Determination of Anti-Phospholipase A2 and Anti-Thrombospondin Type 1 Domain-Containing Protein 7A in Latin Patients with Membranous Nephropathy.

Primary membranous nephropathy (MN) is caused by antibodies against podocyte antigens, especially the type M receptor of phospholipase A2 (PLA2R) and thrombospondin type-1 domain containing 7 A (THSD7A). This study's aim was the determination of anti-PLA2R, anti-THSD7A serum antibodies, and anti-PLA2R renal tissue staining prevalence in a Latin population with MN, as well as evaluating their role as biomarkers for disease activity. The performance of the two anti-PLA2R serum diagnostic methods-ELISA and indirect immunofluorescence (IFI)-was evaluated for the diagnosis of MN. Fifty-nine patients, including 29 with MN, 18 with lupus membranous nephropathy (LMN) and 12 with focal and segmental glomerulosclerosis (FSGS), were evaluated for serum antibodies. Renal biopsies were also evaluated for the presence of anti-PLA2R staining. Twenty-one patients with MN were followed for 1 year. Patients with LMN and FSGS were negative for both antibodies. All 29 MN patients were negative for anti-THSD7A; 16 MN patients were positive for anti-PLA2R by ELISA and/or IFI, and 3 MN patients were positive for anti-PLA2R only by IFI. Thus, the anti-PLA2R ELISA test demonstrated 45% sensitivity and 97% specificity, while the IFI test showed, respectively, 55% and 100% in our MN patients. Among the 28 MN renal biopsies, 20 presented anti-PLA2R positive staining, corresponding to a 72% sensitivity. Positive correlations were observed between the anti-PLA2R ELISA titer and proteinuria. In conclusion, determination of anti-PLA2R antibodies in the MN Latin population showed similar rates to those reported for other populations. The anti-PLA2R serum levels correlated with MN disease activity.

Pulmonary and systemic involvement in COVID-19 patients assessed with ultrasound-guided minimally invasive autopsy.

AIMS: Brazil ranks high in the number of coronavirus disease 19 (COVID-19) cases and the COVID-19 mortality rate. In this context, autopsies are important to confirm the disease, determine associated conditions, and study the pathophysiology of this novel disease. The aim of this study was to assess the systemic involvement of COVID-19. In order to follow biosafety recommendations, we used ultrasound-guided minimally invasive autopsy (MIA-US), and we present the results of 10 initial autopsies. METHODS AND RESULTS: We used MIA-US for tissue sampling of the lungs, liver, heart, kidneys, spleen, brain, skin, skeletal muscle and testis for histology, and reverse transcription polymerase chain reaction to detect severe acute respiratory syndrome coronavirus 2 RNA. All patients showed exudative/proliferative diffuse alveolar damage. There were intense pleomorphic cytopathic effects on the respiratory epithelium, including airway and alveolar cells. Fibrinous thrombi in alveolar arterioles were present in eight patients, and all patients showed a high density of alveolar megakaryocytes. Small thrombi were less frequently observed in the glomeruli, spleen, heart, dermis, testis, and liver sinusoids. The main systemic findings were associated with comorbidities, age, and sepsis, in addition to possible tissue damage due to the viral infection, such as myositis, dermatitis, myocarditis, and orchitis. CONCLUSIONS: MIA-US is safe and effective for the study of severe COVID-19. Our findings show that COVID-19 is a systemic disease causing major events in the lungs and with involvement of various organs and tissues. Pulmonary changes result from severe epithelial injury and microthrombotic vascular phenomena. These findings indicate that both epithelial and vascular injury should be addressed in therapeutic approaches.

Metformin arrests the progression of established kidney disease in the subtotal nephrectomy model of chronic kidney disease.

Metformin, an AMP-activated protein kinase (AMPK) activator, has been shown in previous studies to reduce kidney fibrosis in different models of experimental chronic kidney disease (CKD). However, in all of these studies, the administration of metformin was initiated before the establishment of renal disease, which is a condition that does not typically occur in clinical settings. The aim of the present study was to investigate whether the administration of metformin could arrest the progression of established renal disease in a well-recognized model of CKD, the subtotal kidney nephrectomy (Nx) model. Adult male Munich-Wistar rats underwent either Nx or sham operations. After the surgery (30 days), Nx rats that had systolic blood pressures of >170 mmHg and albuminuria levels of >40 mg/24 h were randomized to a no-treatment condition or to a treatment condition with metformin (300 mg·kg-1·day-1) for a period of either 60 or 120 days. After 60 days of treatment, we did not observe any differences in kidney disease parameters between Nx metformin-treated and untreated rats. However, after 120 days, Nx rats that had been treated with metformin displayed significant reductions in albuminuria levels and in markers of renal fibrosis. These effects were independent of any other effects on blood pressure or glycemia. In addition, treatment with metformin was also able to activate kidney AMPK and therefore improve mitochondrial biogenesis. It was concluded that metformin can arrest the progression of established kidney disease in the Nx model, likely via the activation of AMPK.

