The secreted micropeptide C4orf48 enhances renal fibrosis via an RNA-binding mechanism.

Renal interstitial fibrosis is an important mechanism in the progression of chronic kidney disease (CKD) to end-stage kidney disease. However, we lack specific treatments to slow or halt renal fibrosis. Ribosome profiling identified upregulation of a secreted micropeptide, C4orf48 (Cf48), in mouse diabetic nephropathy. Cf48 RNA and protein levels were upregulated in tubular epithelial cells in human and experimental CKD. Serum Cf48 levels were increased in human CKD and correlated with loss of kidney function, increasing CKD stage, and the degree of active interstitial fibrosis. Cf48 overexpression in mice accelerated renal fibrosis, while Cf48 gene deletion or knockdown by antisense oligonucleotides significantly reduced renal fibrosis in CKD models. In vitro, recombinant Cf48 (rCf48) enhanced TGF-ß1-induced fibrotic responses in renal fibroblasts and epithelial cells independently of Smad3 phosphorylation. Cellular uptake of Cf48 and its profibrotic response in fibroblasts operated via the transferrin receptor. RNA immunoprecipitation-sequencing identified Cf48 binding to mRNA of genes involved in the fibrotic response, including Serpine1, Acta2, Ccn2, and Col4a1. rCf48 binds to the 3'UTR of Serpine1 and increases mRNA half-life. We identify the secreted Cf48 micropeptide as a potential enhancer of renal fibrosis that operates as an RNA-binding peptide to promote the production of extracellular matrix.

Podocyte number and glomerulosclerosis indices are associated with the response to therapy for primary focal segmental glomerulosclerosis.

Corticosteroid therapy, often in combination with inhibition of the renin-angiotensin system, is first-line therapy for primary focal and segmental glomerulosclerosis (FSGS) with nephrotic-range proteinuria. However, the response to treatment is variable, and therefore new approaches to indicate the response to therapy are required. Podocyte depletion is a hallmark of early FSGS, and here we investigated whether podocyte number, density and/or size in diagnostic biopsies and/or the degree of glomerulosclerosis could indicate the clinical response to first-line therapy. In this retrospective single center cohort study, 19 participants (13 responders, 6 non-responders) were included. Biopsies obtained at diagnosis were prepared for analysis of podocyte number, density and size using design-based stereology. Renal function and proteinuria were assessed 6 months after therapy commenced. Responders and non-responders had similar levels of proteinuria at the time of biopsy and similar kidney function. Patients who did not respond to treatment at 6 months had a significantly higher percentage of glomeruli with global sclerosis than responders (p < 0.05) and glomerulosclerotic index (p < 0.05). Podocyte number per glomerulus in responders was 279 (203-507; median, IQR), 50% greater than that of non-responders (186, 118-310; p < 0.05). These findings suggest that primary FSGS patients with higher podocyte number per glomerulus and less advanced glomerulosclerosis are more likely to respond to first-line therapy at 6 months. A podocyte number less than approximately 216 per glomerulus, a GSI greater than 1 and percentage global sclerosis greater than approximately 20% are associated with a lack of response to therapy. Larger, prospective studies are warranted to confirm whether these parameters may help inform therapeutic decision making at the time of diagnosis of primary FSGS.

Selenium binding protein 1 protects renal tubular epithelial cells from ferroptosis by upregulating glutathione peroxidase 4.

