Membrane attack complex (MAC) deposition in renal tubules is associated with interstitial fibrosis and tubular atrophy: a pilot study

IntroductionTreatment failures for lupus nephritis (LN) are high with 10%–30% of patients progressing to end-stage renal disease (ESRD) within 10 years. Interstitial fibrosis/tubular atrophy (IFTA) is a predictor of progression to ESRD. Prior studies suggest that tubulointerstitial injury secondary to proteinuria in LN is mediated by complement activation in the tubules, specifically through the membrane attack complex (MAC). This study aimed to investigate the associations between tubular MAC deposition with IFTA and proteinuria.MethodsIn this cross-sectional study, LN kidney biopsies were assessed for MAC deposition by staining for Complement C9, a component of the MAC. Chromogenic immunohistochemistry was performed on paraffin-embedded human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197). Tubular C9 staining intensity was analysed as present versus absent. IFTA was defined as minimal (<10%), mild (10%–24%), moderate (25%–50%) and severe (>50%).ResultsRenal biopsies from 30 patients with LN were studied. There were 24 (80%) female sex, mean age (SD) was 33 (12) years old and 23 (77%) had pure/mixed proliferative LN. Tubular C9 staining was present in 7 (23%) biopsies. 27 patients had minimal-to-mild IFTA and 3 patients had moderate IFTA. Among the C9 + patients, 3 (43%) had moderate IFTA as compared with none in the C9- group, p=0.009. C9 + patients had higher median (IQR) proteinuria as compared with C9- patients: 6.2 g (3.3–13.1) vs 2.4 g (1.3–4.6), p=0.001 at the time of biopsy. There was no difference in estimated glomerular filtration rate (eGFR) between the C9 + and C9- groups.ConclusionThis study demonstrated that tubular MAC deposition is associated with higher degree of IFTA and proteinuria, which are predictors of progression to ESRD. These results suggest that tubular MAC deposition may be useful in classification of LN. Understanding the role of complement in tubulointerstitial injury will also identify new avenues for LN treatment.

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice

Novel strategies for the prevention and treatment of sepsis-associated acute kidney injury and its long-term outcomes have been required and remain a challenge in critical care medicine. Therapeutic strategies using lipid mediators, such as aspirin-triggered resolvin D1 (ATRvD1), can contribute to the resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called the subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, the urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing proteinuria excretion, the UPC ratio, the glomerular cell number, and extracellular matrix deposition. Pro-fibrotic markers, such as transforming growth factor β (TGFβ), type 3 collagen, and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from the recruitment of IBA1+ cells. The interleukin-1β (IL-1β) levels were increased in the subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-α (TNF-α), IL-10, and IL-4 mRNA expression were increased in the kidney of BSA-challenged post-septic mice, and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult such as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

Dapagliflozin Ameliorates Diabetic Kidney Disease via Upregulating Crry and Alleviating Complement Over-activation in db/db Mice

Sodium-glucose cotransporter 2(SGLT2) inhibitors show prominent renal protective effect in diabetic kidney disease (DKD), anti-inflammatory effect being one of its key mechanisms. Over-activation of the complement system, a crucial part of innate immunity, plays an important role in DKD. We aimed to investigate the effect of SGLT2 inhibitors on alleviating complement over-activation in DKD. Db/db mice were randomly divided into two groups, with 7 mice in each group treated with dapagliflozin and vehicle respectively, and 7 mice in m/m mice group. Laboratory and renal pathological parameters were evaluated. Mouse proximal tubular epithelial cells (MPTECs) were cultured and treated with high glucose. Dapagliflozin and dimethyloxallyl glycine (DMOG) were added as conditional treatment. Dapagliflozin-treated db/db mice showed significantly lower urinary albumin than vehicle-treated ones. Besides typical glomerular and tubulointerstitial injury, both C3b and membrane attack complex (MAC) depositions were significantly attenuated in dapagliflozin-treated db/db mice. The expression of complement receptor type 1-related protein y (Crry), a key complement regulator which inhibits complement over-activation, was significantly upregulated by dapagliflozin. Dapagliflozin-mediated Crry upregulation was associated with inhibition of HIF-1α accumulation under high glucose. When HIF-1α expression was stabilized by DMOG, the protective effect of dapagliflozin via upregulating Crry was blocked. In conclusion, dapagliflozin could attenuate complement over-activation in diabetic mice via upregulating Crry, which is associated with the suppression of HIF-1α accumulation in MPTECs.

