Chronic Deteriorating Renal Function and Renal Fibrosis

2008 ◽  
pp. 109-121 ◽  
Author(s):  
Yoshitaka Isaka ◽  
Shiro Takahara ◽  
Enyu Imai
Keyword(s):  
Renal Function ◽  
Renal Fibrosis

scholarly journals Effects of TRPC6 Inactivation on Glomerulosclerosis and Renal Fibrosis in Aging Rats

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 856
Author(s):  
Eun Young Kim ◽  
Stuart E. Dryer
Keyword(s):  
Renal Function ◽  
Protective Effect ◽  
Renal Fibrosis ◽  
Transient Receptor Potential ◽  
Smooth Muscle Actin ◽  
Receptor Potential ◽  
Sprague Dawley ◽  
Age Related ◽  
Sprague Dawley Rats ◽  
Transient Receptor

Canonical transient receptor potential 6 (TRPC6) channels have been implicated in familial and acquired forms of focal and segmental glomerulosclerosis (FSGS) in patients and animal models, as well as in renal fibrosis following ureteral obstruction in mice. Aging also evokes declines in renal function owing to effects on almost every renal compartment in humans and rodents. Here, we have examined the role of TRPC6 in driving inflammation and fibrosis during aging in Sprague-Dawley rats. This was assessed in rats with non-functional TRPC6 channels owing to CRISPR-Cas9 deletion of a portion of the ankyrin repeat domain required for the assembly of functional TRPC6 channels (Trpc6del/del rats). Wild-type littermates (Trpc6wt/wt rats) were used as controls. Animals were evaluated at 2 months and 12 months of age. There was no sign of kidney disease at 2 months of age, regardless of genotype. However, by 12 months of age, all rats examined showed declines in renal function associated with albuminuria, azotemia and increased urine excretion of β2–microglobulin, a marker for proximal tubule pathology. These changes were equally severe in Trpc6wt/wt and Trpc6del/del rats. We also observed age-related increases in renal cortical expression of markers of fibrosis (α-smooth muscle actin and vimentin) and inflammation (NLRP3 and pro-IL−1β), and there was no detectable protective effect of TRPC6 inactivation. Tubulointerstitial fibrosis assessed from histology also appeared equally severe in Trpc6wt/wt and Trpc6del/del rats. By contrast, glomerular pathology, blindly scored from histological sections, suggested a significant protective effect of TRPC6 inactivation, but only within the glomerular compartment.

scholarly journals Effects of dexmedetomidine on the RhoA /ROCK/ Nox4 signaling pathway in renal fibrosis of diabetic rats

Open Medicine ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. 890-898 ◽  
Author(s):  
Chen Jihua ◽  
Chen Cai ◽  
Bao Xubin ◽  
Yu Yue
Keyword(s):  
Renal Function ◽  
Diabetic Nephropathy ◽  
Signaling Pathway ◽  
Renal Fibrosis ◽  
Rat Kidney ◽  
Diabetic Rats ◽  
Immunohistochemical Method ◽  
Model Group ◽  
Protein Expressions ◽  
Masson Staining

AbstractObjectiveTo investigate the effects and mechanisms of dexmedetomidine (Dex) on model rats of diabetic nephropathy (DN).MethodsRats were divided into NC, model, Dex-L (1μg/ kg), Dex-M (5μg/kg) and Dex-H (10μg/kg) groups. Rats in all groups except in the NC group were injected with streptozotocin (STZ) combined with right nephrectomy. Rats in Dex (1, 5 and 10μg/kg) groups received gavage with Dex (1, 5 and 10μg/kg). After 4 weeks, rats were sacrificed and kidneys were collected. HE staining was performed for a renal injury. Masson staining was applied to detect the fibrotic accumulation in rat kidney. Radioimmunoassay was used to test the renal function. Immunohistochemical method was used to detect protein expressions of RhoA, p-MYPT and Nox4 in rat kidney.ResultsCompared with the NC group, the levels of urine microalbumin in protein, α1-MG and β2-MG, renal fibrotic accumulation, RhoA, p-MYPT, Nox4 and α-SMA in model group increased significantly (P＜0.001, respectively). Compared with the model group, Dex low, medium and high groups improved the deposition of renal fiber in rats, inhibited the expression levels of microalbumin, α1-MG and β2-MG in urine and decreased expression of RhoA, p-MYPT, Nox4 and α-SMA proteins (P＜0.05, P＜0.01).ConclusionDex is possible to inhibit the expression of α-SMA and renal fibrous substance deposition in rat kidney via RhoA/ROCK/Nox4 signaling pathway, thereby reducing early kidney damage in model rats.