Ferroptosis is a form of programmed cell death involved in various types of acute kidney injury (AKI). It is characterized by inactivation of the selenoprotein, glutathione peroxidase 4 (GPX4), and upregulation of acyl-CoA synthetase long-chain family member 4 (ACSL4). Since urinary selenium binding protein 1 (SBP1/SELENBP1) is a potential biomarker for AKI, this study investigated whether SBP1 plays a role in AKI. First, we showed that SBP1 is expressed in proximal tubular cells in normal human kidney, but is significant downregulated in cases of AKI in association with reduced GPX4 expression and increased ACSL4 expression. In mouse renal ischemia-reperfusion injury (I/R), the rapid downregulation of SBP1 protein levels preceded downregulation of GPX4 and the onset of necrosis. In vitro, hypoxia/reoxygenation (H/R) stimulation in human proximal tubular epithelial (HK-2) cells induced ferroptotic cell death in associated with an acute reduction in SBP1 and GPX4 expression, and increased oxidative stress. Knockdown of SBP1 reduced GPX4 expression and increased the susceptibility of HK-2 cells to H/R-induced cell death, whereas overexpression of SBP1 reduced oxidative stress, maintained GPX4 expression, reduced mitochondrial damage, and reduced H/R-induced cell death. Finally, selenium deficiency reduced GPX4 expression and promoted H/R-induced cell death, whereas addition of selenium was protective against H/R-induced oxidative stress. In conclusion, SBP1 plays a functional role in hypoxia-induced tubular cell death. Enhancing SBP1 expression is a potential therapeutic approach for the treatment of AKI.

Intervention treatment reducing cellular senescence inhibits tubulointerstitial fibrosis in diabetic mice following acute kidney injury.

Senescence of kidney tubules leads to tubulointerstitial fibrosis (TIF). Proximal tubular epithelial cells undergo stress-induced senescence during diabetes and episodes of acute kidney injury (AKI), and combining these injuries promotes the progression of diabetic kidney disease (DKD). Since TIF is crucial to progression of DKD, we examined the therapeutic potential of targeting senescence with a senolytic drug (HSP90 inhibitor) and/or a senostatic drug (ASK1 inhibitor) in a model of TIF in which AKI is superimposed on diabetes. After 8 weeks of streptozotocin-induced diabetes, mice underwent bilateral clamping of renal pedicles to induce mild AKI, followed by 28 days of reperfusion. Groups of mice (n=10-12) received either vehicle, HSP90 inhibitor (alvespimycin), ASK1 inhibitor (GS-444217), or both treatments. Vehicle-treated mice displayed tubular injury at day 3 and extensive tubular cell senescence at day 10, which remained unresolved at day 28. Markers of senescence (Cdkn1a and Cdkn2a), inflammation (Cd68, Tnf, and Ccl2), and TIF (Col1a1, Col4a3, α-Sma/Acta2, and Tgfb1) were elevated at day 28, coinciding with renal function impairment. Treatment with alvespimycin alone reduced kidney senescence and levels of Col1a1, Acta2, Tgfb1, and Cd68; however, further treatment with GS-444217 also reduced Col4a3, Tnf, Ccl2, and renal function impairment. Senolytic therapy can inhibit TIF during DKD, but its effectiveness can be improved by follow-up treatment with a senostatic inhibitor, which has important implications for treating progressive DKD.

Smad3 is essential for polarization of tumor-associated neutrophils in non-small cell lung carcinoma.

Neutrophils are dynamic with their phenotype and function shaped by the microenvironment, such as the N1 antitumor and N2 pro-tumor states within the tumor microenvironment (TME), but its regulation remains undefined. Here we examine TGF-ß1/Smad3 signaling in tumor-associated neutrophils (TANs) in non-small cell lung carcinoma (NSCLC) patients. Smad3 activation in N2 TANs is negatively correlate with the N1 population and patient survival. In experimental lung carcinoma, TANs switch from a predominant N2 state in wild-type mice to an N1 state in Smad3-KO mice which associate with enhanced neutrophil infiltration and tumor regression. Neutrophil depletion abrogates the N1 anticancer phenotype in Smad3-KO mice, while adoptive transfer of Smad3-KO neutrophils reproduces this protective effect in wild-type mice. Single-cell analysis uncovers a TAN subset showing a mature N1 phenotype in Smad3-KO TME, whereas wild-type TANs mainly retain an immature N2 state due to Smad3. Mechanistically, TME-induced Smad3 target genes related to cell fate determination to preserve the N2 state of TAN. Importantly, genetic deletion and pharmaceutical inhibition of Smad3 enhance the anticancer capacity of neutrophils against NSCLC via promoting their N1 maturation. Thus, our work suggests that Smad3 signaling in neutrophils may represent a therapeutic target for cancer immunotherapy.

