, we aimed to investigate the therapeutic effect of FGF21 in salt-sensitive hypertension-induced nephropathy. METHODS: Changes of FGF21 expression in deoxycorticosterone acetate (DOCA)-salt-induced hypertensive mice were detected. The influence of FGF21 knockout in mice on DOCA-salt-induced nephropathy were determined. Recombinant human FGF21 (rhFGF21) was intraperitoneally injected into DOCA-salt-induced nephropathy mice, and then the inflammatory factors, oxidative stress levels and kidney injury-related indicators were observed. In vitro, human renal tubular epithelial cells (HK-2) were challenged by palmitate acid (PA) with or without FGF21, and then changes in inflammation and oxidative stress indicators were tested. RESULTS: We observed significant elevation in circulating levels and renal expression of FGF21 in DOCA-salt-induced hypertensive mice. We found that deletion of FGF21 in mice aggravated DOCA-salt-induced nephropathy. Supplementation with rhFGF21 reversed DOCA-salt-induced kidney injury. Mechanically, rhFGF21 induced AMPK activation in DOCA-salt-treated mice and PA-stimulated HK-2 cells, which inhibited NF-κB-regulated inflammation and Nrf2-mediated oxidative stress and thus, is important for rhFGF21 protection against DOCA-salt-induced nephropathy. CONCLUSION: These findings indicated that rhFGF21 could be a promising pharmacological strategy for the treatment of salt-sensitive hypertension-induced nephropathy.