Role of Epidermal Growth Factor Receptor (EGFR) and Its Ligands in Kidney Inflammation and Damage

Chronickidneydisease (CKD)ischaracterized bypersistent inflammationandprogressive fibrosis,ultimatelyleadingto end-stage renal disease. Although many studies have investigated the factors involved in the progressive deterioration of renal function, current therapeutic strategies only delay disease progression, leaving an unmet need for effective therapeutic interventions that target the cause behind the inflammatory process and could slow down or reverse the development and progression of CKD. Epidermal growth factor receptor (EGFR) (ERBB1), a membrane tyrosine kinase receptor expressed in the kidney, is activated after renal damage, and preclinical studies have evidenced its potential as a therapeutic target in CKD therapy. To date, seven official EGFR ligands have been described, including epidermal growth factor (EGF) (canonical ligand), transforming growth factor-α, heparin-binding epidermal growth factor, amphiregulin, betacellulin, epiregulin, and epigen. Recently, the connective tissue growth factor (CTGF/CCN2) has been described as a novel EGFR ligand. The direct activation of EGFR by its ligands can exert different cellular responses, depending on the specific ligand, tissue, and pathological condition. Among all EGFR ligands, CTGF/CCN2 is of special relevance in CKD. This growth factor, by binding to EGFR and downstream signaling pathway activation, regulates renal inflammation, cell growth, and fibrosis. EGFR can also be “transactivated” by extracellular stimuli, including several key factors involved in renal disease, such as angiotensin II, transforming growth factor beta (TGFB), and other cytokines, including members of the tumor necrosis factor superfamily, showing another important mechanism involved in renal pathology. The aim of this review is to summarize the contribution of EGFR pathway activation in experimental kidney damage, with special attention to the regulation of the inflammatory response and the role of some EGFR ligands in this process. Better insights in EGFR signaling in renal disease could improve our current knowledge of renal pathology contributing to therapeutic strategies for CKD development and progression.This work was supported by the Instituto de Salud Carlos III and Fondos FEDER European Union (PI014/00041, PI17/00119), Red de Investigación Renal (REDinREN; RD16/0009), Comunidad de Madrid (B2017/BMD-3751 NOVELREN-CM), and Sociedad Española de Nefrología. The “Juan de la Cierva de Formacion” training program of the Ministerio de Economía, Industria y Competitividad, Gobierno de España supported the salary of SR-M (FJCI-2016-29050)

Oxidative Stress and Cellular Senescence Are Involved in the Aging Kidney

Chronic kidney disease (CKD) can be considered as a clinical model for premature aging. However, non‐invasive biomarkers to detect early kidney damage and the onset of a senescent phenotype are lacking. Most of the preclinical senescence studies in aging have been done in very old mice. Furthermore, the precise characterization and over-time development of age-related senes-cence in the kidney remain unclear. To address these limitations, the age-related activation of cellular senescence-associated mechanisms and their correlation with early structural changes in the kidney were investigated in 3- to 18-month-old C57BL6 mice. Inflammatory cell infiltration was ob-served by 12 months, whereas tubular damage and collagen accumulation occurred later. Early activation of cellular-senescence-associated mechanisms was found in 12-month-old mice, character-ized by activation of the DNA-damage-response (DDR), mainly in tubular cells; activation of the antioxidant NRF2 pathway; and klotho downregulation. However, induction of tubular-cell-cycle-arrest (CCA) and overexpression of renal senescent-associated secretory phenotype (SASP) components was only found in 18-month-old mice. In aging mice, both inflammation and oxidative stress (marked by elevated lipid peroxidation and NRF2 inactivation) remained increased. These findings support the hypothesis that prolonged DDR and CCA, loss of nephroprotective factors (klotho), and dysfunctional redox regulatory mechanisms (NRF2/antioxidant defense) can be early drivers of age-related kidney-damage progressionThis research was funded by grants from the Instituto de Salud Carlos III (ISCIII); Fondos FEDER European Union (PI17/00119, PI20/00140; and DTS20/00083 to M.R.-O.; PI18/01133 to A.M.R.); Sara Borrell’ program from Instituto de Salud Carlos III (ISCIII) (grant number CD20/00042 to R.R.R.-D.); Red de Investigación Renal REDINREN: RD16/0009/0003 and RICORS program to RICORS2040 496 (RD21/0005), to M.R.-O., Sociedad Española de Nefrología; “NOVELREN-CM: Enfermedad renal crónica: nuevas Estrategias para la prevención, Diagnóstico y tratamiento” (B2017/BMD3751 to M.R.-O.); “Convocatoria Dinamización Europa Investigación 2019” MINECO (EIN2019-103294 to M.R.-O.); Juan de la Cierva incorporacion grant: IJC2018-035187-I to S.R.-M.; innovation program under the Marie Skłodowska-Curie grant of the European Union’s Horizon 2020 (IMProvePD ID: 812699) to M.R.-O.; and Fundacion Conchita Rabago to L.T.-