ATRvD1 Attenuates Renal Tubulointerstitial Injury Induced by Albumin Overload in Sepsis-Surviving Mice

Current interventions are not effectives in preventing sepsis-induced acute kidney injury and its long-term outcomes or even after second renal insult. Therapeutic strategies using lipid mediators, as aspirin-triggered resolvin D1 (ATRvD1), can contribute for resolution of acute and chronic inflammation. In this study, we examined the potential effect of ATRvD1 on long-term kidney dysfunction after severe sepsis. Fifteen days after cecal ligation and puncture (CLP), sepsis-surviving BALB/c mice were subjected to a tubulointerstitial injury through intraperitoneal injections of bovine serum albumin (BSA) for 7 days, called subclinical acute kidney injury (subAKI) animal model. ATRvD1 treatment was performed right before BSA injections. On day 22 after CLP, urinary protein/creatinine ratio (UPC), histologic parameters, fibrosis, cellular infiltration, apoptosis, inflammatory markers levels, and mRNA expression were determined. ATRvD1 treatment mitigated tubulointerstitial injury by reducing the proteinuria excretion, UPC ratio, glomerular cell number and extracellular matrix deposition. Pro-fibrotic markers, as transforming growth factor &beta; (TGFb), type 3 collagen and metalloproteinase (MMP)-3 and -9 were reduced after ATRvD1 administration. Post-septic mice treated with ATRvD1 were protected from renal apoptosis and recruitment of F4/80+ cells. Interleukin-1b (IL-1b) levels were increased in subAKI animal model, being attenuated by ATRvD1. Tumor necrosis factor-a (TNF-a), IL-10 and IL-4 mRNA expression was increased in the kidney of BSA-challenged post-septic mice and it was also reduced after ATRvD1. These results suggest that ATRvD1 protects the kidney against a second insult as BSA-induced tubulointerstitial injury and fibrosis by suppressing inflammatory and pro-fibrotic mediators in renal dysfunction after sepsis.

The pathological features of leukemic cells infiltrating the renal interstitium in chronic lymphocytic leukemia/small lymphocytic lymphoma from a large single Chinese center

Background Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is rare in Asians, and patients with CLL/SLL seldomly undergo kidney biopsy. The histopathological features and clinical relevance of tubulointerstitial injury in CLL/SLL have not been extensively characterized. Hence, we attempted to describe the clinical characteristics, renal pathology and clinical outcome of a well-characterized population of CLL/SLL patients with CLL cell infiltration in the renal interstitium from a large single center in China. Methods Between January 1st, 2010 and September 31st, 2020, 31946renal biopsies were performed at Peking University First Hospital, and 10 CLL/SLL patients with CLL cell infiltration in the renal interstitium were included. Complete clinical data were collected from these 10 patients, and renal specimens were examined by routine light microscopy, immunofluorescence and electron microscopy. Results The extent of the infiltrating CLL cells in patients with CLL/SLL varied among different patients and ranged from 10 to 90% of kidney parenchyma. Six (60%) of 10 patients presented with an extent of infiltrating CLL cells ≥50%. Interestingly, we found that three patients (3/10, 30%) expressed monoclonal immunoglobulins in the infiltrating CLL cells, and special cytoplasmic crystalline structures were found in two of the three patients by electron microscopy for the first time. Severe renal insufficiency (Scr ≥200 μmol/L) was associated with ≥50% interstitial infiltration of CLL cells in the renal interstitium. Conclusions The current study confirmed that CLL cells infiltrating the renal interstitium can directly secrete monoclonal immunoglobulins, indicating that the interstitial infiltrating CLL cells possibly cause renal injury directly by secreting monoclonal immunoglobulins in situ. This finding may prove a new clue to elucidate the pathogenetic mechanism of renal injury involved with CLL/SLL.