Mitochondrial micropeptide MOXI promotes fibrotic gene transcription by translocation to the nucleus and bridging N-acetyltransferase 14 with transcription factor c-Jun.

Progressive fibrosis is a hallmark of chronic kidney disease, but we lack effective treatments to halt this destructive process. Micropeptides (peptides of no more than 100 amino acids) encoded by small open reading frames represent a new class of eukaryotic regulators. Here, we describe that the micropeptide regulator of ß-oxidation (MOXI) regulates kidney fibrosis. MOXI expression was found to be up-regulated in human fibrotic kidney disease, and this correlated with the degree of fibrosis and loss of kidney function. MOXI was expressed in the cytoplasm and mitochondria of cultured tubular epithelial cells and translocated to the nucleus upon Transforming Growth Factor-ß1 stimulation. Deletion of Moxi protected mice against fibrosis and inflammation in the folic acid and unilateral ureteral obstruction models. As a potential molecular therapy, treatment with an antisense MOXI oligonucleotide effectively knocked-down MOXI expression and protected against kidney fibrosis in both models. Bimolecular fluorescence complementation identified the enzyme N-acetyltransferase 14 (Nat14) and transcription factor c-Jun as MOXI binding partners. The MOXI/Nat14/c-Jun complex enhances basal and Transforming Growth Factor-ß1 induced collagen I gene promoter activity. Phosphorylation at T49 is required for MOXI nuclear localization and for complex formation with Nat14 and c-Jun. Furthermore, mice with a MoxiT49A point mutation were protected in the models of kidney fibrosis. Thus, our studies demonstrate a key role for the micropeptide MOXI in kidney fibrosis and identify a new function of MOXI in forming a transcriptional complex with Nat14 and c-Jun.

Mizoribine halts kidney fibrosis in childhood IgA nephropathy: association with modulation of M2-type macrophages.

BACKGROUND: The immunosuppressant mizoribine (Miz) can reduce progression of childhood IgA nephropathy (IgAN). This study examined whether Miz affects CD163+ M2-type macrophages which are associated with kidney fibrosis in childhood IgAN. METHODS: A retrospective cohort of 90 children with IgAN were divided into groups treated with prednisolone (PSL) alone (P group; n = 42) or PSL plus Miz (PM group; n = 48) for a 2-year period. Normal human monocyte-derived macrophages were stimulated with dexamethasone (Dex), or Dex plus Miz, and analyzed by DNA microarray. RESULTS: Clinical and histological findings at first biopsy were equivalent between patients entering the P and PM groups. Both treatments improved proteinuria and haematuria, and maintained normal kidney function over the 2-year course. The P group exhibited increased mesangial matrix expansion, increased glomerular segmental or global sclerosis, and increased interstitial fibrosis at 2-year biopsy; however, the PM group showed no progression of kidney fibrosis. These protective effects were associated with reduced numbers of glomerular and interstitial CD163+ macrophages in the PM versus P group. In cultured human macrophages, Dex induced upregulation of cytokines and growth factors, which was prevented by Miz. Miz also inhibited Dex-induced expression of CD300E, an activating receptor which can prevent monocyte apoptosis. CD300e expression by CD163+ macrophages was evident in the P group, which was reduced by Miz treatment. CONCLUSION: Miz halted the progression of kidney fibrosis in PSL-treated pediatric IgAN. This was associated with reduced CD163+ and CD163+CD300e+ macrophage populations, plus in vitro findings that Miz can suppress steroid-induced macrophage expression of pro-fibrotic molecules. A higher resolution version of the Graphical abstract is available as Supplementary information.

ASK1 is a novel molecular target for preventing aminoglycoside-induced hair cell death.

Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides are toxic to the sensory hair cells in the inner ear. As a result, aminoglycoside-treated individuals can develop permanent hearing loss and vestibular impairment. There is considerable evidence that reactive oxygen species (ROS) production and the subsequent phosphorylation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) drives apoptosis in aminoglycoside-treated hair cells. However, treatment strategies that directly inhibit ROS, JNK, or P38 are limited by the importance of these molecules for normal cellular function. Alternatively, the upstream regulator apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) is a key mediator of ROS-induced JNK and P38 activation under pathologic but not homeostatic conditions. We investigated ASK1 as a mediator of drug-induced hair cell death using cochlear explants from Ask1 knockout mice, demonstrating that Ask1 deficiency attenuates neomycin-induced hair cell death. We then evaluated pharmacological inhibition of ASK1 with GS-444217 as a potential otoprotective therapy. GS-444217 significantly attenuated hair cell death in neomycin-treated explants but did not impact aminoglycoside efficacy against P. aeruginosa in the broth dilution test. Overall, we provide significant pre-clinical evidence that ASK1 inhibition represents a novel strategy for preventing aminoglycoside ototoxicity. KEY MESSAGES: ASK1 is an upstream, redox-sensitive regulator of P38 and JNK, which are known mediators of hair cell death. Ask1 knockout does not affect hair cell development in vivo, but significantly reduces aminoglycoside-induced hair cell death in vitro. A small-molecule inhibitor of ASK1 attenuates neomycin-induced hair cell death, and does not impact antibiotic efficacy in vitro. ASK1 may be a novel molecular target for preventing aminoglycoside-induced hearing loss.

Human Kidney Organoids and Tubuloids as Models of Complex Kidney Disease.

Kidney organoids derived from pluripotent stem cells and epithelial organoids derived from adult tissue (tubuloids) have been used to study various kidney disorders with a strong genetic component, such as polycystic kidney disease, Wilms tumor, and congenital nephrotic syndrome. However, complex disorders without clear genetic associations, such as acute kidney injury and many forms of chronic kidney disease, are only just beginning to be investigated using these in vitro approaches. Although organoids are a reductionist model, they contain clinically relevant cell populations that may help to elucidate human-specific pathogenic mechanisms. Thus, organoids may complement animal disease models to accelerate the translation of laboratory proof-of-concept research into clinical practice. This review discusses whether kidney organoids and tubuloids are suitable models for the study of complex human kidney disease and highlights their advantages and limitations compared with monolayer cell culture and animal models.

Role of the adaptive immune system in diabetic kidney disease.

Diabetic kidney disease (DKD) is a highly prevalent complication of diabetes and the leading cause of end-stage kidney disease. Inflammation is recognized as an important driver of progression of DKD. Activation of the immune response promotes a pro-inflammatory milieu and subsequently renal fibrosis, and a progressive loss of renal function. Although the role of the innate immune system in diabetic renal disease has been well characterized, the potential contribution of the adaptive immune system remains poorly defined. Emerging evidence in experimental models of DKD indicates an increase in the number of T cells in the circulation and in the kidney cortex, that in turn triggers secretion of inflammatory mediators such as interferon-γ and tumor necrosis factor-α, and activation of cells in innate immune response. In human studies, the number of T cells residing in the interstitial region of the kidney correlates with the degree of albuminuria in people with type 2 diabetes. Here, we review the role of the adaptive immune system, and associated cytokines, in the development of DKD. Furthermore, the potential therapeutic benefits of targeting the adaptive immune system as a means of preventing the progression of DKD are discussed.

Mice with Established Diabetes Show Increased Susceptibility to Renal Ischemia/Reperfusion Injury: Protection by Blockade of Jnk or Syk Signaling Pathways.

