Renal Interstitium Research Articles

P0482INTERSTITIAL HSP70 EXPRESSION AT DIAGNOSIS ASSOCIATES WITH LONG TERM RENAL SURVIVAL IN AN ANCA VASCULITIS COHORT

Abstract Background and Aims Heat shock proteins(HSP) present an evolving role in immune response. ANCA associated vasculitis(AAV) is often associated with kidney injury and progression to chronic kidney disease(CKD) Method Twenty nine patients with biopsy proven renal AAV were followed(1995-2015). Study end point was renal death. Renal tissue at diagnosis was stained with monoclonal antibodies against HSP60 and HSP70. Interstitial inflammatory cells and interstitial fibrosis graded were evaluated as mild(0-50%) or severe(≥50%). Healthy renal tissue served as control. Survival analysis(Kaplan-Meier), Fisher’s exact test, or Test Chi-Square and bivariate analysis were performed. Statistical significance was set at <0,05. Results Mean age and mean eGFR at baseline was 57,8±19.2 years and 24,47 ml/min/1,73m2 BSA respectively. Interstitial inflammatory infiltrates were present in most patients. Interstitial fibrosis was present in 10,3% of patients. Control biopsies showed a weak HSP expression. AAV-patients presented significantly higher HSP expression within crescents, proximal tubule, distal tubule and renal interstitium. Renal fibrosis associated with intense interstitial HSP70 expression(Pearson´s-R=0.521 p=0,004). Interstitial inflammatory infiltrates associated with increased distal tubular HSP60 expression(Pearson´s-R=0.672 p<0,001). Renal fibrosis associated with renal function loss(Mean time to renal replacement therapy 2.6 years, 95%CI 1.2-4.1 years, Log-Rank Chi-Square=4.33,p=0.038). Strong interstitial HSP70 expression associated with worsened renal survival in AAV-patients even in the absence of severe renal fibrosis(Mean time to renal replacement therapy initiation 0 years ; 95%CI 0, Log-Rank Chi-Square=11.45, p=0.001). Conclusion Interstitial HSP70 overexpression is associated with worse renal outcomes in renal AAV. To our knowledge this is the first study that shows an association of renal heat shock protein expression and renal outcomes in an AAV cohort.

P0377THE RELATIONSHIP BETWEEN SEVERITY OF INTERSTITIAL FIBROSIS AND ANEMIA IN PATIENT WITH PRIMARY GLOMERULONEPHRITIS: THE DATA FROM TSN-GOLD WORKING GROUP

Abstract Background and Aims Anemia is one of the most important complications in chronic renal disease and inadequate erythropoietin production is the most important reason for anemia. Fibrosis of renal interstitium may be associated with inadequate synthesis of erythropoietin and anemia. In our study, we investigated the relationship between the severity of interstitial fibrosis (IF) in patients with primary glomerulonephritis (PGN) and anemia parameters. Method A total of 2794 patients who were recorded to the database of the Turkish Society of Nephrology, Glomerular Diseases (TSN-GOLD) Working Group between May 2009 and June 2019 were included in our national multicenter (44 centers) study. Patients aged 16 years or more with documented biopsy were included in the study. Patients were divided into four groups according to IF severity. IF was not detected in the interstitial area, was defined as none, if fibrosis was present in less than 25% of the area, defined as mild, if fibrosis was present in 25-50%, defined as moderate and if more than 50%, defined as severe. Anemia was defined as hemoglobin <12 g/dl in adult female patients and <13 g/dl in adult male patients, according to Clinical Practice Guidelines for anemia of CKD published by The Kidney Disease: Improving Global Outcomes Foundation. The relationship between IF severity and anemia parameters at the time of biopsy was investigated. Results In the study, 57% of the patients were male and their mean age was 41.0 ± 14.3 years. The mean eGFR was 82.7 ± 36.9 ml / min / 1.73 m2 and the hemoglobin level was 13.1 ± 1.9 g/dl. General characteristics of patients with and without anemia are given in Table 1. While 41.1% of patients had no IF, 41.2% had mild, 14.1% had moderate and 3.5% had severe IF. When the patients were divided into four groups according to the severity of IF, the mean hemoglobin and hematocrit levels were significantly different between the groups (Figure 1). Anemia was present in 960 patients (34.4%) (Figure 2). Anemia rate was significantly higher in the patients with IF than patients without IF (36.8% vs 30.9%, p = 0.001). Anemia rate was %25.1 in patients with eGFH≥60 ml/min/1.73m2 and 56% in patients with eGFR<60 ml/min/1.73m2. Gender, serum albumin, eGFR, proteinuria, presence of diabetes, presence of nephrotic syndrome, and presence of IF were the independent variables in the regression analysis of hemoglobin-related factors (Table 2). Conclusion Approximately one-third of PGN patients had anemia at the time of biopsy and the frequency of anemia increases in patients with IF. The presence of IF is an independent risk factor associated with the development of anemia in PGN. The elucidation of the etiopathogenesis of anemia in PGN patients may play an important role in the follow-up and treatment of patients. Studies on IF can play a critical role.

