Areas Of Interstitial Fibrosis Research Articles

The pathogenic mechanism of monosodium urate crystal-induced kidney injury in a rat model.

(MSU) crystals usually in the kidney tubules especially collecting ducts in the medulla. Previous animal models have not fully reproduced the impact of MSU on kidneys under non-hyperuricemic conditions. In the group treated with MSU, the upper pole of the rat kidney was injected intrarenally with 50 mg/kg of MSU, while the lower pole was injected with an equivalent volume of PBS solution. The body weight and kidney mass of the rats were observed and counted. H&E staining was used to observe the pathological damage of the kidney and to count the number of inflammatory cells. Masoon staining was used to observe the interstitial fibrosis in the kidneys of the rat model. Flow cytometric analysis was used for counting inflammatory cells in rats. ElISA was used to measure the concentration of serum and urine uric acid, creatinine and urea nitrogen in rats. At the MSU injection site, a significantly higher infiltration of inflammatory cells and a substantial increase in the area of interstitial fibrosis compared to the control group and the site of PBS injection were observed. The serum creatinine level was significantly increased in the MSU group. However, there were no significant differences in the rats' general conditions or blood inflammatory cell counts when compared to the control group. The injection of urate crystals into the kidney compromised renal function, caused local pathological damage, and increased inflammatory cell infiltration and interstitial fibrosis. Intrarenal injection of MSU crystals may result in urate nephropathy. The method of intrarenal injection did not induce surgical infection or systemic inflammatory response.

#1682 Myocardial remodeling in Wistar rats with renal dysfunction treated with a high-salt ration

Abstract Background and Aims High food intake of NaCl is the traditional factor of increase in blood pressure (BP) and cardiovascular risks also hurts kidneys. The purpose of the paper is to evaluate the morphological changes in the myocardium of normotensive Wistar rats with an early stage of renal dysfunction treated with a diet with increased concentration of salt for a long time. Method 30 Wistar rats (age 2.5-3 months) were studied and divided into three groups of 10 each. In the first, control, sham-operated (SO) rats, receiving standard diet (0.34% NaCl), in the second, rats subjected to resection of ¾ of the kidney parenchyma, receiving a standard diet (NE group), in the third, rats subjected to ¾ NE and those receiving a high-sodium diet (4% NaCl, high-salt diet, HSD). The feeds differed only in the %NaCl content. The ¾ NE model was created in two stages with an interval of 7 days. 20 mg/kg Zoletil 100 and 0.05 ml of Xylazine were used as anesthesia. At the first stage, the poles of the left kidney (1/2 part of the kidney) were removed, at the second stage, the right kidney was completely removed. The duration of the experiment was 4 months. At the end of the experiment, systolic BP at the tail was measured in awake rats using the cuff method (ELEMA electromanometer, Sweden). After removal from the experiment, blood was collected from the rats for biochemical analysis, and the LVMI was calculated as the ratio of LV mass to the body weight of the animal (mg/g). Myocardial fragments were placed in formalin, and sections 1.5–2 µm thick were prepared from paraffin blocks. The preparations were stained with Hematoxylin and Eosin and Masson's trichrome and used for histological analysis. The statistical significance of intergroup differences was determined using the Mann–Whitney test and Student's t test. Results During the observation period, BP in the SO group of animals did not change: initially - 120 [120;125] mm Hg, after 4 months - 120 [120;125] mmHg, p = 0.527. In the group with NE on a standard diet, an increase in BP was noted - from 120 [120; 125] mmHg up to 135.0 [132.5;137.5] mmHg, p = 0.011. In the group with NE + HSD, the increase in BP levels was more pronounced - up to 140[140;148] mmHg, (p = 0.001, relative to the SO group). The serum urea level in the SO group was 4.7 [4.5;5.4] mmol/l, creatinine −32.5 [29.0;36.0] µmol/l, in the NE group - 10.0 [9.1;10.9] mmol/l, (p = 0.0001, relative to the SO group) and 69.0[64.0;74.0] µmol/l, (p = 0.0001), in the NE+ HSD group – 10.7 [9.8;11.0] mmol/l, (p = 0.0001) and 64.5[46.3;69.5] µmol/l, (p = 0.001), respectively. Thus, the groups of rats with early-stage kidney dysfunction, although they had higher levels of urea and creatinine in the blood serum relative to the SO group, did not differ significantly from each other in these indicators. LVMI increased in rats with NE on a standard diet (LR −2.07±0.05 mg/g, NE −2.14 ± 0.06 mg/g, p = 0.1591). Consumption of a high-salt diet aggravated the growth of myocardial mass in rats with NE (NE + HSD - 2.41 ± 0.14 mg/g, p = 0.0084 and p = 0.0372, respectively). At the histological level, the increase in LV myocardial mass in rats with NE was characterized by hypertrophy of cardiomyocytes and the formation of interstitial fibrosis. The changes were more pronounced in the group with NE + HSD. The diameter of cardiomyocytes in rats of the SO group was 12.9 ± 0.4 µm, in animals with NE on standard diet - 14.5 ± 0.6 µm, in rats with NE + HSD - 15.5 ± 1.6 µm (p<0 .01 and p < 0.02, relative to the SO group). The area of interstitial fibrosis also increased in rats with NE (10.5 ± 2.6%, in the SO group - 0.6 ± 0.2%, p = 0.0000) and was slightly higher in the NE + HSD group (13.6 ± 2.8%, p = 0.0000, relative to the SO group). In rats with NE, both on a standard diet and with HSD, at this stage of the formation of kidney pathology, the thickness of the nutrimyocardial arteries did not differ significantly from the SO indicator rats. Conclusion Long-term salt intake with increased salt addition in Wistar rats in the initial stages of renal dysfunction does not affect the level of urea and creatinine in the blood serum, but promotes an increase in BP and myocardial remodeling, constantly progressing in the progression of cardiomyocyte hypertrophy and interstitial fibrosis.