scholarly journals Indoxyl Sulfate, a Tubular Toxin, Contributes to the Development of Chronic Kidney Disease

Toxins ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 684
Author(s):  
Tong-Hong Cheng ◽  
Ming-Chieh Ma ◽  
Min-Tser Liao ◽  
Cai-Mei Zheng ◽  
Kuo-Cheng Lu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Chronic Kidney Disease ◽  
Renal Function ◽  
Renal Fibrosis ◽  
Tubular Injury ◽  
Ckd Progression ◽  
Renal Function Decline ◽  
Tubular Cells ◽  
All Cause Mortality ◽  
Tgf Β1

Indoxyl sulfate (IS), a uremic toxin, causes chronic kidney disease (CKD) progression via its tubulotoxicity. After cellular uptake, IS directly induces apoptotic and necrotic cell death of tubular cells. Additionally, IS increases oxidative stress and decreases antioxidant capacity, which are associated with tubulointerstitial injury. Injured tubular cells are a major source of transforming growth factor-β1 (TGF-β1), which induces myofibroblast transition from residual renal cells in damaged kidney, recruits inflammatory cells and thereby promotes extracellular matrix deposition in renal fibrosis. Moreover, IS upregulates signal transducers and activators of transcription 3 phosphorylation, followed by increases in TGF-β1, monocyte chemotactic protein-1 and α-smooth muscle actin production, which participate in interstitial inflammation, renal fibrosis and, consequently, CKD progression. Clinically, higher serum IS levels are independently associated with renal function decline and predict all-cause mortality in CKD. The poor removal of serum IS in conventional hemodialysis is also significantly associated with all-cause mortality and heart failure incidence in end-stage renal disease patients. Scavenging the IS precursor by AST-120 can markedly reduce tubular IS staining that attenuates renal tubular injury, ameliorates IS-induced oxidative stress and rescues antioxidant glutathione activity in tubular epithelial cells, thereby providing a protective role against tubular injury and ultimately retarding renal function decline.

ADAM17 upregulation in human renal disease: a role in modulating TGF-α availability?

2009 ◽  
Vol 297 (3) ◽  
pp. F781-F790 ◽  
Author(s):  
W. B. Melenhorst ◽  
L. Visser ◽  
A. Timmer ◽  
M. C. van den Heuvel ◽  
C. A. Stegeman ◽  
...  
Keyword(s):  
Renal Function ◽  
Growth Factor ◽  
Renal Disease ◽  
Renal Fibrosis ◽  
Cellular Proliferation ◽  
Human Kidney ◽  
Macrophage Infiltration ◽  
Peritubular Capillaries ◽  
Egfr Ligand ◽  
Proximal Tubular

