Prothrombin Knockdown Protects Podocytes and Reduces Proteinuria in Glomerular Disease.

Chronic kidney disease (CKD) is a leading cause of death, and its progression is driven by glomerular podocyte injury and loss, manifesting as proteinuria. Proteinuria includes urinary loss of coagulation zymogens, cofactors, and inhibitors. Importantly, both CKD and proteinuria significantly increase the risk of thromboembolic disease. Prior studies demonstrated that anticoagulants reduced proteinuria in rats and that thrombin injured cultured podocytes. Herein we aimed to directly determine the influence of circulating prothrombin on glomerular pathobiology. We hypothesized that (pro)thrombin drives podocytopathy, podocytopenia, and proteinuria. Glomerular proteinuria was induced with puromycin aminonucleoside (PAN) in Wistar rats. Circulating prothrombin was either knocked down using a rat-specific antisense oligonucleotide or elevated by serial intravenous infusions of prothrombin protein, which are previously established methods to model hypo- (LoPT) and hyper-prothrombinemia (HiPT), respectively. After 10 days (peak proteinuria in this model) plasma prothrombin levels were determined, kidneys were examined for (pro)thrombin co-localization to podocytes, histology, and electron microscopy. Podocytopathy and podocytopenia were determined and proteinuria, and plasma albumin were measured. LoPT significantly reduced prothrombin colocalization to podocytes, podocytopathy, and proteinuria with improved plasma albumin. In contrast, HiPT significantly increased podocytopathy and proteinuria. Podocytopenia was significantly reduced in LoPT vs. HiPT rats. In summary, prothrombin knockdown ameliorated PAN-induced glomerular disease whereas hyper-prothrombinemia exacerbated disease. Thus, (pro)thrombin antagonism may be a viable strategy to simultaneously provide thromboprophylaxis and prevent podocytopathy-mediated CKD progression.

Clinical Evaluation and Management of Thrombotic Microangiopathy.

Thrombotic microangiopathy (TMA) refers to a diverse group of diseases that share clinical and histopathologic features. TMA is clinically characterized by microangiopathic hemolytic anemia, consumptive thrombocytopenia, and organ injury that stems from endothelial damage and vascular occlusion. There are several disease states with distinct pathophysiological mechanisms that manifest as TMA. These conditions are associated with significant morbidity and mortality and require urgent recognition and treatment. Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome are traditionally considered to be primary forms of TMA, but TMA more commonly occurs in association with a coexisting condition such as infection, pregnancy, autoimmune disease, or malignant hypertension, among others. Determining the cause of TMA is a diagnostic challenge because of limited availability of disease-specific testing. However, identifying the underlying etiology is imperative as treatment strategies differ. Our understanding of the conditions that cause TMA is evolving. Recent advances have led to improved comprehension of the varying pathogenic mechanisms that drive TMA. Development of targeted therapeutics has resulted in significant improvements in patient outcomes. In this article, we review the pathogenesis and clinical features of the different TMA-causing conditions. We outline a practical approach to diagnosis and management and discuss empiric and disease-specific treatment strategies.

Kidney Biopsy Corner: Amyloidosis.

Amyloidosis is an infiltrative disease caused by misfolded proteins depositing in tissues. Amyloid infiltrates the kidney in several patterns. There are, as currently described by the International Society of Amyloidosis, 14 types of amyloid that can involve the kidney, and these types may have different locations or clinical settings. Herein we report a case of AA amyloidosis occurring in a 24-year-old male with a history of intravenous drug abuse and provide a comprehensive review of different types of amyloids involving the kidney.

Correlation of Red Blood Cell Casts With Renal Dysfunction in Patients With Infection-related Glomerulonephritis.