#1515 Myocardial remodeling is associated with redistribution of phosphate, PIT1/ERK1/2 up-regulation and signaling pathways alterations in mild CKD model

Abstract Background and Aims Patients with CKD have significantly increased cardiovascular risk, and heart failure is a leading cause of morbidity and mortality in these patients. Myocardial hypertrophy is a hallmark of cardiac remodeling and dysfunction in CKD and is known to be mediated by angiotensin II, catecholamines, hyperphosphatemia, hyperparathyroidism, increase in FGF23, decrease in Klotho and alterations in various signaling pathways including calcineurin, mitogen-activated protein kinase ERK1/2, calmodulin-dependent protein kinase II etc. Less well understood are the mechanisms of fibrosis and intramyocardial arterial remodeling in CKD, which may be related to hyperphosphatemia and decreased renal and systemic Klotho. The aim of this study was to evaluate the molecular and cellular features of myocardial remodeling in experimental mild CKD. Method We induced CKD by 3/4 nephrectomy (Nx) in adult male spontaneously hypertensive rats (SHR) with a follow-up period of 4 months. Animals were fed standard chow containing 0.6% phosphate. Sham-operated Wistar rats and SHR served as controls. We measured serum creatinine, renal interstitial fibrosis area, systolic blood pressure (BP), myocardial mass index (MMI), histological indices of myocardial hypertrophy, interstitial fibrosis and intramyocardial artery (IA) remodeling (H&E, Masson's trichrome, von Kossa stain), myocardial phosphorus content, serum levels of inorganic phosphate (Pi), PTH and FGF23. Myocardial expression of phosphate transporters PiT1, ERK1/2, WNT, BMP, Notch, Hedgehog signaling pathways and LRG4 molecules was evaluated by PCR-RT, IHC and IF (Zeiss LSM 710; study performed at the Centre for Collective Use “Confocal Microscopy” of the Pavlov Institute of Physiology, Russian Academy of Sciences). Results Chronic kidney injury and phosphate regulatory factors did not differ, whereas BP and MMI were higher in sham SHR vs. Wistar rats (Fig. 1). Nx rats showed features of mild chronic kidney injury before the elevation of serum PTH, FGF23 levels (Fig. 1). Myocardial P content and serum Pi were higher in Nx and in the combined SHR group and directly correlated with indices of IA remodeling. Cardiomyocyte diameter, myocardial interstitial and perivascular fibrosis were higher in Nx (Figs 1, 2a) and accompanied with upregulation in Ppp3ca mRNA (calcineurin A, Fig. 2b), Mapk1 mRNA (ERK2, Fig. 2b), phospho-ERK1/2 (Figs 1, 3), PiT1 (Figs 1, 2c), Lgr4 (Fig. 2c) and downregulation of Hes1 mRNA (Fig. 2b). PiT1 and pospho-ERK1/2 were co-expressed in IA media from SHR (Fig. 2c). In Nx, nestin and LGR4 were expressed by IA adventitia and media cells, myocardial interstitial cells, and occasionally co-localized (Fig. 2c). Hes1 expression was detected in IA and capillary endothelial cells and co-localised with Gli1 in IA media; Gli1 was also detected in cardiomyocyte nuclei (Fig. 2c). In SHR, beta-catenin expression in cardiomyocytes was more pronounced in Nx (Fig. 2c). Conclusion Mild CKD is associated with histological and molecular features of cardiomyocyte hypertrophy, myocardial fibrosis and intramyocardial arterial remodeling, which are probably modulated by phosphate redistribution and its tissue retention involving upregulation of PiT1 and ERK1/2, as well as alterations in signals related to progenitor regulation and myocyte hypertrophy.

Branched-chain amino acids promote occurrence and development of cardiovascular disease dependent on triglyceride metabolism via activation of the mTOR/SREBP-1/betatrophin pathway

Branched-chain amino acid (BCAA) metabolism is associated with triglyceride (TG) metabolism and the development of cardiovascular disease (CVD). However, the underlying mechanism remains uncertain. This study included 1302 subjects and followed for 4–5 years. A hyperbranched-chain aminoacidemia rat model was induced by high fructose diet (HFTD). The relationship between BCAAs and TG level and its regulatory mechanism was investigated in vitro. As results, as baseline BCAA percentile increased, subjects had higher prevalence and incidence of T2DM, NAFLD, and CVD risk (P < 0.05). In animal model, the accumulation of BCAAs and TG and betatrophin expression were significantly elevated in the HFTD group when comparing with those in the SD group(P < 0.05). Immunofluorescence and Masson's trichrome staining revealed that the area of interstitial fibrosis was significantly increased in the HFTD group compared with control group. Met treatment significantly decreased TG levels and betatrophin expression and reversed myocardial fibrosis (P < 0.05). In vitro, LO2 cells, stimulated with 0.1–5 mM BCAAs, displayed a significant dose-dependent increase in betatrophin expression (P < 0.05). And 5 mM BCAAs stimulation significantly increased the p-mTOR and SREBP-1 expression (P < 0.05). However, this effect could be reversed by using the corresponding inhibitor or siRNAs. In conclusions, BCAAs promote occurrence and development of cardiovascular disease dependent on TG metabolism via activation of the mTOR/SREBP-1/betatrophin pathway. The study provides a new theory for the pathogenesis of CVD caused by amino acid metabolism disorders.