A challenge-based interdisciplinary undergraduate concept fostering translational medicine

Translational medicine (TM) is an interdisciplinary branch of biomedicine that bridges the gap from bench-to-bedside to improve global health. Fundamental TM skills include interdisciplinary collaboration, communication, critical thinking, and creative problem-solving (4Cs). TM is currently limited in undergraduate biomedical education programs, with little patient contact and opportunities for collaboration between different disciplines. In this study, we developed and evaluated a novel interdisciplinary challenge-based educational concept, grounded in the theoretical framework of experimental research-based education, to implement TM in undergraduate biomedicine and medicine programs. Students were introduced to an authentic clinical problem through an interdisciplinary session with patients, medical doctors, and scientists. Next, students collaborated in groups to design unique laboratory-based research proposals addressing this problem. Stakeholders subsequently rewarded the best proposal with funding to be executed in a consecutive interdisciplinary laboratory course, in which mixed teams of biomedicine and medicine students performed the research in a fully equipped wet laboratory. Written questionnaires and focus groups revealed that students developed 4C skills and acquired a 4C mindset. Working on an authentic patient case and the interdisciplinary setting positively contributed to communication, collaboration, critical thinking, and creative problem-solving skills. Furthermore, students were intrinsically motivated by (i) the relevance of their work that made them feel taken seriously and competent, (ii) the patient involvement that highlighted the societal relevance of their work, and (iii) the acquisition of a realistic view of what doing science in a biomedical research laboratory is. In conclusion, we showcase a widely applicable interdisciplinary challenge-based undergraduate concept fostering TM

Acute Kidney Injury is Aggravated in Aged Mice by the Exacerbation of Proinflammatory Processes

Acute kidney injury (AKI) is more frequent in elderly patients. Mechanisms contributing to AKI (tubular cell death, inflammatory cell infiltration, impaired mitochondrial function, and prolonged cell-cycle arrest) have been linked to cellular senescence, a process implicated in regeneration failure and progression to fibrosis. However, the molecular and pathological basis of the age-related increase in AKI incidence is not completely understood. To explore these mechanisms, experimental AKI was induced by folic acid (FA) administration in young (3-months-old) and old (1-year-old) mice, and kidneys were evaluated in the early phase of AKI, at 48 h. Tubular damage score, KIM-1 expression, the recruitment of infiltrating immune cells (mainly neutrophils and macrophages) and proinflammatory gene expression were higher in AKI kidneys of old than of young mice. Tubular cell death in FA-AKI involves several pathways, such as regulated necrosis and apoptosis. Ferroptosis and necroptosis cell-death pathways were upregulated in old AKI kidneys. In contrast, caspase-3 activation was only found in young but not in old mice. Moreover, the antiapoptotic factor BCL-xL was significantly overexpressed in old, injured kidneys, suggesting an age-related apoptosis suppression. AKI kidneys displayed evidence of cellular senescence, such as increased levels of cyclin dependent kinase inhibitors p16ink4a and p21cip1, and of the DNA damage response marker γH2AX. Furthermore, p21cip1 mRNA expression and nuclear staining for p21cip1 and γH2AX were higher in old than in young FA-AKI mice, as well as the expression of senescence-associated secretory phenotype (SASP) components (Il-6, Tgfb1, Ctgf, and Serpine1). Interestingly, some infiltrating immune cells were p21 or γH2AX positive, suggesting that molecular senescence in the immune cells (“immunosenescence”) are involved in the increased severity of AKI in old mice. In contrast, expression of renal protective factors was dramatically downregulated in old AKI mice, including the antiaging factor Klotho and the mitochondrial biogenesis driver PGC-1α. In conclusion, aging resulted in more severe AKI after the exposure to toxic compounds. This increased toxicity may be related to magnification of proinflammatory-related pathways in older mice, including a switch to a proinflammatory cell death (necroptosis) instead of apoptosis, and overactivation of cellular senescence of resident renal cells and infiltrating inflammatory cells