A disintegrin and metalloproteinase (ADAM)17 sheds growth factors from the cell membrane, including epidermal growth factor receptor (EGFR) ligand transforming growth factor (TGF)-α. In mice, angiotensin II infusion induces renal fibrosis via ADAM17-mediated TGF-α shedding and subsequent EGFR activation. Pharmacological ADAM17 inhibition reduced renal fibrotic lesions and improved renal function, positioning ADAM17 as a promising target of intervention in renal disease. We studied ADAM17 expression in the human kidney. ADAM17 mRNA was constitutively expressed in normal adult kidneys, with highest expression in distal tubules. In human renal disease, ADAM17 was de novo expressed in proximal tubules, peritubular capillaries, and glomerular mesangium and upregulated in podocytes. Glomerular mesangial and endothelial ADAM17 were associated with mesangial matrix expansion, focal glomerulosclerosis, and glomerular macrophage infiltration ( P < 0.01). Peritubular capillary and proximal tubular ADAM17 were associated with interstitial fibrosis and interstitial macrophage infiltration ( P < 0.05). Both glomerular and interstitial ADAM17 were associated with decreased renal function ( P < 0.05). In renal fibrosis, ADAM17 colocalized with TGF-α. Moreover, in cultured human podocytes and proximal tubular cells, pharmacological ADAM17 inhibition reduced constitutive TGF-α shedding by 78% ( P < 0.005) and 100% ( P < 0.05), respectively, and phorbol ester-induced TGF-α shedding by 84% ( P < 0.005) and 92% ( P = 0.005), respectively. Finally, ADAM17 inhibition reduced cellular proliferation. In conclusion, the ADAM17 expression pattern and its role in shedding TGF-α from cultured human kidney cells suggest a role in the development of fibrosis. Since EGFR signaling is implicated in renal fibrosis, targeting ADAM17 to reduce availability of EGFR ligand TGF-α may represent a promising way of intervention in human renal disease.

scholarly journals Buyang Huanwu Decoction protects against STZ-induced diabetic nephropathy by inhibiting TGF-β/Smad3 signaling-mediated renal fibrosis and inflammation

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Weifeng Wu ◽  
Yifan Wang ◽  
Haidi Li ◽  
Haiyong Chen ◽  
Jiangang Shen
Keyword(s):  
Renal Function ◽  
Diabetic Nephropathy ◽  
High Glucose ◽  
Renal Fibrosis ◽  
Mesangial Cells ◽  
Buyang Huanwu Decoction ◽  
Active Compounds ◽  
Renal Inflammation ◽  
Pas Staining ◽  
Tgf Β1

Abstract Background Buyang Huanwu Decoction (BHD) is a classical Chinese Medicine formula empirically used for diabetic nephropathy (DN). However, its therapeutic efficacies and the underlying mechanisms remain obscure. In our study, we aim to evaluate the renoprotective effect of BHD on a streptozotocin (STZ)-induced diabetic nephropathy mouse model and explore the potential underlying mechanism in mouse mesangial cells (MCs) treated with high glucose in vitro, followed by screening the active compounds in BHD. Methods Mice were received 50 mg/kg streptozotocin (STZ) or citrate buffer intraperitoneally for 5 consecutive days. BHD was intragastrically administrated for 12 weeks starting from week 4 after the diabetes induction. The quality control and quantitative analysis of BHD were studied by high-performance liquid chromatography (HPLC). Renal function was evaluated by urinary albumin excretion (UAE) using ELISA. The mesangial matrix expansion and renal fibrosis were measured using periodic acid-schiff (PAS) staining and Masson Trichrome staining. Mouse mesangial cells (MCs) were employed to study molecular mechanisms. Results We found that the impaired renal function in diabetic nephropathy was significantly restored by BHD, as indicated by the decreased UAE without affecting the blood glucose level. Consistently, BHD markedly alleviated STZ-induced diabetic glomerulosclerosis and tubulointerstitial injury as shown by PAS staining, accompanied by a reduction of renal inflammation and fibrosis. Mechanistically, BHD inhibited the activation of TGF-β1/Smad3 and NF-κB signaling in diabetic nephropathy while suppressing Arkadia expression and restoring renal Smad7. We further found that calycosin-7-glucoside (CG) was one of the active compounds from BHD, which significantly suppressed high glucose-induced inflammation and fibrosis by inhibiting TGF-β1/Smad3 and NF-κB signaling pathways in mesangial cells. Conclusion BHD could attenuate renal fibrosis and inflammation in STZ-induced diabetic kidneys via inhibiting TGF-β1/Smad3 and NF-κB signaling while suppressing the Arkadia and restoring renal Smad7. CG could be one of the active compounds in BHD to suppress renal inflammation and fibrosis in diabetic nephropathy.

scholarly journals P0678RENAL SUBCAPSULAR ADMINISTRATION OF ADIPOSE DERIVED MESENCHYMAL STEM CELLS PREVENTED THE PROGRESSION OF RENAL DAMAGE IN AN EXPERIMENTAL MODEL OF CKD