Chinese Medicine Supplementing Qi and Activating Blood Circulation Relieves the Progression of Diabetic Cardiomyopathy

Diabetic cardiomyopathy (DCM) is the leading cause of diabetic death as the final occurrence of heart failure and arrhythmia. Traditional Chinese medicine is usually used to treat various diseases including diabetes. This study sought to investigate the effects of Traditional Chinese medicine supplementing Qi and activating blood circulation (SAC) in DCM. After the construction of the DCM model by streptozotocin (STZ) injection and high glucose/fat diet feeding, rats were administered intragastrically with SAC. Then, cardiac systolic/diastolic function was evaluated by detecting left ventricular systolic pressure (LVSP), maximal rate of left ventricular pressure rise (+LVdp/dtmax), and fall (-LVdp/dtmax), heart rate (HR), left ventricular ejection fraction (EF), LV fractional shortening (FS) and left ventricular end-diastolic pressure (LVEDP). Masson’s and TUNEL staining were used to assess fibrosis and cardiomyocyte apoptosis. DCM rats exhibited impaired cardiac systolic/diastolic function manifested by decreasing LVSP, + LVdp/dtmax, -LVdp/dtmax, HR, EF and FS, and increasing LVEDP. Intriguingly, traditional Chinese medicine SAC alleviated the above-mentioned symptoms, indicating a potential role in improving cardiac function. Masson’s staining substantiated that SAC antagonized the increased collagen deposition and interstitial fibrosis area and the elevations in protein expression of fibrosis-related collagen I and fibronectin in heart tissues of DCM rats. Furthermore, TUNEL staining confirmed that traditional Chinese medicine SAC also attenuated cardiomyocyte apoptosis in DCM rats. Mechanically, DCM rats showed the aberrant activation of the TGF-β/Smad signaling, which was inhibited after SAC. SAC may exert cardiac protective efficacy in DCM rats via the TGF-β/Smad signaling, indicating a new promising therapeutic approach for DCM.

Inhibition of PFKP in renal tubular epithelial cell restrains TGF-β induced glycolysis and renal fibrosis

Metabolic reprogramming to glycolysis is closely associated with the development of chronic kidney disease (CKD). Although it has been reported that phosphofructokinase 1 (PFK) is a rate-limiting enzyme in glycolysis, the role of the platelet isoform of PFK (PFKP) in kidney fibrosis initiation and progression is as yet poorly understood. Here, we investigated whether PFKP could mediate the progression of kidney interstitial fibrosis by regulating glycolysis in proximal tubular epithelial cells (PTECs). We induced PFKP overexpression or knockdown in renal tubules via an adeno-associated virus (AAV) vector in the kidneys of mice following unilateral ureteral occlusion. Our results show that the dilated tubules, the area of interstitial fibrosis, and renal glycolysis were promoted by proximal tubule-specific overexpression of PFKP, and repressed by knockdown of PFKP. Furthermore, knockdown of PFKP expression restrained, while PFKP overexpression promoted TGF-β1-induced glycolysis in the human PTECs line. Mechanistically, Chip-qPCR revealed that TGF-β1 recruited the small mothers against decapentaplegic (SMAD) family member 3-SP1 complex to the PFKP promoter to enhance its expression. Treatment of mice with isorhamnetin notably ameliorated PTEC-elevated glycolysis and kidney fibrosis. Hence, our results suggest that PFKP mediates the progression of kidney interstitial fibrosis by regulating glycolysis in PTECs.

A diagnostically challenging case of Lymphoplasmacytic lymphoma involving the kidney clinically mimicking benign hypertension associated chronic kidney disease

Abstract Introduction/Objective Lymphomas involving the kidneys are under-recognized and often do not present clinically with signs and symptoms raising suspicion for a hematological malignancy. Worsening renal function may necessitate renal biopsy, but often the clinical picture may not warrant a biopsy. We herein present a case of lymphoplasmacytic lymphoma involving the kidney clinically mimicking benign renal vascular disease associated chronic kidney disease. Methods/Case Report Elderly male with past medical history of stage 3 chronic kidney disease presumed to be due to benign hypertension and undergoing treatment for the same presented to his nephrologist for routine follow up. His renal parameters were stable, but his high blood pressure seemed to be difficult to control despite maximum medical intervention. Upon the patient’s request to know exact etiology of his kidney problem, a renal biopsy was performed. Histomorphological evaluation revealed multifocal areas of renal parenchymal infiltration by atypical lymphocytic aggregates with background renal parenchyma showing moderate arteriosclerosis, mild interstitial fibrosis, tubular atrophy and no significant glomerulopathy. Further workup of the lymphoid aggregates led to the finding of a low grade CD5 negative, CD10 negative B-cell lymphoma with plasmacytic differentiation. Molecular testing for final characterization revealed a MYD88 mutation, which along with the immunomorphological findings confirmed the diagnosis of lymphoplasmacytic lymphoma (LPL). The patient was referred to hematology-oncology and was recommended to start chemotherapy due to renal involvement. Results (if a Case Study enter NA) n/a Conclusion In medical renal biopsies, lymphocytic infiltrates in areas of interstitial fibrosis are very common, and an atypical lymphocytic infiltrate can be easily overlooked. Pathologists evaluating these biopsies should have a high degree of suspicion for a hematological malignancy if they encounter atypical lymphocytic infiltrates in the renal parenchyma. Accurate diagnosis is essential, as renal lymphoma whether primary or metastatic, requires initiation of timely systemic chemotherapy.

Subcutaneous Infusion of DNA-Aptamer Raised against Advanced Glycation End Products Prevents Loss of Skeletal Muscle Mass and Strength in Accelerated-Aging Mice.