CONTEXT.­: Infection-related glomerulonephritis (IRGN) usually manifests as a proliferative immune-complex glomerulonephritis. The degree of renal dysfunction at presentation can vary. Association with histologic features on kidney biopsy remains unknown. OBJECTIVE.­: To study the correlation between renal function in IRGN at the time of biopsy and the severity of histologic features. DESIGN.­: Culture-proven IRGN cases at our facility were included and divided based on estimated glomerular filtration rate (eGFR) 15 ml/min/1.73 m2. Patients' demographic and pathologic findings were obtained from electronic medical records and kidney biopsy reports. RESULTS.­: In total, 104 cases were diagnosed with IRGN on biopsy (mean age, 55.6 ± 15.6 years; male, n = 79 [76%]; median eGFR, 14.5 mL/min/1.73 m2), and 51 of 104 showed eGFR <15 mL/min/1.73 m2. Among all the histologic features assessed, only percent tubules with red blood cell (RBC) casts showed statistical difference, being significantly higher in the lower eGFR group (P = .004). Multivariable logistic regression analysis also showed that %tubules with RBC casts were associated with lower eGFR (odds ratio, 1.12; 95% CI, 1.01-1.24; P = .01). Patients with 5% or more RBC casts (n = 31) showed a lower eGFR (P = .02) and a higher %cellular crescent (P < .001) compared with those with less than 5% RBC casts. Patients with concomitant anticoagulant therapy (n = 11) showed higher percentages of RBC casts than those without anticoagulants (P = .02). CONCLUSIONS.­: Particular attention to the extent of RBC casts on kidney biopsy is recommended in patients with IRGN because these portend worse renal dysfunction, more so in patients requiring anticoagulation (including for hemodialysis) because they are especially vulnerable to developing anticoagulant-related nephropathy.

N-acetylcysteine ameliorates hematuria-associated tubulointerstitial injury in 5/6 nephrectomy mice.

Chronic kidney disease (CKD) is characterized by increased interstitial fibrosis and tubular atrophy (IFTA) in the kidney. Chronic hematuria is a hallmark of several human kidney diseases and often is seen in patients on anticoagulation therapy. We had previously demonstrated that chronic hematuria associated with warfarin increases IFTA in 5/6 nephrectomy (5/6NE) rats, and such treatment increases reactive oxygen species (ROS) in the kidney. The goal of this study was to evaluate the effects of the antioxidant N-acetylcysteine (NAC) on the progression of IFTA in 5/6NE mice. 5/6NE C57BL/6 and 5/6NE 129S1/SvImJ mice were treated with warfarin alone or with warfarin and NAC for 23 weeks. Serum creatinine (SCr), hematuria, blood pressure (BP), and ROSs in the kidney were measured; kidney morphology was evaluated. Warfarin doses were titrated to achieve prothrombin time (PT) increase to the levels seen with therapeutic human doses. Warfarin treatment resulted in an increased SCr, systolic BP, hematuria, expression of TGF-ß and ROS in the kidney in both mouse strains. Tumor necrosis factor alpha (TNF-ɑ) levels in the serum were increased in 5/6NE mice treated with warfarin. IFTA was increased as compared with control 5/6NE mice, and this increase in IFTA was more prominent in 129S1/SvImJ than in C57BL/6 mice. NAC ameliorated the warfarin-associated increase in SCr and BP but not hematuria. IFTA, TGF-ß, and ROS in the kidney as well as TNF-ɑ levels in the serum were reduced in mice treated with NAC and warfarin as compared to mice treated with warfarin alone. NAC mitigates the increase in SCr and IFTA in mice with chronic hematuria by reducing oxidative stress in the kidney. This data open novel possibilities for treatments in CKD patients.

Isolated arterial mucoid intimal thickening lesion in early post-transplant kidney allograft biopsies.

INTRODUCTION: Thrombotic microangiopathy (TMA) on kidney biopsy shows a variable combination of features: arterial mucoid intimal thickening, acellular closure of glomerular capillary loops, fragmented red blood cells, fibrin thrombi, and arterial fibrinoid necrosis. However, some early post-transplant kidney biopsies show only arterial mucoid intimal thickening. We aimed to elucidate the importance of this finding. METHODS: We identified 19 biopsies showing isolated arterial mucoid intimal thickening and compared them with 22 bona fide TMA biopsies identified based on the pathological findings (excluding rejection) (2011-2020). Additionally, delayed graft function (DGF) (n = 237), and no DGF (control, n = 1314) groups were included for survival analysis. RESULTS: Seven of 19 cases with isolated arterial mucoid intimal thickening showed peripheral blood schistocytes but no other systemic features of TMA. Eight patients underwent adjustments in maintenance immunosuppression (mainly calcineurin inhibitors). None of the cases progressed to full-blown TMA on consecutive biopsies. The overall and death-censored graft survival rates in this group were comparable to the DGF group, but significantly better than the TMA group (P = .005 and .04, respectively). CONCLUSIONS: Isolated arterial mucoid intimal thickening in early post-transplant biopsies may be an early/mild form of TMA, probably requiring adjustment in immunosuppressive regimen. Careful exclusion of known causes of TMA, and donor-derived arterial injury are important.

The Diagnostic Conundrum of Glomerular Crescents With IgA Deposits.