Patients with diabetes are at an increased risk for acute kidney injury (AKI) after renal ischemia/reperfusion injury (IRI). However, there is a lack preclinical models of IRI in established diabetes. The current study characterized renal IRI in mice with established diabetes and investigated potential therapies. Diabetes was induced in C57BL/6J mice by low-dose streptozotocin injection. After 7 weeks of sustained diabetes, mice underwent 13 minutes of bilateral renal ischemia and were euthanized after 24 hours of reperfusion. Age-matched, nondiabetic controls underwent the same surgical procedure. Renal IRI induced two- and sevenfold increases in plasma creatinine level in nondiabetic and diabetic mice, respectively (P < 0.001). Kidney damage, as indicated by histologic damage, tubular cell death, tubular damage markers, and inflammation, was more severe in the diabetic IRI group. The diabetic IRI group showed greater accumulation of spleen tyrosine kinase (Syk)-expressing cells, and increased c-Jun N-terminal kinase (Jnk) signaling in tubules compared to nondiabetic IRI. Prophylactic treatment with a Jnk or Syk inhibitor substantially reduced the severity of AKI in the diabetic IRI model, with differential effects on neutrophil infiltration and Jnk activation. In conclusion, established diabetes predisposed mice to renal IRI-induced AKI. Two distinct proinflammatory pathways, JNK and SYK, were identified as potential therapeutic targets for anticipated AKI in patients with diabetes.

Corrigendum: c-Jun amino terminal kinase signaling promotes aristolochic acid-induced acute kidney injury.

[This corrects the article DOI: 10.3389/fphys.2021.599114.].

Cyclophilin D Promotes Acute, but Not Chronic, Kidney Injury in a Mouse Model of Aristolochic Acid Toxicity.

The plant-derived toxin, aristolochic acid (AA), is the cause of Chinese Herb Nephropathy and Balkan Nephropathy. Ingestion of high dose AA induces acute kidney injury, while chronic low dose ingestion leads to progressive kidney disease. Ingested AA is taken up by tubular epithelial cells of the kidney, leading to DNA damage and cell death. Cyclophilin D (CypD) participates in mitochondrial-dependent cell death, but whether this mechanism operates in acute or chronic AA-induced kidney injury is unknown. We addressed this question by exposing CypD-/- and wild type (WT) mice to acute high dose, or chronic low dose, AA. Administration of 5 mg/kg AA to WT mice induced acute kidney injury 3 days later, characterised by loss of kidney function, tubular cell damage and death, and neutrophil infiltration. All of these parameters were significantly reduced in CypD-/- mice. Chronic low dose (2 mg/kg AA) administration in WT mice resulted in chronic kidney disease with impaired renal function and renal fibrosis by day 28. However, CypD-/- mice were not protected from AA-induced chronic kidney disease. In conclusion, CypD facilitates AA-induced acute kidney damage, but CypD does not contribute to the transition of acute kidney injury to chronic kidney disease during ongoing AA exposure.

c-Jun Amino Terminal Kinase Signaling Promotes Aristolochic Acid-Induced Acute Kidney Injury.

Aristolochic acid (AA) is a toxin that induces DNA damage in tubular epithelial cells of the kidney and is the cause of Balkan Nephropathy and Chinese Herb Nephropathy. In cultured tubular epithelial cells, AA induces a pro-fibrotic response via the c-Jun amino terminal kinase (JNK) signaling pathway. This study investigated the in vivo role of JNK signaling with a JNK inhibitor (CC-930) in mouse models of acute high dose AA-induced kidney injury (day 3) and renal fibrosis induced by chronic low dose AA exposure (day 22). CC-930 treatment inhibited JNK signaling and protected from acute AA-induced renal function impairment and severe tubular cell damage on day 3, with reduced macrophage infiltration and expression of pro-inflammatory molecules. In the chronic model, CC-930 treatment inhibited JNK signaling but did not affect AA-induced renal function impairment, tubular cell damage including the DNA damage response and induction of senescence, or renal fibrosis; despite a reduction in the macrophage pro-inflammatory response. In conclusion, JNK signaling contributes to acute high dose AA-induced tubular cell damage, presumably via an oxidative stress-dependent mechanism, but is not involved in tubular atrophy and senescence that promote chronic kidney disease caused by ongoing DNA damage in chronic low dose AA exposure.