We have developed DNA aptamers that can inhibit the toxic effects of advanced glycation end products (AGE-Apts). We herein evaluated the effects of AGE-Apts on muscle mass and strength in senescence-accelerated mouse prone 8 (SAMP8) mice. Eight-month-old male SAMP8 mice received subcutaneous infusion of control DNA aptamers (CTR-Apts) or AGE-Apts. Mice in an age-matched senescence-accelerated mouse resistant strain 1 (SAMR1) group were treated with CTR-Apts as controls. The soleus muscles were collected after the 8-week intervention for weight measurement and histological, RT-PCR, and immunofluorescence analyses. Grip strength was measured before and after the 8-week intervention. AGE-Apt treatment inhibited the progressive decrease in the grip strength of SAMP8 mice. SAMP8 mice had lower soleus muscle weight and fiber size than SAMR1 mice, which was partly restored by AGE-Apt treatment. Furthermore, AGE-Apt-treated SAMP8 mice had a lower interstitial fibrosis area of the soleus muscle than CTR-Apt-treated SAMP8 mice. The soleus muscle levels of AGEs, oxidative stress, receptor for AGEs, and muscle ring-finger protein-1 were increased in the CTR-Apt-treated mice, all of which, except for AGEs, were inhibited by AGE-Apt treatment. Our present findings suggest that the subcutaneous delivery of AGE-Apts may be a novel therapeutic strategy for aging-related decrease in skeletal muscle mass and strength.

Early progression of chronic histologic lesions in kidney transplant biopsies is not associated with HLA histocompatibility.

Early progression of chronic histologic lesions in kidney allografts represents the main finding in graft attrition. The objective of this retrospective cohort study was to elucidate whether HLA histocompatibility is associated with progression of chronic histologic lesions in the first year post-transplant. Established associations of de novo donor-specific antibody (dnDSA) formation with HLA mismatch and microvascular inflammation (MVI) were calculated to allow for comparability with other study cohorts. We included 117 adult kidney transplant recipients, transplanted between 2016 and 2020 from predominantly deceased donors, who had surveillance biopsies at 3 and 12 months. Histologic lesion scores were assessed according to the Banff classification. HLA mismatch scores [i.e. eplet, predicted indirectly recognizable HLA-epitopes algorithm (PIRCHE-II), HLA epitope mismatch algorithm (HLA-EMMA), HLA whole antigen A/B/DR] were calculated for all transplant pairs. Formation of dnDSAs was quantified by single antigen beads. More than one-third of patients exhibited a progression of chronic lesion scores by at least one Banff grade in tubular atrophy (ct), interstitial fibrosis (ci), arteriolar hyalinosis (ah) and inflammation in the area of interstitial fibrosis and tubular atrophy (i-IFTA) from the 3- to the 12-month biopsy. Multivariable proportional odds logistic regression models revealed no association of HLA mismatch scores with progression of histologic lesions, except for ah and especially HLA-EMMA DRB1 [odds ratio (OR)=1.10, 95% confidence interval (CI) 1.03-1.18]. Furthermore, the established associations of dnDSA formation with HLA mismatch and MVI (OR=5.31, 95% CI 1.19-22.57) could be confirmed in our cohort. These data support the association of HLA mismatch and alloimmune response, while suggesting that other factors contribute to early progression of chronic histologic lesions.

#6508 THE EFFECTS AND MECHANISM OF BRAIN-TARGETING DRUG-LOADED EXOSOMES ON ADRIAMYCIN NEPHROPATHY MODEL

Abstract Background and Aims Chronic kidney disease (CKD) is a common clinical syndrome, and the current clinical efficacy is still limited. Our previous study confirmed that a small amount of losartan injected into the central lateral ventricle can effectively protect the kidneys, but the route of administration limits its clinical application. As a drug carrier, exosomes have several advantages and are expected to support drug delivery. Therefore, this study aims to develop a brain-targeting exosome complex to provide a new idea for the treatment of CKD. Method In this study, the brain-targeting exosome complex (ExoACP) was synthesized by co-cultivation. The markers of ExoACP were detected by immunoblotting. The structure and particle size distribution were observed by TEM and NTA. Angiotensin II receptor antagonist losartan (ExoACP@Los) was loaded on ExoACP by electroporation. The mice of Adriamycin (ADR)-induced nephropathy were divided into 5 groups: Control, ADR, ADR + Los (IG), ADR + ExoACP (iv.), ADR + ExoACP@Los (iv.). Corresponding treatment was given every other day from the 3rd week after modeling, and all mice were sacrificed at the 5th week, serum and renal tissue was collected for the following experiments. Results This study found that the exosome markers of ExoACP were highly expressed. The Exo and Ang-CP05-Exo were disc-like structures, and the size of the latter was larger than that of the former, suggesting that ExoACP was successfully modified on the surface of Exo. The modification efficiency of ExoACP on Exo was as high as 96.1%. Compared with free Exo, the distribution of ExoACP in brain was increased. Blood biochemical results showed that ExoACP and ExoACP@Los had no apparent side-effects on mice (Figure 1). In-vivo, ExoACP@Los treatment significantly improved the levels of kidney function in mice with ADR nephropathy. ExoACP@Los could significantly reduce renal tubular damage, interstitial fibrosis area and degree of glomerular sclerosis, suggesting that ExoACP@Los could improve the injury of renal tissue (Figure 2). Conclusion This study synthesized a drug-loaded exosome complex with brain targeting and good biocompatibility, and significantly improved renal function, renal tissue damage and collagen fiber deposition in the ADR model.