Introduction: Glomerulonephritis (GN) with crescents and IgA deposits in kidney biopsy poses a frequent diagnostic and therapeutic dilemma because of multiple possibilities. Methods: Native kidney biopsies showing glomerular IgA deposition and crescents (excluding lupus nephritis) were identified from our biopsy archives between 2010 and 2021. Detailed clinicopathologic features were assessed. One-year clinical follow-up on a subset of cases was obtained. Results: A total of 285 cases were identified, and these clustered into IgA nephropathy (IgAN, n = 108), Staphylococcus or other infection-associated GN/infection-related GN (SAGN/IRGN, n = 43), and antineutrophil cytoplasmic antibody-associated GN (ANCA-GN, n = 26) based on a constellation of clinicopathologic features, but 101 cases (group X) could not be definitively differentiated. The reasons have been elucidated, most important being atypical combination of clinicopathologic features and lack of definitive evidence of active infection. Follow-up (on 72/101 cases) revealed that clinicians' working diagnosis was IgAN in 43%, SAGN/IRGN in 22%, ANCA-GN in 28%, and others in 7% of the cases, but treatment approach varied from supportive or antibiotics to immunosuppression in each subgroup. Comparing these cases as "received immunosuppression" versus "non-immunosuppression," only 2 features differed, namely C3-dominant staining, and possibility of recent infection (both higher in the no-immunosuppression group) (P < 0.05). Renal loss was higher in the non-immunosuppression subgroup, but not statistically significant (P = 0.11). Conclusion: Diagnostic overlap may remain unresolved in a substantial number of kidney biopsies with glomerular crescents and IgA deposits. A case-by-case approach, appropriate antibiotics if infection is ongoing, and consideration for cautious immunosuppressive treatment for progressive renal dysfunction may be needed for best chance of renal recovery.

Triple hit to the kidney-dual pathological crescentic glomerulonephritis and diffuse proliferative immune complex-mediated glomerulonephritis: A case report.

BACKGROUND: Anti-glomerular basement membrane (GBM) disease is a rare rapidly progressive glomerulonephritis, frequently associated with alveolar hemorrhage in the lungs and involving the kidney by crescentic glomerulonephritis. It has been described in association with other glomerulonephritides [such as anti-neutrophilic antibody (ANCA)-glomerulonephritis, membranous nephropathy, and immunoglobulin (Ig)A nephropathy]. CASE SUMMARY: Herein we present an unusual case of concurrent anti-GBM disease, ANCA-associated crescentic glomerulonephritis and diffuse proliferative immune complex mediated glomerulonephritis with predominant staining for IgA and C3 by immunofluorescence. The patient is a 46-year-old Caucasian male who presented to the emergency department with acute onset of flank pain and was found to have high serum creatinine levels of 15 mg/dL, proteinuria, and hematuria. He rapidly deteriorated and became anuric. He was found to have high anti-GBM antibodies titers (151 units) and high anti-neutrophil cytoplasmic-ANCA. Despite prompt and early treatment, the patient's condition worsened, and he succumbed to his illness. CONCLUSION: Our case emphasizes the importance of a renal biopsy in anti-GBM disease, even in the presence of positive serum anti-GBM antibodies, to identify other potential causes of rapidly progressive glomerulonephritis. The challenge in treating such cases lies in the different therapy modalities.

Genomic Study on Blood Culture Isolates From Patients With Staphylococcus Infection-associated Glomerulonephritis.

Introduction: Staphylococcus infection-associated glomerulonephritis (SAGN), is an autoimmune sequela of infection affecting a subset of infected patients without specific predictive factors, frequently presenting with acute nephritic syndrome and propensity for chronic kidney disease. We performed a comparative genotypic and phenotypic analysis of S. aureus isolates from patients that did and those that did not develop SAGN. Methods: We had 22 culture-proven cases of SAGN from Ohio State University Wexner Medical Center (OSUWMC) from 2004 to 2016, 9 of 22 being blood cultures, with archived isolates. These, along with blood culture isolates from 12 patients with no clinical evidence of SAGN (between ages 40 to 80 years) over the same period were used for genotyping. For host demographic comparison, we used all available SAGN cases (n = 85, including those with positive cultures other than blood; and patients with kidney biopsies received from referring hospitals) and all OSUWMC patients with positive Staphylococcus cultures without glomerulonephritis (GN) (n = 23,496). Results: Multiple sequence types (STs) suggesting strain diversity was seen in the GN isolates with mainly clonal complexes (CC) 5 and 59. Mutations in the agr operon were identified in significantly higher number of the GN isolates (83%) than non-GN isolates (16%). Significant differences in ß-hemolysis and biofilm formation was also observed between the groups. Conclusion: The functionality of these agr mutants remains to be seen, but the presently known effects of reduced agr function, namely increased surface adhesins, biofilm formation, and persistent bacteremia could be important microbial factors predisposing to SAGN and testing for them early during infection could help to predict its development.