COVID-19 nephropathy: probable mechanisms of kidney failure

The mechanistic understanding of signaling pathways that contributes to the kidney involvement by coronavirus disease 2019 (COVID-19) is an extremely critical point for improving therapeutic options. These pathways consist of the production of pro-inflammatory factors, the infiltration of pro-inflammatory cells into the renal interstitium, the activation of C5b-9 complexes, and receptorangiotensin converting enzyme 2 (ACE2). Cytopathogenic effects and invasion into renal tubular cells have been confirmed.

Eosinophilic peritonitis and nephrotic syndrome in Kimura\u2019s disease: a case report and literature review

BackgroundEosinophilic peritonitis is a relatively rare entity. Kimura’s disease is a rare chronic inflammatory disorder of unknown etiology, characterized by subcutaneous nodules mainly in the head and neck region, regional lymphadenopathy and occasional involvement of kidney. There is currently no report of eosinophilic peritonitis in Kimura’s disease.Case presentationA 44-year-old Chinese man presented with abdominal distention, nausea, vomiting and edema in lower limbs for 1 month. Laboratory data showed elevated eosinophils in peripheral blood and ascites, nephrotic syndrome with progressively renal dysfunction, and elevated IgE. Ultrasonography of lymph nodes showed multiple lymphadenopathy in bilateral inguinal regions. Surgical excision was performed for one of the enlarged lymph nodes and histopathology revealed diagnosis of Kimura’s disease. Renal biopsy indicated focal segmental glomerulosclerosis (FSGS) and acute tubulointerstitial nephritis with infiltration of eosinophils in renal interstitium. The patient was prescribed with oral prednisolone therapy (30 mg/day), and underwent continuous ambulatory peritoneal dialysis (CAPD). The peripheral and peritoneal eosinophil count decreased rapidly and normalized within 2 days. Forty-five days after prednisolone therapy, partial remission of nephrotic syndrome and decrease of serum creatinine were achieved while peritoneal dialysis dosage had decreased. Inguinal lymph nodes gradually shrunk in size. The overall conditions remain stable afterwards.ConclusionsThis rare case highlighted the clinical conundrum of a patient presenting with eosinophilic peritonitis, lymphadenopathy, nephrotic syndrome and renal failure associated with Kimura’s disease. The remarkable eosinophilia, pathology of lymph node and kidney, as well as significant response to steroids should guide towards the diagnosis.

Beyond a Passive Conduit: Implications of Lymphatic Biology for Kidney Diseases.

The kidney contains a network of lymphatic vessels that clear fluid, small molecules, and cells from the renal interstitium. Through modulating immune responses and via crosstalk with surrounding renal cells, lymphatic vessels have been implicated in the progression and maintenance of kidney disease. In this Review, we provide an overview of the development, structure, and function of lymphatic vessels in the healthy adult kidney. We then highlight the contributions of lymphatic vessels to multiple forms of renal pathology, emphasizing CKD, transplant rejection, and polycystic kidney disease and discuss strategies to target renal lymphatics using genetic and pharmacologic approaches. Overall, we argue the case for lymphatics playing a fundamental role in renal physiology and pathology and treatments modulating these vessels having therapeutic potential across the spectrum of kidney disease.