#4488 OLD BIOPSY IS ALWAYS GOLD, REVISING MEMBRANOPROLIFERATIVE GN DIAGNOSIS IN A RENAL TRANSPLANT PATIENT

Abstract Background and Aims MPGN resulting from hepatitis C virus (HCV) infection typically shows granular deposition of immunoglobulin M (IgM), C3, and both kappa and lambda light chains. Immunoglobulin G (IgG) may or may not be present, and C1q is typically negative, the same pathology can be found in other viral infections. The recurrence rate post-transplantation is high, with 33% of immune-complex MPGN. Graft loss is even higher 65%, especially among patients with idiopathic MPGN. Treatment of a patient with significant proteinuria can be tricky, as no proven treatment is described yet, but plasmapheresis and induction with cyclophosphamide were described for heavy proteinuria &amp;gt;3.5 g/day. Method 79 year old female patient, known case of live unrelated renal transplantation in December 2013, primary renal disease is unknown. She had a history of equivocal hepatitis C virus antibodies, but Hepatitis C virus RNA was not detected. She is also known to have Type II Diabetes Mellitus and hypertension. The patient had baseline creatinine of 100-120 micromol/L. 24 hr urine protein was 0.8 g/Day but started rising quickly to reach a peak of 9.2 g/g seven months post-transplantation to nephrotic range proteinuria but her serum albumin level was always above 35 g/L. Serum protein electrophoresis showed no M band, a kappa/ Lambda ratio 1.21 within a normal range (0.260-1.65). Anti- nuclear antibody (ANA), P-ANCA, C-ANCA, and Double-stranded DNA all were negative. Complements were within normal range C3 1.39 g/L (0.79 -1.52), C4 0.44 g/L(0.17-0.57)m rheumatoid factor and Anti- citrullinated peptide antibodies were negative. Hepatitis b Virus surface antigen and core antibodies were not reactive, HIV screening was negative. Urine analysis showed RBCs but no RBC casts. Renal biopsy showed mild mesangial expansion and mild l increase in mesangial cells and matrix. Some glomeruli show increased lobulation with increase in mesangial cells and matrix with segmental endocapillary proliferation There is double contour of the glomerular basement membrane with scattered spikes and holes within the basement membrane seen best with silver stain. There is mild increase in the thickness of glomerular basement membrane. There is focal mild lymphocytic infiltration. Tubulitis is not seen in the examined material. One Focal area of mild interstitial fibrosis. Sampled blood vessels are unremarkable. The stain for C4d is negative. Immunofluorescence showed granular capillary wall immunoreactivity to IgM (+2), C4 (+1), C1q (+1), and Lambda light chain (+2). The glomeruli show no immunoreactivity to IgG, IgA, C3, Kappa and fibrin. The glomeruli show (+) staining for albumin. Tubular protein reabsorption droplets showed immunoreactivity for albumin, IgG, IgM, C3, kappa and lambda light chains. Electron microscopy showed many subendothelial, and mesangial electron-dense deposits. The patient was on maintenance immunosuppression medications inform of mycophenolic acid 720 mg, cyclosporine; 250mg BID, and Prednisolone 5 mg daily.Revisiting the new classification of membranoproliferative glomerulonephritis is challenging in this patient, since there is the dominance of IgM deposition by immunofluorescence and C4 but not C3 makes it challenging, the negative monoclonal band in serum electrophoresis and negative autoimmune workup excludes immune complex mediate MPGN, leave idiopathic MPGN a possibility, keeping in mind that hepatitis C virus infection was never confirmed. On the other hand, IF showed IgM with C4 subendothelial and mesangial dense deposits that excludes away the C4 glomerulopathy. Results Whether this is a recurrence of glomerular disease early on post-transplantation or denovo membranoproliferative disease, she responded well to the addition of losartan 100 mg daily, with a reduction of her urinary protein to less than 1 g/g. the patient never received treatment for the hepatitis C virus. Conclusion In our patient, it is difficult to conclude if the cause of MPGN is recurrence or denovo GN, especially with severe proteinuria that responded well to ACEi treatment. The pathological findings can be tricky.

#4032 RAPAMYCIN AGGRAVATES KIDNEY LESIONS IN RATS FED HIGH PHOSPHATE

Abstract Background and Aims Rapamycin is currently used to prevent rejection after kidney transplantation. mTOR blockade by rapamycin may slow the progression of diabetic kidney disease. Rapamycin promotes phosphaturia by inhibiting renal tubular transport of phosphate. Excessive tubular load of phosphate produces kidney damage by inducing tubular injury and interstitial fibrosis. The aim of this work is to evaluate the effect of rapamycin on kidney pathology in rats fed different phosphate levels. Method Thirty four female Wistar rats were used in this experiment. Rats, aged 2 months at the beginning of the studies, were allotted to 6 groups (n = 4-6). Rats with intact renal function were fed a standard diet containing either nomal (0.6%, NP), low (0.2%, LP) or high (1.2%, HP) phosphate. Half of the groups received placebo treatment (NP, LP, HP) and the other half were treated with rapamycin (NP-Rapa, LP-Rapa, HP-Rapa) at a dose of 1.3 mg/kg/day, for 22 days. Plasma concentrations of urea, creatinine, and phosphate as well as urine creatinine and phosphate were measured by spectrophotometry. Microscopic evaluation of the kidneys was performed in tissue sections fixed with formaldehyde solution and embedded in paraffin. Kidney sections were stained with hematoxylin-eosin, Masson Trichrome, periodic acid Schiff, and Von Kossa staining. Thirty non overlapping fields were evaluated. Chronic interstitial fibrosis and renal calcification were measured with a point counting grid and the result was expressed in percentage. Atrophic tubules were counted and the result was expressed as number of atrophic tubuli/field. Values are expressed as the mean ± standard error (SE). The difference between means was assessed by ANOVA. Fisher LSD test was used as a post-hoc procedure. A correlation study was carried out using the Pearson test. p&amp;lt;0.05 was considered significant. Results Kidney lesions were more severe in rats treated with rapamycin and fed high phosphate diet. Areas of chronic interstitial fibrosis, with interstitial fibrosis and lymphoplasmacytic infiltrate, as well as atrophic tubules with thickened and wrinkled tubular basement membrane, loss of the brush border and flattening of the tubular cell cytoplasm, were observed. Calcification foci, mainly located in the corticomedullary junction, were identified with Von Kossa staining. Treatment with rapamycin did not influence renal function parameters in NP and LP rats but increased plasma creatinine and urea in HP rats. Plasma P concentration tended to decrease after rapamycin treatment. Fractional excretion of P was higher in all the groups treated with rapamycin, and the differences reached significance in HP-Rapa rats. Fractional excretion of P showed an excellent correlation with kidney lesions: chronic interstitial fibrosis (r = 0.822), calcification (r = 0.827) and tubular atrophy (r = 0.855). Conclusion The phosphaturic action of rapamycin aggravates renal lesions in rats fed high phosphate diets. Thus, rapamycin could be deleterious for the kidneys in the context of high phosphorus intake.