Mesenchymal Stromal Cells Induce Podocyte Protection in the Puromycin Injury Model.

Podocytes are specialized cells with a limited capacity for cell division that do not regenerate in response to injury and loss. Insults that compromise the integrity of podocytes promote proteinuria and progressive renal disease. The aim of this study was to evaluate the potential renoprotective and regenerative effects of mesenchymal stromal cells (mSC) in a severe form of the podocyte injury model induced by intraperitoneal administration of puromycin, aggravated by unilateral nephrectomy. Bone derived mSC were isolated and characterized according to flow cytometry analyses and to their capacity to differentiate into mesenchymal lineages. Wistar rats were divided into three groups: Control, PAN, and PAN+ mSC, consisting of PAN rats treated with 2 × 105 mSC. PAN rats developed heavy proteinuria, hypertension, glomerulosclerosis and significant effacement of the foot process. After 60 days, PAN rats treated with mSC presented a significant amelioration of all these abnormalities. In addition, mSC treatment recovered WT1 expression, improved nephrin, podocin, synaptopodin, podocalyxin, and VEGF expression, and downregulated proinflammatory Th1 cytokines in the kidney with a shift towards regulatory Th2 cytokines. In conclusion, mSC administration induced protection of podocytes in this experimental PAN model, providing new perspectives for the treatment of renal diseases associated with podocyte damage.

Pathogenic role of angiotensin II and the NF-κB system in a model of malignant hypertensive nephrosclerosis.

We previously reported that rats treated with an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), during lactation developed hypertension in adult life, without apparent functional or structural damage to kidneys, providing a new model of essential hypertension. Here, we investigated whether uninephrectomy associated with salt overload would unveil a latent renal dysfunction in this model, aggravating arterial hypertension and promoting renal injury. Male Munich-Wistar rat pups received PDTC from maternal milk (PDTCLact) from 0 to 20 days after birth. Another group received no treatment during lactation. All offspring underwent uninephrectomy (UNx) at 10 weeks of age and then were subdivided into NS, receiving a normal salt (0.5% Na+) diet, PDTCLact + NS, HS, receiving a high-salt diet (2% Na+ chow + 0.5% saline to drink), and PDTCLact+HS. Twelve weeks later, HS rats were moderately hypertensive with mild albuminuria and renal injury. In contrast, severe hypertension, glomerulosclerosis, and cortical collagen deposition were prominent in PDTCLact + HS animals, along with "onion-skin" arteriolar lesions, evidence of oxidative stress and intense renal infiltration by macrophages, and lymphocytes and angiotensin II-positive cells, contrasting with low circulating renin. The NF-κB pathway was also activated. In a separate set of PDTCLact+HS rats, Losartan treatment prevented NF-κB activation and strongly attenuated glomerular injury, cortical fibrosis, and renal inflammation. NF-κB activity during late nephrogenesis is essential for the kidneys to properly maintain sodium homeostasis in adult life. Paradoxically, this same system contributed to renal injury resembling that caused by malignant hypertension when renal dysfunction caused by its inhibition during lactation was unmasked by uninephrectomy associated with HS.

Klotho deficiency aggravates sepsis-related multiple organ dysfunction.

Sepsis-induced organ failure is characterized by a massive inflammatory response and oxidative stress. Acute kidney injury (AKI) occurs in approximately half of patients in septic shock, and the mortality associated with sepsis-induced AKI is unacceptably high. Klotho is a protein expressed by renal cells and has anti-senescence properties. Klotho has also been shown to protect the kidneys in ischemia-reperfusion injury and to have antioxidant properties. To analyze the role of Klotho in sepsis-related organ dysfunction and AKI, we used a cecal ligation and puncture (CLP) model of sepsis in heterozygous Klotho-haploinsufficient mice and their wild-type littermates (CLP- Kl/+ and CLP-WT mice, respectively). In comparison with the CLP-WT mice, CLP- Kl/+ mice showed lower survival, impaired renal function, impaired hepatic function, greater oxidative stress, upregulation of inflammatory pathways (at the systemic and kidney tissue levels), and increased NF-κB activation. It is noteworthy that CLP- Kl/+ mice also showed lower heart-rate variability, less sympathetic activity, impaired baroreflex sensitivity to sodium nitroprusside, and a blunted blood pressure response to phenylephrine. We also demonstrated that sepsis creates a state of acute Klotho deficiency. Given that low Klotho expression exacerbates sepsis and multiple organ dysfunction, Klotho might play a protective role in sepsis, especially in elderly individuals in whom Klotho expression is naturally reduced.