GRAPE SEED EXTRACT VERSUS CAPTOPRIL IN AMELIORATING 5-FLUOROURACIL-INDUCED NEPHROTOXICITY IN ADULT MALE ALBINO RATS: BIOCHEMICAL, HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY

Background: 5-Fluorouracil (5-FU) is one of the extensively used chemotherapeutic agents for different human malignancies. It has severe side effects and is considered a nephrotoxic agent. Captopril is one of the angiotensin-converting enzyme inhibitors (ACEI) used in treatment of hypertension and congestive heart failure. It is also an effective radical scavenger and antioxidant due to its free thiol group. Grape seeds extract is one of the effective radical scavengers with antioxidant, anti-proliferative and anti-inflammatory properties. Objective: The present study was designed to assess the probable protective role of captopril and grape seeds extract against 5-FU-induced nephrotoxicity in adult male albino rats. Materials and Methods: Forty eight adult male albino rats were divided into 8 equal groups; Group I kept as the control group, Group II (grape seeds consumed group), received aqueous grape seeds extract (500 mg/kg/day) orally by gastric tube. Group III (captopril group), injected intraperitoneally by captopril solution (60 mg/kg) once daily. Group IV (grape seeds & captopril), Group V (5-Fluorouracil group), injected intraperitoneally by 5-FU solution (20 mg/kg) once daily. Group VI (grape seeds/5-FU), Group VII (captopril/5-FU) and Group VIII (grape seeds & captopril/5-FU). Treatment was continued at the same time daily for 4 weeks. At the end of the experiment, final body weight, blood urea nitrogen (BUN), creatinine (Cr) and uric acid levels were measured. Also, specimens of right kidneys were taken for histological and immunohistochemical studies with anti-inducible nitric oxide synthase (iNOS). Results: 5-FU resulted in a significant reduction in final body weight and a significant elevation of (BUN), (Cr) and uric acid levels. Moreover, glomerular and tubular distortion, vacuolated epithelial lining, intraluminal acidophilic hyaline casts, congested peritubular capillaries, interstitial inflammatory infiltration, and increased fibrosis within the renal interstitium. The immunohistochemical expression of iNOS supported kidney impairment in the 5-FU group. Treatment with either grape seeds or captopril revealed some improvement in the final body weight, biochemical markers and histological changes. The best effect was encountered when grape seeds and captopril were combined. Conclusion: and Recommendation: Grape seeds and captopril succeeded in ameliorating 5-FU induced nephrotoxicity with a better effect by their combination. So, it is recommended to use grape seeds extract and captopril during chemotherapy with 5 fluorouracil to reduce its renal toxicity.

GRAPE SEED EXTRACT VERSUS CAPTOPRIL IN AMELIORATING 5-FLUOROURACIL-INDUCED NEPHROTOXICITY IN ADULT MALE ALBINO RATS: BIOCHEMICAL, HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY

Background: 5-Fluorouracil (5-FU) is one of the extensively used chemotherapeutic agents for different human malignancies. It has severe side effects and is considered a nephrotoxic agent. Captopril is one of the angiotensin-converting enzyme inhibitors (ACEI) used in treatment of hypertension and congestive heart failure. It is also an effective radical scavenger and antioxidant due to its free thiol group. Grape seeds extract is one of the effective radical scavengers with antioxidant, anti-proliferative and anti-inflammatory properties. Objective: The present study was designed to assess the probable protective role of captopril and grape seeds extract against 5-FU-induced nephrotoxicity in adult male albino rats. Materials and Methods: Forty eight adult male albino rats were divided into 8 equal groups; Group I kept as the control group, Group II (grape seeds consumed group), received aqueous grape seeds extract (500 mg/kg/day) orally by gastric tube. Group III (captopril group), injected intraperitoneally by captopril solution (60 mg/kg) once daily. Group IV (grape seeds & captopril), Group V (5-Fluorouracil group), injected intraperitoneally by 5-FU solution (20 mg/kg) once daily. Group VI (grape seeds/5-FU), Group VII (captopril/5-FU) and Group VIII (grape seeds & captopril/5-FU). Treatment was continued at the same time daily for 4 weeks. At the end of the experiment, final body weight, blood urea nitrogen (BUN), creatinine (Cr) and uric acid levels were measured. Also, specimens of right kidneys were taken for histological and immunohistochemical studies with anti-inducible nitric oxide synthase (iNOS). Results: 5-FU resulted in a significant reduction in final body weight and a significant elevation of (BUN), (Cr) and uric acid levels. Moreover, glomerular and tubular distortion, vacuolated epithelial lining, intraluminal acidophilic hyaline casts, congested peritubular capillaries, interstitial inflammatory infiltration, and increased fibrosis within the renal interstitium. The immunohistochemical expression of iNOS supported kidney impairment in the 5-FU group. Treatment with either grape seeds or captopril revealed some improvement in the final body weight, biochemical markers and histological changes. The best effect was encountered when grape seeds and captopril were combined. Conclusion: and Recommendation: Grape seeds and captopril succeeded in ameliorating 5-FU induced nephrotoxicity with a better effect by their combination. So, it is recommended to use grape seeds extract and captopril during chemotherapy with 5 fluorouracil to reduce its renal toxicity.