Cellular senescence in kidney biopsies is associated with tubular dysfunction and predicts CKD progression in childhood cancer patients with karyomegalic interstitial nephropathy

Karyomegalic interstitial nephropathy (KIN) has been reported as an incidental finding in patients with childhood cancer treated with ifosfamide. It is defined by the presence of tubular epithelial cells (TECs) with enlarged, irregular, and hyperchromatic nuclei. Cellular senescence has been proposed to be involved in kidney fibrosis in hereditary KIN patients. We report that KIN could be diagnosed 7-32 months after childhood cancer diagnosis in 6/6 consecutive patients biopsied for progressive chronic kidney disease (CKD) of unknown cause between 2018 and 2021. The morphometry of nuclear size distribution and markers for DNA damage (γH2AX), cell-cycle arrest (p21+, Ki67-), and nuclear lamina decay (loss of lamin B1), identified karyomegaly and senescence features in TECs. Polyploidy was assessed by chromosome fluorescence in situ hybridization (FISH). In all six patients the number of p21-positive TECs far exceeded the typically small numbers of truly karyomegalic cells, and p21-positive TECs contained less lysozyme, testifying to defective resorption, which explains the consistently observed low-molecular-weight (LMW) proteinuria. In addition, polyploidy of TEC was observed to correlate with loss of lysozyme staining. Importantly, in the five patients with the largest nuclei, the percentage of p21-positive TECs tightly correlated with estimated glomerular filtration rate loss between biopsy and last follow-up (R 2  = 0.93, p < 0.01). We conclude that cellular senescence is associated with tubular dysfunction and predicts CKD progression in childhood cancer patients with KIN and appears to be a prevalent cause of otherwise unexplained CKD and LMW proteinuria in children treated with DNA-damaging and cell stress-inducing therapy including ifosfamide. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland

Acute Kidney Injury is Aggravated in Aged Mice by the Exacerbation of Proinflammatory Processes

Acute kidney injury (AKI) is more frequent in elderly patients. Mechanisms contributing to AKI (tubular cell death, inflammatory cell infiltration, impaired mitochondrial function, and prolonged cell-cycle arrest) have been linked to cellular senescence, a process implicated in regeneration failure and progression to fibrosis. However, the molecular and pathological basis of the age-related increase in AKI incidence is not completely understood. To explore these mechanisms, experimental AKI was induced by folic acid (FA) administration in young (3-months-old) and old (1-year-old) mice, and kidneys were evaluated in the early phase of AKI, at 48 h. Tubular damage score, KIM-1 expression, the recruitment of infiltrating immune cells (mainly neutrophils and macrophages) and proinflammatory gene expression were higher in AKI kidneys of old than of young mice. Tubular cell death in FA-AKI involves several pathways, such as regulated necrosis and apoptosis. Ferroptosis and necroptosis cell-death pathways were upregulated in old AKI kidneys. In contrast, caspase-3 activation was only found in young but not in old mice. Moreover, the antiapoptotic factor BCL-xL was significantly overexpressed in old, injured kidneys, suggesting an age-related apoptosis suppression. AKI kidneys displayed evidence of cellular senescence, such as increased levels of cyclin dependent kinase inhibitors p16ink4a and p21cip1, and of the DNA damage response marker γH2AX. Furthermore, p21cip1 mRNA expression and nuclear staining for p21cip1 and γH2AX were higher in old than in young FA-AKI mice, as well as the expression of senescence-associated secretory phenotype (SASP) components (Il-6, Tgfb1, Ctgf, and Serpine1). Interestingly, some infiltrating immune cells were p21 or γH2AX positive, suggesting that molecular senescence in the immune cells (“immunosenescence”) are involved in the increased severity of AKI in old mice. In contrast, expression of renal protective factors was dramatically downregulated in old AKI mice, including the antiaging factor Klotho and the mitochondrial biogenesis driver PGC-1α. In conclusion, aging resulted in more severe AKI after the exposure to toxic compounds. This increased toxicity may be related to magnification of proinflammatory-related pathways in older mice, including a switch to a proinflammatory cell death (necroptosis) instead of apoptosis, and overactivation of cellular senescence of resident renal cells and infiltrating inflammatory cells