Cannabidiol alleviates right ventricular fibrosis by inhibiting the transforming growth factor β pathway in monocrotaline-induced pulmonary hypertension in rats

Cannabidiol (CBD) is a non-intoxicating compound of Cannabis with anti-fibrotic properties. Pulmonary hypertension (PH) is a disease that can lead to right ventricular (RV) failure and premature death. There is evidence that CBD reduces monocrotaline (MCT)-induced PH, including reducing right ventricular systolic pressure (RVSP), vasorelaxant effect on pulmonary arteries, and decreasing expression of profibrotic markers in the lungs. The aim of our study was to investigate the effect of chronic administration of CBD (10 mg/kg daily for 21 days) on profibrotic parameters in the RVs of MCT-induced PH rats. In MCT-induced PH, we found an increase in profibrotic parameters and parameters related to RV dysfunction, i.e. plasma pro-B-type natriuretic peptide (NT-proBNP), cardiomyocyte width, interstitial and perivascular fibrosis area, amount of fibroblasts and fibronectin, as well as overexpression of the transforming growth of factor β1 (TGF-β1), galectin-3 (Gal-3), suppressor of mothers against decapentaplegic 2 (SMAD2), phosphorylated SMAD2 (pSMAD2) and alpha-smooth muscle actin (α-SMA). In contrast, vascular endothelial cadherin (VE-cadherin) levels were decreased in the RVs of MCT-induced PH rats. Administration of CBD reduced the amount of plasma NT-proBNP, the width of cardiomyocytes, the amount of fibrosis area, fibronectin and fibroblast expression, as well as decreased the expression of TGF-β1, Gal-3, SMAD2, pSMAD2, and increased the level of VE-cadherin. Overall, CBD has been found to have the anti-fibrotic potential in MCT-induced PH. As such, CBD may act as an adjuvant therapy for PH, however, further detailed investigations are recommended to confirm our promising results.

Visualization of Activated Fibroblasts in Heart Failure with Preserved Ejection Fraction with [18F]AlF-NOTA-FAPI-04 PET/CT Imaging.

During the pathogenesis of heart failure with preserved ejection fraction (HFpEF), fibroblasts are activated and express the fibroblast activation protein (FAP). Targeted imaging of FAP can qualitatively and quantitatively assess the fibroblast activity. This study aimed to use [18F]AlF-NOTA-FAPI-04 (AlF = aluminum fluoride; NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid; FAPI = FAP inhibitor) positron emission tomography/computed tomography (PET/CT) imaging to detect activated fibroblasts in a rat HFpEF model. The rat HfpEF model was established by feeding a high-fat diet plus l-NAME (Nω-nitro-l-arginine methyl ester) for 10 weeks. Blood pressure, echocardiography, and [18F]AlF-NOTA-FAPI-04 PET/CT were used to assess the progression of HfpEF. The biodistribution of [18F]AlF-NOTA-FAPI-04 in healthy rats was obtained. Cardiac tissue sections were also analyzed using Masson's, hematoxylin and eosin (H&E), and FAP immunohistochemistry (IHC) staining. The echocardiography and blood pressure data indicated that the rat HfpEF model was successfully established. [18F]AlF-NOTA-FAPI-04 PET/CT imaging showed obvious radiotracer accumulation in the left ventricular wall of the HfpEF rats from the seventh week. A biodistribution test showed that the tracer was cleared mainly via renal and intestinal excretion. Percentage of injected dose per gram tissue (% ID) of the heart and its surrounding organs was lower in normal rats, which was conducive to image analysis. Masson's and H&E stainings showed large areas of vascular and interstitial fibrosis in the HfpEF rat hearts. IHC staining also confirmed the presence of FAP-positive cardiac fibroblasts of the HfpEF rat hearts, with a good correlation with FAPI PET. Thus, [18F]AlF-NOTA-FAPI-04 PET/CT is a promising and non-invasive method to assess the progression of fibrosis in HfpEF to facilitate the clinical management.

Clinical Relevance of Computationally Derived Attributes of Peritubular Capillaries from Kidney Biopsies.

The association between peritubular capillary (PTC) density and disease progression has been studied in a variety of kidney diseases using immunohistochemistry. However, other PTC attributes such as PTC shape have not been explored yet. The recent development of computer vision techniques provides the opportunity for the quantification of PTC attributes using conventional stains and whole slide images. To explore the relationship between PTC characteristics and clinical outcome, n=280 periodic acid Schiff-stained kidney biopsies (88 minimal change disease, 109 focal segmental glomerulosclerosis, 46 membranous nephropathy, and 37 IgA nephropathy) from the NEPTUNE digital pathology repository were computationally analyzed. A previously validated deep learning model was applied to segment cortical PTCs. Average PTC aspect ratio (the PTC major to minor axis ratio), size (PTC pixels per PTC segmentation), and density (PTC pixels per unit cortical area) were computed for each biopsy. Cox proportional hazards models were used to assess associations between these PTC parameters and outcome (40% eGFR decline or kidney failure). Cortical PTC characteristics, and interstitial fractional space PTC density were compared between areas of interstitial fibrosis and tubular atrophy (IFTA) and areas without IFTA. When normalized PTC aspect ratio was below 0.6, a 0.1 increase in normalized PTC aspect ratio was significantly associated with disease progression, with a hazard ratio (95% confidence interval) of 1.28 (1.04-1.59) (p=0.019), while PTC density and size were not significantly associated with outcome. Interstitial fractional space PTC density was lower in areas of IFTA compared to non-IFTA areas. Computational image analysis enables quantification of the status of the kidney microvasculature and the discovery of a previously unrecognized PTC biomarker (aspect ratio) of clinical outcome.