Endothelin Receptor A and B Co‐operate to Mediate GPER‐induced Natriuresis

G protein‐coupled estrogen receptors (GPER) are membrane‐associated receptors that mediate rapid estrogenic signaling. We recently found that activation of GPER in the renal medulla of female rats, but not males, evokes endothelin‐dependent natriuresis. Endothelin‐1 (ET‐1) inhibits tubular reabsorption of Na+ via stimulating ET receptor B (ETB). Evidence points that ET receptor A (ETA) may contribute to natriuresis in female rats only. The current study was designed to identify whether GPER‐induced natriuresis is mediated via activation of renal medullary ETA and/or ETBreceptors in female rats. We determined the effect of stimulating GPER in the renal medulla on urine flow and urinary Na+ excretion in anesthetized female ETB deficient rats (ETB def) and transgenic (TG) controls. We infused saline to the renal medullary interstitium during an equilibration period of 60–80 min at a flow rate of 500 μl/h, followed by vehicle, then the GPER selective agonist, G1 (5 pmol/kg/min). Infusion of G1 to the renal medulla increased urine flow and Na+excretion in TG control animals (from 4.77 ± 1.19 to 7.36 ± 2.41 μl/min and from 0.47 ± 0.09 to 0.91 ± 0.27 μmol/min, respectively, n=7, p<0.05). Similarly, medullary GPER activation evoked a diuretic and natriuretic response in ETB def rats (urine flow: from 4.07 ± 0.47 to 6.52 ± 1.01 μl/min and Na+ excretion: from 0.29 ± 0.06 to 0.64 ± 0.16 μmol/min, respectively, n=8, p<0.05). G1 infusion did not change blood pressure or urinary K+ excretion in ETB def or TG control animals. Mean arterial blood pressure was higher in ETB def rats, compared to TG controls (117 ± 6 vs. 99 ± 3 mmHg, n=7–8, p<0.05). We also determined the physiological response to medullary GPER activation in anesthetized female Sprague Dawley rats after individual blockade of ETA or ETB receptors, achieved by an intravenous bolus injection of ABT‐627 (5mg/kg) or A‐192621 (10 mg/kg), respectively, or vehicle. Neither selective antagonism of ETA nor ETB antagonism impacted the diuretic or natriuretic actions of G1. However, simultaneous blockade of ETA and ETB receptors abolished G1‐evoked diuresis and natriuresis. Blood pressure and urinary K+ excretion remained unchanged during G1 infusion in the presence or absence of ET receptor antagonists. Thus, using pharmacological and genetic approaches, our study reveals that ETA and ETB receptors in the renal medulla work co‐operatively to mediate the diuretic and natriuretic response to GPER activation.Support or Funding InformationFunded by AHA 18CDA34110010 and K99DK119413 to EYG and P01 HL136267 to DMP.

The role of matrix metalloproteinases and their inhibitors in physiopathological processes in children with kidney diseases

The review of studies of international and national researchers on the impact of matrix metalloproteinases (matrix metalloproteinases, ММРs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) on physiological and pathological processes in children with kidney diseases. It is shown that MMPs play a significant role in organogenesis, i.e. nephrogenesis. Thus, MMP-2, MMP-9 and TIMPs play a large role in basal membranes remodeling associated with epithelial structures in a developing kidney. Immunohistochemistry assay showed that MMP-2 was localized in structures of immature nephrons undergoing epithelial differentiation, MMP-9 – only in vascular structures included in immature glomeruli. Decreased activity of MMP and /or increased synthesis of TIMPs in nephrocytes contribute to the reduction of intercellular substance components catabolism and serve as a basis for glomerular fibrosis and renal interstitium. MMPs are the major group of proteases which regulate metabolism in extracellular matrix and serve as the most important parameters in tissue remodeling observed in acute and chronic inflammatory processes in kidneys. The literature review gives an opportunity to assess the importance of drug design preventing and delaying the progression of nephrosclerosis.