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Marina P Claro ◽  
Krislley R Pereira ◽  
Everidiene K V B Silva ◽  
Flavio Teles ◽  
Paulyana F Barbosa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Renal Function ◽  
Adipose Tissue ◽  
Survival Rate ◽  
Renal Fibrosis ◽  
Wistar Rats ◽  
Tubulointerstitial Fibrosis ◽  
Renal Inflammation ◽  
Male Wistar Rats

Abstract INTRODUCTION / AIMS Chronic kidney disease (CKD) is a progressive, debilitating condition of high lethality, which prevalence have been increasing considerably in recent decades. CKD can be triggered by many different factors, such as genetic predisposition, systemic hypertension, diabetes mellitus and autoimmune diseases. It is characterized by the gradual loss of renal function, leading to kidney failure and the need for renal replacement therapy for the maintenance of life. Regardless of the etiology of CKD, the establishment of local renal inflammation, with leucocyte recruitment, cell proliferation, extracellular matrix accumulation, glomerular and tubulointerstitial fibrosis, contribute significantly to its establishment and evolution. Due to its known pathophysiology, the primary aim when clinically treating CKD is to slow the progression of renal function loss and the advance of inflammation. However, until the present moment, there is no efficient pharmacological treatment to completely arrest the aggravation of renal inflammation and, specially, renal fibrosis. This motivates the scientific community to develop experimental research in order to test new therapeutic approaches to stunt renal fibrosis. In this context, experimental application of mesenchymal stem cells (mSC) as a treatment to control renal inflammation have been showing promising results in studies with animal models of CKD. The aim of the present study was to analyze the renoprotective effects of subcapsular application of Adipose Tissue-derived mSC (ASC), in rats submitted to 5/6 nephrectomy, after the establishment of the disease (15 days after CKD induction), in order to more closely resemble the clinical settings in humans. METHODS ASC were obtained from gonadal adipose tissue from healthy male Wistar rats. These cells were cultured until P4 when characterization by flow cytometry and in vitro differentiation were performed. Male Wistar rats underwent 5/6 nephrectomy and were followed for 15 days until the complete establishment of CKD (group CKD 15d). At this time, animals underwent a new surgery in which they received a subcapsular injection of 2x106 ASC diluted in 10 μL of sterile PBS (group CKD + ASC 30d), or only 10 μL of sterile PBS (group CKD 30d). Sham-operated rats, euthanized at day 15 (Sham 15d) and 30 (Sham 30d) were used as controls. Survival rate, body weight (BW), 24h urinary protein (24h UPE) and albumin (24h UAE) excretion serum creatinine (SCr) and blood urea nitrogen (BUN) concentration, percentage (GS%) and index (GSI) of glomerulosclerosis, tubulointerstitial fibrosis (INT%) and renal infiltration by macrophages (CD68) were studied at 15 and 30 days after 5/6 nephrectomy. Our results are presented as Mean ± SE. Differences among groups were analyzed by one-way ANOVA. RESULTS ASC injection significantly improved the survival rate of CKD + ASC 30d animals, compared to the observed in the untreated group. Moreover, ASC treatment markedly reduced protein and albumin urinary excretion, prevented the development of glomerulosclerosis, both the percentage of sclerotic glomeruli and the index of glomerular damage, numerically reduced interstitial fibrosis and significantly avoided renal inflammation by halting the progression of renal cortical macrophage infiltration. CONCLUSIONS According to our results, subcapsular ASC application promoted considerable renoprotection in the 5/6 renal ablation model, even after the complete establishment of severe CKD, suggesting that experimental therapy with these cells could be associated to the current pharmacological treatments employed to detain the progression of CKD. Figure

P1751THE GUANYLATE CYCLASE C AGONIST LINACLOTIDE LIMITS PROGRESSION TO RENAL FIBROSIS AND IMPROVE RENAL FUNCTION IN MOUSE MODEL OF CHRONIC KIDNEY DISEASE AFTER ISCHEMIA-REPERFUSION INJURY