Senescent cell accumulation & Research-based undergraduate education

In part 1 of this thesis, the accelerated aging process of damaged kidney cells (“cellular senescence) in Chronic Kidney Disease (CKD), and targets for intervention are explored. Senescent kidney cells contribute to renal failure through scarring of kidney tissue and loss of function. We show that renal senescent cells accumulate in scarred kidney tissue in two groups of CKD patients: kidney transplant recipients and childhood cancer survivors (as complication of their treatment). From the reviewed literature and these observations, we conclude that CKD patients may benefit from elimination of renal senescent cells by so called “senotherapeutics”. We identified two important factors that contribute to senescent cell accumulation in the context of renal injury. First, aging magnifies senescent cell numbers after renal injury, suggesting that especially elderly may benefit from senotherapeutic therapy. Next, the molecule CCN2 plays an important role in renal senescent cell accumulation after kidney transplantation. Our findings suggest that an inhibitor of CCN2 is a promising senotherapeutic CKD treatment. For identifying other factors or targets for senescence in the kidney, we developed a screening tool using kidney cells that are in a state that resembles senescence. In part 2 of this thesis, implementation of a new translational medicine educational concept in which students conduct research into a real medically relevant problem, in biomedical bachelor education is evaluated. We show that bachelor students successfully design studies, acquire funding and execute laboratory-studies. Further, they develop important academic skills. This provides a new insight into the involvement of bachelor students in biomedical research

Senescent cell accumulation & Research-based undergraduate education

In part 1 of this thesis, the accelerated aging process of damaged kidney cells (“cellular senescence) in Chronic Kidney Disease (CKD), and targets for intervention are explored. Senescent kidney cells contribute to renal failure through scarring of kidney tissue and loss of function. We show that renal senescent cells accumulate in scarred kidney tissue in two groups of CKD patients: kidney transplant recipients and childhood cancer survivors (as complication of their treatment). From the reviewed literature and these observations, we conclude that CKD patients may benefit from elimination of renal senescent cells by so called “senotherapeutics”. We identified two important factors that contribute to senescent cell accumulation in the context of renal injury. First, aging magnifies senescent cell numbers after renal injury, suggesting that especially elderly may benefit from senotherapeutic therapy. Next, the molecule CCN2 plays an important role in renal senescent cell accumulation after kidney transplantation. Our findings suggest that an inhibitor of CCN2 is a promising senotherapeutic CKD treatment. For identifying other factors or targets for senescence in the kidney, we developed a screening tool using kidney cells that are in a state that resembles senescence. In part 2 of this thesis, implementation of a new translational medicine educational concept in which students conduct research into a real medically relevant problem, in biomedical bachelor education is evaluated. We show that bachelor students successfully design studies, acquire funding and execute laboratory-studies. Further, they develop important academic skills. This provides a new insight into the involvement of bachelor students in biomedical research

CCN2 Aggravates the Immediate Oxidative Stress&ndash;DNA Damage Response following Renal Ischemia&ndash;Reperfusion Injury

AKI, due to the fact of altered oxygen supply after kidney transplantation, is characterized by renal ischemia&ndash;reperfusion injury (IRI). Recent data suggest that AKI to CKD progression may be driven by cellular senescence evolving from prolonged DNA damage response (DDR) following oxidative stress. Cellular communication factor 2 (CCN2, formerly called CTGF) is a major contributor to CKD development and was found to aggravate DNA damage and the subsequent DDR&ndash;cellular senescence&ndash;fibrosis sequence following renal IRI. We therefore investigated the impact of CCN2 inhibition on oxidative stress and DDR in vivo and in vitro. Four hours after reperfusion, full transcriptome RNA sequencing of mouse IRI kidneys revealed CCN2-dependent enrichment of several signaling pathways, reflecting a different immediate stress response to IRI. Furthermore, decreased staining for &gamma;H2AX and p-p53 indicated reduced DNA damage and DDR in tubular epithelial cells of CCN2 knockout (KO) mice. Three days after IRI, DNA damage and DDR were still reduced in CCN2 KO, and this was associated with reduced oxidative stress, marked by lower lipid peroxidation, protein nitrosylation, and kidney expression levels of Nrf2 target genes (i.e., HMOX1 and NQO1). Finally, silencing of CCN2 alleviated DDR and lipid peroxidation induced by anoxia-reoxygenation injury in cultured PTECs. Together, our observations suggest that CCN2 inhibition might mitigate AKI by reducing oxidative stress-induced DNA damage and the subsequent DDR. Thus, targeting CCN2 might help to limit post-IRI AKI