Coronavirus-nephropathy; renal involvement in COVID-19

Renal disturbances by coronavirus disease 2019 (COVID-19), consisted of acute kidney injury, due to acute tubular necrosis induced by sepsis, hydration, cytokine storm syndrome, rhabdomyolysis and hypoxia. As the direct cytopathic effect of virus on various renal cells has been detected in previous studies, direct virus invasion to the renal tubular cells and interstitium or glomeruli is possible. Previous studies showed that coronavirus enters into the cells by angiotensin-converting enzyme II receptors that are extensively presented in the renal cells. Further, acute kidney injury in COVID-19 is strongly associated with higher mortality and morbidity and is an indicator for survival with Coronavirus infection. In the overall approach to patients with COVID-19 infection, special attention should be paid to control of classical risk factors of kidney injury.

SAT-393 The profile of biopsy-proven renal tubulointerstitial lesions in patients with Glomerular disease

Renal tubules and interstitium are vulnerable to injury and play a central role in the progression of various CKDs. However, high quality study on the profiles of biopsy-proven tubulointerstitial lesions (TIL) is extremely limited. We conducted a retrospective renal biopsy series including 62,521native biopsies at 1211 hospitals across China from 2015 to 2017. The TILs, including the shedding of tube epithelial, renal tubular atrophy, renal interstitial fibrosis, edema and inflammatory infiltration, were identified from the pathological report. We analyzed the severity and chronicity of TILs stratified by gender, age groups, biopsy indications, and concurrent glomerular diseases. We also examined the effect of intrarenal arterial lesion on the risk of TIL. Of the 56,701 patients with exact pathological diagnosis, 79.95% of the patients had tubulointerstitial lesions, renal interstitial inflammatory infiltration was the most common type of TIL (77.70%), followed by renal tubular atrophy (56.36%) and renal interstitial fibrosis (27.84%). Severe and chronic TIL was more common in adults than in children. The prevalence of moderate to severe TIL was 74.14%, 26.00%, and 18.84% in patients with diabetic nephropathy (DN), IgA nephropathy (IgAN) and focal segmental glomerulosclerosis (FSGS), respectively. Similarly, patients with DN, IgAN, focal segmental glomerulosclerosis (FSGS) were more likely to have chronic TIL. Moderate to severe TIL was more common in the south of China. After adjusting for age, sex, hospital level, region, biopsy indication and type of concurrent glomerular diseases, patients with renal arteriole injury had a six-fold higher risk of TIL (OR 7.22, 95% CI, 6.48 to 8.05). In this large, multicenter renal biopsy series, the type and severity of TILs varied with age, region and concurrent glomerular diseases. Renal arteriole injury was associated with a significantly increased risk of TIL.

SUN-432 HEMATOLOGIC MALIGNANCIES IN RENAL BIOPSIES- EXPERIENCE IN AN ACADEMIC CENTER

It is rare to have involvement of renal biopsies done for glomerulonephritis by hematologic malignancies. We present 4 diverse cases of hematologic malignancies with histologic and immunohistochemical evidence of renal involvement. These cases highlight the importance of a thorough evaluation of cellular infiltrates in renal biopsies along with clinical history. Electronic records for consecutive native and transplant renal biopsies from year 2015 to 2018 were reviewed. We noted 4 cases with non-myeloma malignancies. These included B-cell lymphomas and one case of acute myeloid leukemia. On evaluation, patients were noted to have systemic involvement. Routine evaluation of the kidney showed the renal tubulo-interstitium to be involved by B-cell lymphomas including CLL/SLL, mantle cell lymphoma and diffuse large B-cell lymphoma. In one case myeloid leukemic blasts were noted. Image from patient 4, shows presence of myeloid blasts in the renal biopsy that stain with CD34 and myeloperoxidase (MPO).Patient 4 : Myeloid blasts on H+E stain with large nuclei and prominent nucleoli. The blasts stain with CD34 and MPO. Key clinical and biopsy findings are presented below in Table 1. This case series demonstrates that malignant infiltration of the renal interstitium may result in acute kidney injury. The commonest causes are low-grade non-Hodgkin's lymphoma and acute lymphocytic leukemia. Renal biopsy findings may sometimes direct evaluation for an unsuspected hematologic malignancy, possibly allowing for earlier detection and improving patient outcome.