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Yuki Nakayama ◽  
Naohito Isoyama ◽  
Kimihiko Nakamura ◽  
Toshiya Hiroyoshi ◽  
Kouki Fujikawa ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Renal Function ◽  
Renal Transplantation ◽  
Kidney Disease ◽  
Mouse Model ◽  
Guanylate Cyclase ◽  
Renal Fibrosis ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Guanylate Cyclase C

Abstract Background and Aims Renal ischemia-reperfusion injury (IRI) is a clinically significant condition that leads to acute kidney injury (AKI). AKI is followed by tissue repair characterized by collagen deposition and fibrosis which ultimately results in progression to chronic kidney disease (CKD). Especially in renal transplantation, the degree of IRI has directly led to poor long-term graft survival. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine, has been linked with progression of CKD. Linaclotide, a guanylate cyclase C agonist, has been reported decrease the plasma levels of TMAO. We investigated whether the reduction of TMAO by linaclotide protect renal function after IRI using an experimental mouse model. Method Linaclotide (100μg/kg) was administered for 2weeks before IRI and continued for 2weeks after IRI. After 2weeks since IRI, the renal function was evaluated by serum creatinine level and removed kidneys sections were performed Azan stain to evaluate the level of fibrosis. Results The administration of linaclotide before IRI significantly improved renal function. (Fig.1) Histological examination of kidneys showed linaclotide limits to expand fibrosis area after I/R injury. (Fig.2) Conclusion The reduction of TMAO by linaclotide before renal IRI could prevent renal fibrosis and improve renal function. Linaclotide may be useful for the patient expected to suffer renal IRI for example renal transplantation and partial nephrectomy. Fig2

Abstract 250: 6β-hydroxytestosterone, A Cytochrome P450 1b1-derived Metabolite Of Testosterone, Mediates Increase In Thirst, Renal Dysfunction, And End Organ Damage Associated With Angiotensin II-induced Hypertension In Male Mice

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Ajeeth K Pingili ◽  
Mehmet Kara ◽  
Brett L Jennings ◽  
Anne M Estes ◽  
Kafait U Malik
Keyword(s):  
Oxidative Stress ◽  
Renal Function ◽  
Cytochrome P450 ◽  
Renal Fibrosis ◽  
Organ Damage ◽  
Ang Ii ◽  
Male Mice ◽  
End Organ Damage ◽  
Cytochrome P450 1B1 ◽  
Induced Hypertension

Recently, we showed that 6β-hydroxytestosterone (6β-OHT), a cytochrome P450 1B1 (CYP1B1)-derived metabolite of testosterone, contributes to the development of angiotensin II (Ang II)-induced hypertension and associated cardiovascular pathophysiology. In view of the critical role of Ang II in renal homeostasis and end organ damage, we determined the contribution of 6β-OHT to Ang II actions on water consumption and renal function in male Cyp1b1 +/+ and Cyp1b1 -/- mice. Eight weeks old male Cyp1b1 +/+ and Cyp1b1 -/- intact or castrated mice were injected with 6β-OHT (15 μg/g, i.p. every 3 rd day) or vehicle (DMSO, 50 μl), and infused with Ang II (700 ng/kg/min) or vehicle for 2 weeks. Urine was collected for 24 hours on the final day of experiment. Castration attenuated Ang II-induced increase in water consumption and urine output, proteinuria and decrease in osmolality in both Cyp1b1 +/+ , and Cyp1b1 -/- mice (Table 1). 6β-OHT did not alter Ang II-induced increase in water intake, urine output, proteinuria and decrease in osmolality in Cyp1b1 +/+ mice, but restored these effects of Ang II in Cyp1b1 -/- or castrated mice (Table 1). Cyp1b1 gene disruption or castration prevented Ang II-induced renal fibrosis, inflammation, and oxidative stress. 6β-OHT did not alter Ang II-induced renal fibrosis, inflammation or oxidative stress in Cyp1b1 +/+ mice, however in Cyp1b1 -/- or castrated mice it restored these effects of Ang II. These data suggest that 6β-OHT, contributes to increased thirst, impairment of renal function and end organ damage associated with Ang II-induced hypertension in male mice, and that CYP1B1 could serve as a novel target for the treatment of renal disease and hypertension.