JUN Amino-Terminal Kinase 1 Signaling in the Proximal Tubule Causes Cell Death and Acute Renal Failure in Rat and Mouse Models of Renal Ischemia/Reperfusion Injury.

Activation of the JUN amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including acute renal ischemia/reperfusion injury (IRI). Two forms of JNK, JNK1 and JNK2, are expressed in the kidney. Systemic administration of pan-JNK inhibitors suppresses renal IRI; however, the contribution of JNK1 versus JNK2, and the specific role of JNK activation in the proximal tubule in IRI, remains unknown. These questions were addressed in rat and mouse models of acute bilateral renal IRI. Administration of the JNK inhibitor, CC-930, substantially reduced the severity of renal failure, tubular damage, and inflammation at 24 hours in a rat IRI model. Additionally, Jnk1-/- mice, but not Jnk2-/- mice, were shown to be significantly protected against acute renal failure, tubular damage, and inflammation in the IRI model. Furthermore, mice with conditional Jnk1 deletion in the proximal tubule also showed considerable protection from IRI-induced renal failure, tubular damage, and inflammation. Finally, primary cultures of Jnk1-/-, but not Jnk2-/-, tubular epithelial cells were protected from oxidant-induced cell death, in association with preventing phosphorylation of proteins (receptor interacting serine/threonine kinase 3 and mixed lineage kinase domain-like pseudokinase) in the necroptosis pathway. In conclusion, JNK1, but not JNK2, plays a specific role in IRI-induced cell death in the proximal tubule, leading to acute renal failure.

Steroid treatment promotes an M2 anti-inflammatory macrophage phenotype in childhood lupus nephritis.

BACKGROUND: M1-type proinflammatory macrophages (MΦ) promote glomerular injury in lupus nephritis (LN). However, whether this phenotype is altered by steroid therapy is unclear. Therefore, we investigated the effect of steroid treatment on MΦ phenotype in LN. METHODS: Patients with LN (7-18 years old) were divided into 2 groups: those with no treatment (N) before biopsy (n = 17) and those who underwent steroid (S) treatment (3-73 days) before biopsy (n = 15). MΦ number and phenotype were assessed by immunofluorescence. In vitro studies used monocyte-derived MΦ from healthy volunteers. RESULTS: Age at biopsy, urine findings, and kidney function (eGFR) were comparable between the two groups. Biopsies in N group had higher levels of active lesions such as endocapillary hypercellularity, necrosis, and cellular crescent formation (p < 0.05). The total CD68+ MΦ infiltrate was comparable between N and S groups. However, N group had more M1 MΦ (CD68+ CD86+ cells) (p < 0.05) and fewer M2 MΦ (CD68+ CD163+ cells) (p < 0.05), giving a 6-fold increase in the M2/M1 ratio in S vs. N groups. Dexamethasone treatment of cultured MΦ induced upregulation of CD163 expression, increased production of anti-inflammatory (IL-10, IL-19) and profibrotic factors (FGF-22, PDGF), and upregulated the scavenger receptor, stabilin-1. Upregulation of stabilin-1 in CD163+ M2 MΦ was confirmed in biopsies from S group. CONCLUSIONS: Initial steroid treatment induces MΦ phenotypic change from proinflammatory M1 to anti-inflammatory or profibrotic M2 in LN with acute/active lesions. Although steroid treatment is effective for resolution of M1-medated injury, promotion of fibrotic lesions via M2 MΦ is a potential downside of steroid single therapy in LN.

Neural transcription factor Pou4f1 promotes renal fibrosis via macrophage-myofibroblast transition.

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, α-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGF-ß1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-ß1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.

Omics technologies for kidney disease research.

Omics-based technologies in the areas of genomics, transcriptomics, and proteomics are revolutionizing research into many types of human and experimental disease. This review focuses on the potential of omics technologies to identify pathogenic mechanisms and biomarkers of disease in order to improve the diagnosis, prognosis, monitoring, and treatment of kidney disease.