Innate And Adaptive Immunity are Progressively Activated in Parallel with Renal Injury in the 5/6 Renal Ablation Model.

The mechanisms triggering renal inflammation in chronic kidney disease (CKD) are unclear. We performed a detailed analysis of the time course of innate and adaptive immunity activation in the 5/6 renal ablation (Nx) model. Munich-Wistar rats undergoing Nx were studied 15, 60 and 120 days after ablation. Hypertension, albuminuria, creatinine retention, interstitial expansion and infiltration by macrophages and T-lymphocytes were already evident 15 days after Nx. PCR-array was used to screen for altered gene expression, whereas gene and protein expressions of TLR4, CASP1, IL-1ß and NLRP3 were individually assessed. Tlr4, Tlr5, Lbp, Nlrp3, Casp1, Irf7 and Il1b were already upregulated 15 days after Nx, while activation of Tlr2, Tlr7, Tlr9, Nod2, Tnf and Il6 was seen after 60 days post-ablation. The number of genes related to innate or adaptive immunity grew steadily with time. These observations indicate that parallel activation of innate and adaptive immunity antecedes glomerular injury and involves a growing number of intricate signaling pathways, helping to explain the difficulty in detaining renal injury in Nx as CKD advances, and, stressing the need for early treatment. Additionally, these findings may contribute to the search of therapeutic targets specific for advanced phases of CKD.

Caloric Restriction Is More Efficient than Physical Exercise to Protect from Cisplatin Nephrotoxicity via PPAR-Alpha Activation.

The antineoplastic drug cisplatin promotes renal injury, which limits its use. Protocols that reduce renal cisplatin toxicity will allow higher doses to be used in cisplatin treatment. Here, we compare physical exercise and caloric restriction (CR) as protocols to reduce cisplatin renal injury in mice. Male C57BL/6 were divided into four groups: Control, cisplatin, exercise + cisplatin, and 30% CR + cisplatin. Animals were injected with a single dose of cisplatin (20 mg/kg i.p.) and sacrificed 96 h after injection. Quantitative real time PCR, histological analyses, immunohistochemistry, and biochemical measurements were performed to investigate renal injury, necrosis, apoptosis, and inflammatory mechanisms. Both protocols protected against cisplatin renal injury, but CR was more effective in reducing uraemia and renal necrosis. The CR + Cisplatin group exhibited reduced serum IL-1ß and TNF-α levels. No differences were noted in the renal mRNA expression of cytokines. Both interventions reduced apoptosis, but only the CR + Cisplatin group decreased TNFR2 protein expression. PPAR-α was activated in mice after CR. An antagonist of PPAR-α blocked the protective effect of CR. Both interventions attenuated the nephrotoxicity caused by cisplatin injection, but CR + Cisplatin showed a better response by modulating TNFR2. Moreover, part of the CR benefit depends on PPAR-α activation.

Catheter-based induction of renal ischemia/reperfusion in swine: description of an experimental model.

Several techniques to induce renal ischemia have been proposed: clamp, PVA particles, and catheter-balloon. We report the development of a controlled, single-insult model of unilateral renal ischemia/reperfusion (I/R) without contralateral nephrectomy, using a suitable model, the pig. This is a balloon-catheter-based model using a percutaneous, interventional radiology procedure. One angioplasty balloon-catheter was placed into the right renal artery and inflated for 120 min and reperfusion over 24 h. Serial serums were sampled from the inferior vena cava and urine was directly sampled from the bladder throughout the experiment, and both kidneys were excised after 24 h of reperfusion. Analyses of renal structure and function were performed by hematoxylin-eosin/periodic Acid-Schiff, serum creatinine (SCr), blood urea nitrogen (BUN), fractional excretion of ions, and glucose, SDS-PAGE analysis of urinary proteins, and serum neutrophil gelatinase-associated lipocalin (NGAL). Total nitrated protein was quantified to characterize oxidative stress. Acute tubular necrosis (ATN) was identified in every animal, but only two animals showed levels of SCr above 150% of baseline values. As expected, I/R increased SCr and BUN. Fractional sodium, potassium, chloride, and bicarbonate excretion were modulated during ischemia. Serum-nitrated proteins and NGAL had two profiles: decreased with ischemia and increased after reperfusion. This decline was associated with increased protein excretion during ischemia and early reperfusion. Altogether, these data show that the renal I/R model can be performed by percutaneous approach in the swine model. This is a suitable translational model to study new early renal ischemic biomarkers and pathophysiological mechanisms in renal ischemia.