scholarly journals P0541KLOTHO IS INVOLVED IN EARLY AND LONG-TERM PROTECTION AGAINST RHABDOMYOLYSIS ASSOCIATED AKI

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Melania Guerrero Hue ◽  
Cristina García Caballero ◽  
Alfonso Rubio Navarro ◽  
Juan Manuel Amaro Villalobos ◽  
Raul Rodrigues Diez ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Renal Function ◽  
Protein Expression ◽  
Renal Fibrosis ◽  
Tubular Cell ◽  
Tubular Injury ◽  
Tubular Cells ◽  
Glycerol Injection

Abstract Background and Aims Rhabdomyolysis is characterized by the breakdown of the skeletal muscle and the subsequent myoglobin (Mb) release into the bloodstream. A common complication of this syndrome is acute renal injury (AKI). Once filtered by the kidney, Mb causes oxidative stress, inflammation and tubular cell death. There is no specific treatment for rhabdomyolysis-AKI, so it is crucial a better understanding of this syndrome to identify new therapeutic targets. Klotho is an anti-aging protein mostly expressed by the kidney. In addition to its functions in the regulation of mineral metabolism, Klotho protects from AKI-harmful effects. However, no previous studies analyzed the role of Klotho in rhabdomyolysis. Method We performed a pre-clinical model of rhabdomyolysis in C57BL/6J mice (male, 12 weeks old, n=30) by intramuscular injection of 10 ml/kg of 50% glycerol (≥99.5% m/v). Mice were sacrificed 3 and 6 hours or 1, 3, 7 and 30 days after glycerol administration. To evaluate to beneficial effect of Klotho in rhabdomyolysis, C57BL/6J mice were injected intraperitoneally with 0.1 mg/kg recombinant mouse Klotho (1819-KL, R&D Systems), or vehicle (PBS) 30 minutes before and 1, 3 and 5 days after glycerol injection. Blood, urine and renal samples were collected to analyze renal function, Klotho/FGF23 levels, oxidative stress, inflammation, fibrosis and cell death, all of them pathological processes affecting Klotho expression. In addition, we carried out studies in murine tubular cells (MCTs) to study the molecular mechanisms involved in Klotho regulation. Results Our results indicate that rhabdomyolysis induces an early decrease in Klotho renal mRNA and protein expression as well as Klotho serum levels. Klotho levels decreased in line with augmentation of creatinine concentration, kidney inflammation (CCL2 and IL-6 mRNA expression) and tubular injury marker NGAL. Moreover, patients with rhabdomyolysis-AKI also showed lower plasma Klotho levels and increased FGF23 plasma concentration than age-matched healthy individuals. Renal klotho protein expression remained reduced one month after rhabdomyolysis-induction, in line with long term renal fibrosis and pro-inflammatory macrophage accumulation (F4/80+ cells). Exogenous recombinant Klotho administration ameliorated renal function and reduced rhabdomyolysis-mediated tubular cell death oxidative stress (4-HNE staining) and tubular injury 24h after glycerol injection. In the same line, Klotho administration during AKI development reduced long term renal fibrosis and macrophage infiltration one month later. Antioxidant therapies with N-acetylcysteine (NAC) and sulforaphane, a potent Nuclear factor erythroid-2-related factor 2 (Nrf2) inducer, reduced Mb-mediated Klotho decrease in cultured tubular cells. Inhibition of TNF-α and IL-6 with infliximab and tocilizumab, respectively, also reverted Mb-mediated Klotho decrease. Inhibition of the inflammatory NFkB and p38 pathways also prevented Mb-mediated Klotho reduction. Conclusion Our findings are the first to demonstrate decreased renal and soluble Klotho levels not only in the early phases of rhabdomyolysis-induced AKI, but also when renal function was recovered, indicating that long-term consequences of AKI, such as inflammation and fibrosis, are also involved in Klotho downregulation. In addition, our results also indicate that Klotho administration may be a potential strategy to decrease rhabdomyolysis- long term negative effects.

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