JAK/STAT signaling controls the fate of CD8+CD103+ tissue-resident memory T cell in lupus nephritis

Autoimmune mediated inflammation and renal damage in lupus nephritis (LN) depends partly on the infiltration of lymphocytes in glomeruli and renal interstitium. Here we identified a population of CD8+ T cells with a CD103+-phenotype in the healthy kidneys of human and mouse. These cells were typically CD69+CD103+ tissue-resident memory T cells (TRM) in the kidney. CD8+ TRM cells were expanded in the kidneys of patients with LN or MRL/lpr mice. The expansion of renal CD8+ TRM cells correlated significantly with kidney disease activity. These cells were active in producing cytokines, perforin and granzyme B in the kidney of MRL/lpr mice. Importantly, renal CD8+ TRM cells underwent proliferation and self-renewal to maintain a stable TRM pool in the kidney of MRL/lpr mice, contributing to renal inflammation and damage. JAK/STAT signaling in the MRL/lpr mice was required for renal TRM self-renewal as well as maintenance of effector functions. Targeting JAK/STAT signaling by tofacitinib effectively suppressed effector functions and impaired the survival of renal TRM cells in the kidney, contributing to improved kidney function in MRL/lpr mice. These results provided evidences that renal CD8+ TRM cells play a role in the pathogenesis of LN. They could serve as a therapeutic target for LN.

Renal leukocyte chemotactic factor 2 (ALECT2)-associated amyloidosis in Chinese patients

Background: Leukocyte chemotactic factor 2 (ALECT2) amyloidosis is one of the recently described types of amyloidosis. In this study, we reported the first large case series of renal ALECT2 amyloidosis in Chinese patients.Methods: We studied the prevalence, clinical characteristics, renal pathology, outcome and genetic features among seven patients diagnosed with renal ALECT2 amyloidosis at Peking University First Hospital of China from 2000 to 2018.Results: Seven patients were diagnosed with ALECT2 amyloidosis, representing 1.9% of the renal biopsy-proven amyloidosis cases. The mean age at diagnosis was 68 years without gender preference. The patients mainly manifested with varying impaired kidney function with a mean estimated glomerular filtration rate of 42.7 mL/min/1.73 m2 (range 8.0–80.5) and proteinuria at 3.9 g/24 h (range 0.4–11.3). There were four ALECT2 amyloidosis patients with concurrent membranous nephropathy (MN), who presented a higher proteinuria (6.4 ± 4.0 g/24 h vs. 2.0 ± 1.8 g/24 h) and higher frequency of nephrotic syndrome (50% vs. 0) than patients with isolated ALECT2 amyloidosis. Renal biopsy showed strongly congophilic amyloid deposits distributed mainly in the renal cortical interstitium, as well as the glomerular mesangium, the inner layer of glomerular basement membrane (GBM), and vascular walls. Two patients with concurrent ALECT2 amyloidosis and MN showed mild amyloid deposits, which have not been identified as ALECT2 amyloidosis by IHC and LMD/MS methods. All patients were corroborated by immunoelectron microscopy to exhibit the specific location of LECT2 in the amyloid fibrils. Genetic analysis revealed no mutations but homozygosity for the G allele encoding valine at position 40 in the mature protein in all patients. Except for one patient who died 8 years later after he was diagnosed with ALECT2 amyloidosis, the others presented with relatively stable renal function during the mean follow-up period of 12.5 months.Conclusions: ALECT2 amyloidosis was the third most common type of renal amyloidosis in Chinese patients from a single centre. The majority of ALECT2 amyloidosis patients were of Han ethnicity, with a high rate of concurrent MN. The recognition and accurate diagnosis of renal ALECT2 amyloidosis should be considered in Chinese patients.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Tubule-derived lactate is required for fibroblast activation in acute kidney injury.