Therapeutic effects of lisinopril and empagliflozin in a mouse model of hypertension-accelerated diabetic kidney disease

Hypertension is a critical comorbidity for progression of diabetic kidney disease (DKD). To facilitate development of novel therapeutic interventions with the potential to control disease progression, there is a need to establish translational animal models that predict treatment effects in human DKD. The present study aimed to characterize renal disease and outcomes of standard of medical care in a mouse model of advanced DKD facilitated by adeno-associated virus (AAV)-mediated renin overexpression in uninephrectomized (UNx) db/db mice. Five weeks after single AAV administration and four weeks after UNx, female db/db UNx-ReninAAV mice received (PO, QD) vehicle, lisinopril (40 mg/kg), empagliflozin (20 mg/kg) or combination treatment for 12 weeks (n=17 per group). Untreated db/+ mice (n=8) and vehicle-dosed db/db UNx-LacZAAV mice (n=17) served as controls. Endpoints included plasma, urine and histomorphometric markers of kidney disease. Total glomerular numbers and individual glomerular volume was evaluated by whole-kidney 3D imaging analysis. db/db UNx-ReninAAV mice developed hallmarks of progressive DKD characterized by severe albuminuria, advanced glomerulosclerosis and glomerular hypertrophy. Lisinopril significantly improved albuminuria, glomerulosclerosis, tubulointerstitial injury and inflammation. While empagliflozin alone had no therapeutic effect on renal endpoints, lisinopril and empagliflozin exerted synergistic effects on renal outcomes. In conclusion, the db/db UNx-ReninAAV mouse demonstrates good clinical translatability with respect to the physiological and histological hallmarks of progressive DKD. Efficacy of standard of care to control hypertension and hyperglycemia provides proof-of-concept for testing novel drug therapies in the model.

The Herbal Formula Granule Prescription Mahuang Decoction Ameliorated Chronic Kidney Disease Which Was Associated with Restoration of Dysbiosis of Intestinal Microbiota in Rats

Chronic kidney disease (CKD) has become a global health issue, and there is increasing evidence showing the beneficial roles of traditional Chinese medicine (TCM) in CKD treatment. Here, we studied the renoprotective role of Mahuang decoction, a famous TCM prescription, in a rat CKD model induced with the combination of doxorubicin and adenine. Our data showed that intragastric administration of Mahuang decoction inhibited the loss of bodyweight and attenuated proteinuria, serum creatinine, and blood urea nitrogen in CKD rats. Kidney histological analysis revealed decreased tubulointerstitial injury and fibrosis in CKD rats treated with Mahuang decoction accompanied with suppressed expression of TGF-β1 and phosphorylated NF-κB/P65 (p-P65) as indicated by immunohistochemistry. ELISA analysis demonstrated reduced serum levels of proinflammatory cytokines TNFα and IL-6. Most importantly, intestinal microbiota analysis by 16s rRNA-seq showed that Mahuang decoction restored the impaired richness and diversity of intestinal microflora and recovered the disrupted microbial community through reducing the abundance of deleterious microbes and promoting the expansion of beneficial microbes in CKD rats. Collectively, our findings demonstrated that Mahuang decoction mitigated kidney functional and structural impairment in CKD rats which were associated with the restoration of dysbiosis of intestinal microbiota, implying its potential in clinical CKD treatment.