Role of protease-activated receptor-1 (PAR-1) in the glomerular filtration barrier integrity.

Protease-activated receptors (PAR) play an important role in the regulation of cellular function by the coagulation system, and they are activated by thrombin. PAR-1 is expressed in both endothelial cells and podocytes in the kidney. The role of PAR1 in the maintenance of the glomerular filtration barrier is not clear. Anticoagulant-related nephropathy (ARN) is a kidney disease with glomerular hematuria and red blood cell tubular casts. We validated 5/6 nephrectomy (5/6NE) in rats as a model of ARN and had demonstrated that direct thrombin inhibitor (dabigatran) induces ARN. The aim of this study was to investigate the role of PAR-1 in the ARN pathogenesis. 5/6NE rats were treated with dabigatran (150 mg/kg/day), PAR-1 inhibitor SCH79797 (1 and 3 mg/kg/day) and PAR-1 agonist TFLLR-NH2 (0.25 and 0.50 µmol/kg/day) for 7 days. Serum creatinine and hematuria were assessed daily. Kidney morphology was evaluated at the end of the study. In 5/6NE rats treated with either dabigatran or combination with a PAR-1 modulator, there was an elevation in serum creatinine, glomerular hematuria, red blood casts in the tubules, and acute tubular epithelial cell injury. Interestingly, both PAR-1 modulators in a dose-depended manner had similar effects on the serum creatinine levels and hematuria as those of dabigatran. Dabigatran-induced increase in the systolic blood pressure was not affected by PAR-1 modulators. In conclusion, the normal function of PAR-1 is crucial to maintain the glomerular filtration barrier integrity. Either activation or blockage of PAR-1 leads to glomerular hematuria and subsequent acute tubular epithelial cell injury.

Autopsy Techniques in Patients With History of Radiofrequency Ablation for Atrial Fibrillation.

CONTEXT.­: Esophageal fistula formation is one of the most feared complications of radiofrequency catheter ablation. This procedure and its many variations, such as the "maze," are becoming the mainstream treatment for atrial fibrillation owing to limitations of antiarrhythmic drugs. The incidence of this complication rate has been reported to be from 0.01% to 1%. OBJECTIVE.­: To delineate the importance of using the en bloc Letulle method of dissection for identifying esophageal fistulas for cases with a history of radiofrequency catheter ablation. DESIGN.­: Six autopsy cases with a history of radiofrequency catheter ablation for atrial fibrillation were selected from 1736 autopsies performed between 2009 and 2020. RESULTS.­: The initial presenting symptoms included neurologic symptoms, chest pains, epigastric discomfort, and sepsis. Transesophageal echocardiogram in 4 cases showed no evidence of thrombus or vegetation, however, 2 cases had evidence of atrial esophageal fistula. The autopsy findings included 5 atrial esophageal fistulas and 1 esophagopericardial fistula. Atrial esophageal fistulas were small and could be detected without difficulty when the en bloc Letulle technique was used and would have been easily missed by the Virchow method. The immediate causes of the deaths were myocardial ischemia, septic emboli to brain and heart, hypovolemic shock secondary to exsanguination, stroke, and coagulopathy. CONCLUSIONS.­: To date, this is the largest collection of autopsy cases showing esophageal fistula associated with prior radiofrequency catheter ablation. The Letulle dissection method is preferable in this setting.

Chronic Hematuria Increases Chronic Kidney Injury and Epithelial-Mesenchymal Transition in 5/6 Nephrectomy Rats.