Acute kidney injury (AKI) is a highly prevalent medical syndrome associated with high mortality and morbidity. Several types of cells, including epithelial cells, vascular endothelial cells, pericytes, and macrophages, participate in the development of AKI. Recently, renal fibroblasts were found to play an important role in the regulation of tubular injury, repair, and recovery after AKI. However, the mechanisms underlying fibroblast activation and proliferation during the progression of AKI remain unclear. In the present study, we found many activated myofibroblasts located in the renal interstitium with an abundance of extracellular matrix deposition following folic acid-induced AKI. The proliferative pattern of tubular epithelial cells and interstitial cells following acute injury was different, indicating that the proliferation of fibroblasts followed the proliferation of tubular epithelial cells. Furthermore, we observed that proliferative tubular epithelial cells preferred aerobic glycolysis as the dominating metabolic pathway in the progression of AKI. Lactate generated from injured tubules was taken up by interstitial fibroblasts in the later stages of AKI, which induced fibroblast activation and proliferation in vitro. Early inhibition of lactate production in tubules by glycolytic inhibitors suppressed fibroblast activation after folic acid-induced injury. Collectively, these results support the important role of fibroblasts in the development of AKI and suggest that lactate produced by glycolysis in tubular epithelial cells is a novel regulator of fibroblast activation and proliferation.

Deletion of FHL2 in fibroblasts attenuates fibroblasts activation and kidney fibrosis via restraining TGF-β1-induced Wnt/β-catenin signaling.

Four-and-a-half LIM domains protein 2 (FHL2) has been proposed involving in β-catenin activity. We previously reported that FHL2 mediates TGF-β1-induced tubular epithelial-to-mesenchymal transition through activating Wnt/β-catenin signaling. However, the potential role and mechanism for FHL2 in TGF-β1-induced fibroblast activation and kidney fibrosis remains unknown. Here, we initially observed higher levels of FHL2 expression in fibrotic kidneys from both patients and mice, especially in α-smooth muscle actin (α-SMA)-positive cells in the interstitium. In cultured interstitial fibroblasts, FHL2 expression was induced by TGF-β1. Knockdown of FHL2 remarkably suppressed TGF-β1-induced α-SMA, type I collagen, and fibronectin expression, while overexpression of FHL2 was sufficient to activate fibroblasts. In mice, fibroblast-specific deletion of FHL2 diminished renal induction of α-SMA, type I collagen, and fibronectin and interstitial extracellular matrix deposition at 2weeks after ureteral obstruction. We next investigated Wnt/β-catenin activity and found that β-catenin was activated in most FHL2-positive cells in renal interstitium from mice with obstructive nephropathy. In vitro, TGF-β1 induced a physical interaction between FHL2 and β-catenin, especially in the nucleus. Downregulation of FHL2 inhibited TGF-β1-induced active β-catenin upregulation, β-catenin nuclear translocation, and β-catenin-mediated transcription, whereas overexpression of FHL2 was able to activate Wnt/β-catenin signaling. FHL2 overexpression-induced β-catenin-mediated gene transcription could be hindered by ICG-001, but FHL2 overexpression-induced upregulation of active β-catenin could not be. Collectively, this study reveals that the signal regulatory effect of FHL2 on β-catenin plays an important role in TGF-β1-induced fibroblast activation and kidney fibrosis.

Management of Drug-Associated Acute Interstitial Nephritis.