Complement Components C3 and C4 Indicate Vasculitis Manifestations to Distinct Renal Compartments in ANCA-Associated Glomerulonephritis

Background: Acute kidney injury (AKI) is a common and severe complication of antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) causing progressive chronic kidney disease (CKD), end-stage renal disease (ESRD) or death. Pathogenic ANCAs, in particular proteinase 3 (PR3) and myeloperoxidase (MPO), trigger a deleterious immune response resulting in pauci-immune necrotizing and crescentic glomerulonephritis (GN), a common manifestation of glomerular injury in AAV. However, there is growing evidence that activation of the complement pathway contributes to the pathogenesis and progression of AAV. We here aimed to compare glomerular and tubulointerstitial lesions in ANCA GN and extrarenal manifestation of AAV in association with levels of circulating complement components C3c and C4. Methods: Plasma levels of C3c and C4 in a total number of 53 kidney biopsies with ANCA GN were retrospectively included between 2015 and 2020. Glomerular and tubulointerstitial lesions were evaluated according to established scoring systems for ANCA GN and analogous to the Banff classification. Results: We here show that circulating levels of C3c and C4 in ANCA GN were comparable to the majority of other renal pathologies. Furthermore, hypocomplementemia was only detectable in a minor subset of ANCA GN and not correlated with renal or extrarenal AAV manifestations. However, low levels of circulating C3c correlated with AKI severity in ANCA GN independent of systemic disease activity or extrarenal AAV manifestation. By systematic scoring of glomerular and tubulointerstitial lesions, we provide evidence that low levels of circulating C3c and C4 correlated with vasculitis manifestations to distinct renal compartments in ANCA GN. Conclusions: We here expand our current knowledge about distinct complement components in association with vasculitis manifestations to different renal compartments in ANCA GN. While low levels of C4 correlated with glomerulitis, our observation that low levels of circulating complement component C3c is associated with interstitial vasculitis manifestation reflected by intimal arteritis implicates that C3c contributes to tubulointerstitial injury in ANCA GN.

A Higher Foci Density of Interstitial Fibrosis and Tubular Atrophy Predicts Progressive Chronic Kidney Disease after a Radical Nephrectomy for Tumor

Background Chronic tubulointerstitial injury on kidney biopsy is usually quantified by the percentage of cortex with interstitial fibrosis/tubular atrophy (IF/TA). Whether other patterns of IF/TA or inflammation in the tubulointerstitium have prognostic importance beyond percentage IF/TA is unclear. Methods We obtained, stained, and digitally scanned full cortical thickness wedge sections of renal parenchyma from patients who underwent a radical nephrectomy for tumor in 2000 to 2015 and morphometrically analyzed the tubulointerstitium of the cortex for percentage IF/TA, IF/TA density (foci per mm2 cortex), percentage subcapsular IF/TA, striped IF/TA, percentage inflammation (both within and outside IF/TA regions), and percentage subcapsular inflammation. Patients were followed with visits every 6-12 months. Progressive chronic kidney disease (CKD) was defined as dialysis, kidney transplantation, or 40% decline from the postnephrectomy estimated glomerular filtration rate (eGFR). Cox models assessed risk of CKD or noncancer mortality with morphometric measures of tubulointerstitial injury after adjustment for percentage IF/TA and clinical characteristics. Results Among 936 patients (mean age, 64 years; postnephrectomy baseline eGFR, 48 ml/min per 1.73m2), 117 progressive CKD events and 183 noncancer deaths occurred over a median 6.4 years. Higher IF/TA density predicted both progressive CKD and noncancer mortality after adjustment for percentage IF/TA and predicted progressive CKD after further adjustment for clinical characteristics. Independent of percentage IF/TA, age, and sex, higher IF/TA density correlated with lower eGFR, smaller nonsclerosed glomeruli, more global glomerulosclerosis, and smaller total cortical volume. Conclusions Higher density of IF/TA foci (a more scattered pattern with more and smaller foci) predicts higher risk of progressive CKD after radical nephrectomy compared with the same percentage of IF/TA but with fewer and larger foci.