Effects of 1,25-(OH)2-D3 on Oxidative Stress and Inflammation of Contrast

Contrast-induced nephropathy (CIN) is a common complication of clinical diagnosis and treatment of coronary atherosclerotic heart disease. This study focused on the role of 1,25-(OH)2-D3 (D3) in contrast-induced renal tissue inflammation and oxidative stress. Thirty C57BL/6 mouse underwent unilateral renal pedicle ligation. One week after surgery, they were randomly divided into three groups, 10 mice in control group (CON group), 10 mice injected with iodixanol in contrast-induced nephropathy model group (CM group), 10 mice injected with contrast agent +in 1,25-(OH)2-D3 group (D3 group). The effects of 1,25-(OH)2-D3 on contrast nephropathy were studied by Hematoxylin-Eosin (HE) observation of kidney structure, detection of serum creatinine, urea nitrogen observation of kidney function, and Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-PCR). Under the light microscope, the renal tissue structure of the CM group was disordered, hypertrophic glomeruli, renal tubular cells were significantly edematous, and the interstitial fibrosis area was significantly increased. The serum creatinine (SCr) and blood urea nitrogen (BUN) in CM group were significantly increased compared with CON group, the antioxidant proteins GPX1 and GPX3 were significantly decreased, while the apoptosis proteins Bax and Caspase-3 were significantly increased, and the anti-apoptotic protein Bcl-2 was significantly decreased. The expression of inflammatory factors IL-1β, IL-6 and TNF-α was significantly increased. However, the D3 can effectively inhibit apoptosis through inhibiting oxidative stress and inflammatory response. In addition, the expression levels of Toll Like Receptor 4 (TLR4) and /NF-κB-P65 in the D3 group were lower than those in the CM group. It is indicated that 1,25-(OH)2-D3 has an inhibitory effect on TLR4/NF-κB signaling pathway. 1,25-(OH)2-D3 can reduce contrast-induced SCr and BUN and alleviate renal structural damage by inhibiting the TLR4/NF-κB pathway, which has an effect of reducing inflammation, increasing tissue antioxidant levels, and delaying cellular senescence.

Effect of Youthful Blood Environment and Its Key Stem Cell Factor on Renal Interstitial Fibrosis in Elderly Mice

Introduction: Youthful blood environment was shown to decelerate the aging process of the kidney and to attenuate senile renal fibrosis in a young-old parabiotic animal model; in addition, we identified a stem cell factor (SCF) that is closely linked with the process. This research was to investigate the effect of youthful blood environment on senile renal interstitial fibrosis and the role of SCF. Methods: We bred SCF receptor c-Kit gene loss-of-function Wps/Wps mice and established a combination mice model that was subjected to unilateral ureteral obstructive (UUO) and parabiotic surgeries. Parabiotic mice were divided into isochronic parabiotic (young-young [Y-IP] and old-old [O-IP]) and heterochronic parabiotic (young-old [HP]) groups. UUO surgery was performed in one of the parabiotic pairs in the IP group (Y-IPuuo and O-IPuuo) and in the elderly mice in the HP group (O-HPuuo). In order to study the role of SCF/c-kit on renal interstitial fibrosis, UUO surgery was performed in wildtype (WT) and Wps/Wps mice. Results: Fourteen days after UUO surgery, the kidney interstitial fibrosis area, kidney function, and the expressions of SCF/c-Kit, pNF-κB, and fibrosis-related proteins in the O-HPuuo group were significantly lower than those in the Ouuo and O-IPuuo groups. Compared with WT UUO mice, the expressions of pNF-κB and fibrosis-related proteins and the kidney function were all significantly decreased in Wps/Wps UUO mice. Conclusion: Youthful blood environment downregulated the expressions of SCF/c-Kit in elderly UUO mice and ameliorated UUO-induced kidney fibrosis and function loss.

Biochemical evidence of PM2.5 critical components for inducing myocardial fibrosis in vivo and in vitro.

PM2.5 constituents are tightly linked to the initiation of many cardiovascular diseases (CVDs). Little is known, however, about the events which critical components of PM2.5 can induce the initiating events in CVDs. C57BL/6 female mice were exposed to PM2.5 (3 mg/kg b.w.) from four different cities (Taiyuan, Beijing, Hangzhou, and Guangzhou) by oropharyngeal aspiration every other day. PM2.5 from Taiyuan increased the diastolic function of the hearts and induced myocardial fibrosis with increased areas of interstitial fibrosis through the NOX4/TGF-β1/Smad 3/Col1a1 pathways. Pb, Cr, Mn, Zn, and most of the polycyclic aromatic hydrocarbons (PAHs) were positively associated with the related indicators of cardiac diastolic function and myocardial fibrosis by using Pearson correlation (R2 = 0.9085-0.9897). To determine the critical components in PM2.5 that can induce the occurrence of myocardial fibrosis, BEAS-2b cells were treated with one or more of five candidate components with/without Guangzhou PM2.5, and then the conditioned medium of BEAS-2b was used to culture AC16 cells. The results showed that Zn + Pb + Mn + BaP with PM2.5 from Guangzhou exposure significantly increased reactive oxygen species production of BEAS-2b cells and induced a dramatic increase of myocardial fiber-related gene expression (Col1a1 and TGF-β) in AC16 cells. It indicated that the different mass concentrations of Zn, Pb, Mn, and ΣPAHs in PM2.5 might be the critical factors that modulated myocardial fibrosis induction by targeted. Our study provided a novel avenue for further elucidation of molecular mechanisms of PM2.5 components-induced myocardial fibrosis.