Drug-associated acute interstitial nephritis (AIN) is characterized by acute or subacute loss of kidney function, interstitial infiltrates, edema, and tubulitis on kidney biopsy, with a relative sparing of the glomerulus and vasculature (1). Estimates indicate that AIN is the primary finding in 2%–5% of all native kidney biopsies. In those biopsied for acute kidney dysfunction, the frequency of AIN is closer to 10%–30%, although the true burden of AIN likely remains underestimated (1). Due to procedural risk, biopsies are often deferred in favor of empirical discontinuation of the suspected offending agent. Several AIN etiologies have been identified, but the majority of cases (60%–70%) have been linked to drugs and toxins. Drug-associated AIN is thought to be a cell-mediated immune response, akin to a type 4 (delayed-type) hypersensitivity reaction (2). Mechanistic explanations for AIN include that drugs could act as haptens to modify the endogenous response to native renal proteins or induce an autoimmune reaction to the tubular basement membrane through molecular mimicry. Drugs could also rarely lead to systemic immune activation that contributes to deposition or sequestration of immune complexes in the renal interstitium (2,3). As early as 7 days after drug exposure, the early inflammatory lesions in AIN can begin to evolve into irreversible interstitial fibrosis. This likely explains why, even with the best available management, only 40%–50% of patients with AIN experience complete renal recovery (4). As more and diverse cases of drug-associated AIN have been reported, it is apparent that the “classic triad” of extrarenal manifestations—which include fever, rash, and eosinophilia—only occur in about 10% of patients (5,6). Rather, drug-associated AIN has a highly variable presentation. In 133 cases of biopsy-confirmed AIN (95 of which were drug-related), fever, rash, and eosinophilia individually occurred in 20%–23% of patients. Microscopic hematuria, sterile pyuria, and …

Generating Kidney Organoids from Human Pluripotent Stem Cells Using Defined Conditions.

The ultimate goal of regenerative medicine is to have access to an unlimited supply of specific cell types on demand, which can be used as effective therapies for a wide range of intractable disorders. With the availability of human pluripotent stem cells (hPSCs) and greatly improved protocols for their directed differentiation into specific cell types, including kidney, this prospect could soon become a reality. We have previously described the generation of kidney organoids from hPSCs. This chapter describes our latest differentiation protocol for generating kidney tissue, which uses a cost-effective and completely defined, xeno-free medium. As with our previous protocol, these complex, multicellular three-dimensional structures are composed of all anticipated kidney cell types including nephrons segmented into the glomerulus, proximal and distal tubule as well as an extensive endothelial network, and renal interstitium. As such, kidney organoids provide useful tools for understanding human development, disease modeling, drug screening/toxicology studies and tissue engineering applications, and may facilitate the development of transplantable hPSC-derived kidney tissue for regenerative medicine purposes in the future.

Hirudin Ameliorates Renal Interstitial Fibrosis via Regulating TGF-β1/Smad and NF-κB Signaling in UUO Rat Model.

Purpose Hirudin, a polypeptide structure containing 65 amino acids, is a potent natural thrombin inhibitor with anticoagulant property extracted from Hirudo medicinalis. It has been reported to have anti-inflammatory and antifibrotic property. Here we explored the renoprotective effect of hirudin on unilateral ureteral obstruction (UUO) induced renal interstitial fibrosis (RIF). Methods Rats were randomly divided into five groups: sham group, UUO alone group, and three UUO + hirudin-treatment groups (10, 20, or 40 IU/kg/d, for 14 continuous days). At the end of the experiment period, animals were sacrificed. Pathologic changes in renal specimens were observed using hematoxylin and eosin (HE) staining and Masson staining. The expressions of collagen III (Col III), fibronectin (FN), α-smooth muscle actin (α-SMA), protease-activated receptor 1 (PAR-1), and proteins in the TGF-β1/Smad and NF-κB pathways in renal tissues were examined by immunohistochemistry and/or Western blotting. Results HE and Masson staining showed that hirudin-treated UUO rats had lower extent of renal injury and deposition of extracellular matrix (ECM) in renal interstitium than those in the UUO group. The results of immunohistochemistry and WB indicated decreased protein expressions of Col III, FN, α-SMA, PAR-1, and inflammatory markers such as tumor necrosis factor-α and interleukin-6 after hirudin treatment. Furthermore, hirudin reduced the expressions of transforming growth factor β1 (TGF-β1), phosphorylated-Smad2, and phosphorylated-Smad3 in the UUO model. In parallel, we found inhibited nuclear factor-κB (NF-κB) signaling after hirudin treatment, with downregulated protein expressions of P65, phosphorylated-P65, and phosphorylated-iκBα and increased iκBα. Conclusion Hirudin improves kidney injury and suppresses inflammatory response and ECM accumulation in UUO rats; its underlying mechanism may be associated with the inhibition of TGF-β1/Smad and NF-κB signaling.