Chronic kidney disease (CKD) is a common outcome of many kidney diseases. Interstitial fibrosis and tubular atrophy (IFTA) is a histologic hallmark of CKD. Hematuria is a common symptom in many human kidney diseases. Free hemoglobin may affect tubular epithelial cells by generating reactive oxygen species (ROS). Epithelial-mesenchymal transition (EMT) of the tubular epithelial cells has been shown to play an important role in the IFTA development. The aim of this study was to determine the effects of chronic hematuria on the CKD progression in 5/6 nephrectomy (5/6NE) rat model of CKD. 5/6 NE rats were treated with oral warfarin (0.5 mg/kg/day) or vehicle (control). The animals were monitored for 26 weeks, while prothrombin time (PT), serum creatinine (SCr), and hematuria were measured weekly. Staining for iron, trichrome, and EMT (vimentin, E-cadherin, smooth muscle actin) markers was performed on the remnant kidneys. ROS were detected in the kidneys by protein carbonyl assay and immunohistochemistry for heme oxygenase 1 (HMOX1), at the end of the study. Apoptosis was detected by TUNEL assay. Warfarin treatment resulted in a PT increase 1.5-2.5 times from control and an increase in hematuria and SCr. Histologically, warfarin-treated animals had more iron-positive tubular epithelial cells and increased IFTA as compared to control (42.9 ± 17% vs. 18.3 ± 2.6%). ROS were increased in the kidney in warfarin-treated rats. The number of tubules that show evidence of EMT was significantly higher in warfarin-treated 5/6NE as compared to control 5/6NE rats. The number of apoptotic tubular epithelial cells was higher in warfarin-treated 5/6 NE rats. Chronic hematuria results in increased iron-positive tubular epithelial cells, EMT, apoptosis, and more prominent IFTA in CKD rats. Our data suggest an important role of chronic hematuria in the progression of CKD.

Can Antinuclear Antibodies (ANA) be Monoclonal?

BACKGROUND: Nuclear staining by immunofluorescence in a kidney biopsy is often seen in patients with positive antinuclear antibodies (ANA) in the serum. These ANA are usually polyclonal, but herein we report 9 cases with an unusual finding of monoclonal nuclear staining by immunofluorescence on kidney biopsy. Case Presentation. Nine cases with predominant stain for kappa or lambda light chain were identified by searching the renal pathology laboratory database for the past 10 years. All cases had positive stain for only kappa (six cases) or lambda (three cases) light chain in the nuclei. Eight out of nine cases had positive nuclear IgG stain, and one case had positive nuclear IgA stain. Among cases with positive nuclear IgG staining, six cases were positive for IgG1 subclass, one case was positive for IgG2 subclass, and one case was positive for IgG3 subclass. All patients with positive IgG nuclear stain, who had testing for ANA, had positive ANA. Patients with positive IgG1 subclass did not have monoclonal protein in the serum or urine, but the patient with positive IgG2 subclass and lambda light chain stain in the nuclei had IgG lambda monoclonal gammopathy. CONCLUSIONS: We identified a new unique pattern of nuclear stain by immunofluorescence in kidney biopsies that suggests the presence of monoclonal ANA. Workup for underlying monoclonal gammopathy is warranted in such patients.

Hypertension and the Kidney: Reduced Kidney Mass Is Bad for Both Normotensive and Hypertensive Rats.

BACKGROUND: Hypertension is a leading cause of chronic kidney disease worldwide. Early studies demonstrated the short-term effects of hypertension on kidney function and morphology in ablative nephropathy. The aim of this study was to investigate the long-term consequences of hypertension in 5/6 nephrectomy (5/6NE) model. METHODS: Reduction of the kidney mass by 5/6NE was created in spontaneous hypertensive rats (SHR) and genetically similar normotensive Wistar Kyoto (WKY) rats. Blood pressure, serum creatinine (SCr), hematuria, and proteinuria were monitored weekly for 23 weeks. Kidney morphology was assessed at the end of the study. Sham-operated rats from both strains were used as controls. RESULTS: Rats with 5/6NE had increased SCr, blood pressure, hematuria, and proteinuria in both SHR and WKY. Even though the SCr levels and blood pressure were greater in 5/6NE SHR as compared with 5/6NE WKY rats, absolute changes from sham-operated rats were not statistically significant between these 2 groups. 5/6NE SHR had earlier onset and higher proteinuria than 5/6NE WKY rats. Hematuria was similar in 5/6NE SHR and 5/6NE WKY rats. However, 5/6NE SHR had enlarged glomeruli, increased interstitial fibrosis, and prominent intimal thickening in the small arteries/arterioles as compared with 5/6NE WKY rats. CONCLUSIONS: The long-term severity of kidney injury correlated with higher blood pressure. Reduction of the kidney mass increases SCr, hematuria, proteinuria, and blood pressure in both normotensive and hypertensive rats. Histological assessment provides better information about underlying chronic kidney injury than actual changes in SCr and urinalysis.