Effect of growth hormone on endometrium growth of intrauterine adhesion and the underlying mechanism.

The main treatment for intrauterine adhesion (IUA) is hysteroscopic adhesiolysis (HA), which most of treatment frequently employs estrogen and progesterone cycle therapy. The growth and coverage of endometrium after operation is a difficult problem, and several hospitals in China have performed growth hormone (GH) in empirically treating IUA, which has achieved excellent curative effects. Unfortunately, the mechanism of action has not yet been clearly elucidated. In previous study, an IUA animal model after surgical abortion and curettage in pregnant rats has been successfully established. In this experiment, the IUA animal model after surgical abortion and curettage in pregnant rats, which is more in line with the mechanism of human intrauterine adhesion, was used for the first time to investigate the therapeutic effect of GH on IUA in the pregnant rat curettage model. The expression of signal transducers and activators of transcription 3(STAT3), phosphorylated STAT3 (p-STAT3), STAT5 and p-STAT5 content were detected by immunohistochemistry to preliminarily explore the possible mechanism of GH involving in promoting endometrial growth of IUA, and to provide a theoretical basis for clinical medication and treatment. Pregnant rats were anesthetized, and the bilateral embryos were removed completely. Then the rat endometrium was scraped with a curette in 4 different directions (front, back, left, and right). After the IUA animal model was established, the rats were randomly divided into 3 groups (n=5): a control group, a GH group, and a GH + AG490 group. Normal saline (0.4 mL/100 g) was injected subcutaneously at the 7th day after curettage in the control group；0.15 U/100 g of GH was injected subcutaneously at the 7th day after curettage in the GH group; 0.15 U/100 g of GH was injected subcutaneously and 1 mg/100 g AG490 was injected intraperitoneally at the 7th day after curettage in the GH+ AG490 group. All the rats were injected continuously for 5 days. The rats in each group were sacrificed at the 14th day. The uterus of rats in each group was stained with HE staining to explore the endometrial morphology and the number of endometrial glands in each group, and Masson staining was utilized to observe the degree of endometrial fibrosis. The levels of STAT3, p-STAT3, STAT5 and p-STAT5 were detected by immunohistochemistry. 1) The number of glands in the GH group was more than that in the control group on the 14th day, with statistical difference (P<0.05). However, the number of endometrial glands in the AG490+GH group was decreased compared with the GH group on the 14th day (P<0.05). 2) The fibrosis ratio in the GH group was less than that in the control group at the 14th day after operation (P<0.05). However, the area of endometrial interstitial fibrosis in the AG490+GH group was much higher than that in the GH group 14 days after operation (P<0.05). 3) Compared with the control group, there was not significant difference in the levels of STAT3 and STAT5 in GH group (both P>0.05), while the levels of protein p-STAT3 and p-STAT5 were increased in the GH group (both P<0.05). Compared with the GH group, there was not significant difference in the levels of STAT3 and STAT5 in the AG490+GH group (both P>0.05), while the levels of p-STAT3 and p-STAT5 were decreased in the AG490+GH group (both P<0.05). GH can not only promote the growth of endometrial glands in the IUA model, but also reduce the degree of fibrosis and play a role in the treatment of IUA, which may be related to the activation of the Janus kinase (JAK), JAK/STAT3 and STAT5 signaling pathways.

Abstract 10279: Three-Dimensional Deep-Tissue Imaging of the Right Ventricle Reveals the Complex Remodeling of the Microvascular Network in Right Heart Failure

Introduction: Right ventricular (RV) failure is the leading cause of death in patients with pulmonary arterial hypertension (PAH). Capillary rarefaction has been proposed as the hallmark of RV failure, yet our understanding of the capillary architecture is limited by 2D image analysis. Three-dimensional (3D) confocal deep tissue imaging offers the unparalleled opportunity to characterize the architectural microvascular changes in relationship to cardiomyocytes in pressure-overloaded RV failure. Methods and Results: 250 μm heart tissue slices from mice with RV failure, 7-weeks after Pulmonary Artery Banding (PAB) were harvested and the microvascular network was assessed for density, segment length, diameter, orientation, as well as microvasculature-cardiomyocyte contact area in regions with and without interstitial fibrosis using confocal 3D imaging. Human heart tissue from end-stage PAH patients was compared to controls with normal RV function. In PAB mice, 3D imaging of the RV free wall revealed tortuous, shorter, thicker and higher-branched microvascular segments as well as development of fibrosis compared to Sham animals (Fig A). When corrected for fibrosis, the microvascular density was preserved even in advanced stages of RV failure. Physical contact between the microvasculature and cardiomyocytes was preserved in areas without fibrosis, yet significantly impaired in areas with interstitial fibrosis (Fig B), accompanied by an overexpression of β-myosin heavy chain suggesting local ischemia (Fig C). 3D imaging of human RV tissue of end-stage PAH patients revealed architectural microvascular remodeling that varied depending on etiologies and length of disease yet showed preserved microvascular density. (Fig. D). Conclusions: 3D deep tissue imaging revealed compensatory changes of the microvascular network to maintain a stable cardiomyocyte coverage in pressure-overloaded RV failure, which was impaired in areas of interstitial fibrosis.