Grande, Joseph P.

Key Points In heme protein–induced AKI, mitochondrial functional integrity, as reflected by ATP and NAD + content and NAD + /NADH ratio, is impaired. Mitochondrial quality control is compromised as reflected by impaired biogenesis, exaggerated fission, and marked ultrastructural damage. Modern concepts regarding mitochondria and AKI apply to heme protein–induced AKI, with the possibility of novel therapeutic strategies. Background Mitochondrial injury occurs in and underlies acute kidney injury (AKI) caused by ischemia-reperfusion and other forms of renal injury. However, to date, a comprehensive analysis of this issue has not been undertaken in heme protein–induced AKI (HP-AKI). We examined key aspects of mitochondrial function, expression of proteins relevant to mitochondrial quality control, and mitochondrial ultrastructure in HP-AKI, along with responses to heme in renal proximal tubule epithelial cells. Methods The long-established murine glycerol model of HP-AKI was examined at 8 and 24 hours after HP-AKI. Indices of mitochondrial function (ATP and NAD + ), expression of proteins relevant to mitochondrial dynamics, mitochondrial ultrastructure, and relevant gene/protein expression in heme-exposed renal proximal tubule epithelial cells in vitro were examined. Results ATP and NAD + content and the NAD + /NADH ratio were all reduced in HP-AKI. Expression of relevant proteins indicate that mitochondrial biogenesis (PGC-1 α , NRF1, and TFAM) and fusion (MFN2) were impaired, as was expression of key proteins involved in the integrity of outer and inner mitochondrial membranes (VDAC, Tom20, and Tim23). Conversely, marked upregulation of proteins involved in mitochondrial fission (DRP1) occurred. Ultrastructural studies, including novel 3D imaging, indicate profound changes in mitochondrial structure, including mitochondrial fragmentation, mitochondrial swelling, and misshapen mitochondrial cristae; mitophagy was also observed. Exposure of renal proximal tubule epithelial cells to heme in vitro recapitulated suppression of PGC-1 α (mitochondrial biogenesis) and upregulation of p-DRP1 (mitochondrial fission). Conclusions Modern concepts pertaining to AKI apply to HP-AKI. This study validates the investigation of novel, clinically relevant therapies such as NAD + -boosting agents and mitoprotective agents in HP-AKI.

AbstractLafora disease is a rare inherited neurodegenerative disease with onset in adolescence. Patients present with progressive myoclonic seizures and cognitive decline. The disease is linked to mutations in either of the two genes encoding malin and laforin, and it is associated with the accumulation of polyglucosan inclusions (Lafora bodies [LBs]) in various tissues, such as brain, liver, muscle, and skin, with the skin being particularly accessible for biopsy. Histopathologic examination of affected tissue with demonstration of LBs, together with the presence of pathologic mutation in EPM2A or NHLRC1 genes, is sufficient for diagnosis of this neurologic disorder when clinically suspected. Here, we report the case of a 16‐year‐old female with progressive neurologic symptoms and homozygous mutation in the NHLRC1 gene encoding malin. The skin biopsy was instrumental in reaching the final diagnosis by showing LBs in sweat glands by histopathologic and electron microscopic examination.

ABSTRACT Background Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. Method This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. Results RAS demonstrated a loss of medium-sized vessels (0.2–0.3 mm) compared with donor kidneys (P = 0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. Conclusions These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Introduction: The estimated prevalence of diabetes mellitus (DM) is 8.4% among adults in the U.S. Cardiovascular disease is a major complication of DM. We have observed that diabetic mice, but not non-diabetic mice, undergoing 2kidney-1clip (2K1C) surgery inducing renovascular hypertension had high mortality attributed to ruptured aortic aneurysm. However, the effect of diabetes on formation of aortic aneurysm remains unclear. Method: In our previous experiment, 163 mice were divided into 4 groups: leptin KO 2K1C (db 2K1C, n=52), leptin KO Sham (db Sham, n=20), wild type 2K1C (dbc 2K1C, n=62), and wild type Sham (dbc Sham, n=29), respectively. We found that 42% (22/52) of db 2K1C died, from ruptured aortic aneurysms examined by gross examination, within 120 days, while others survived 100%. In this study, available H&E aortic sections of survived mice (n=74): db 2K1C (n=30), db Sham (n=15), dbc 2K1C (n=20), and dbc Sham (n=9) were examined 2K1C surgery in a blinded fashion. Aortic lesions defined as necrosis, thrombosis and dropped-out smooth muscle cells, which are signs of early formation of aortic aneurysm, were identified. Inflammation was graded as mild, moderate and severe inflammation. Result: The db 2K1C mice had a significantly greater aortic lesions (16/30, 53%) compared to the db Sham (13/15, 20%); and dbc 2K1C mice (1/20, 5%), p=0.002 and 0.0001, respectively. The number of aortic lesions in the dbc 2K1C group was not statistically different from that of the dbc Sham group (p=0.5). Similarly, the number of aortic lesions in the db Sham group was not statistically different from that of the dbc Sham group (0/9, 0%), p=0.125. Moderate-to-severe inflammation in the tunica media was more prevalence in db mice than the dbc mice. Conclusion: We report for the first time an increased incidence of aortic lesions in db 2K1C compared to dbc and sham groups. This finding suggests that diabetes contributes to aortic aneurysm formation in mice with renal artery stenosis, potentially due to induction of inflammation. Further studies to confirm a mechanism are warranted.

Background: Combined renovascular hypertension (RVH) and type II diabetes (DM II) are a major health problem. Both share common risk factors and contribute to cardiovascular morbidity and mortality. We recently observed a high mortality rate in DM II mice (dbdb) with RVH. Purpose: We sought to determine the interactions between renovascular hypertension and DM II on the cardiovascular phenotype in dbdb mice with RVH. Methods and Materials: RVH was established by placing a polytetrafluoroethylene cuff on the right renal artery of C57BL/KS (wild type, WT) or dbdb mice. We divided the mice into four groups: WT sham, WT type RAS, dbdb sham, dbdb RAS. We examined the blood pressure, plasma angiotensin I, renin, heart weight and myocyte diameter. Histologic parameters including inflammation, necrosis and fibrosis were assessed semiquantitively. Results: Blood pressure, Renin, Plasma angiotensin I: RAS produced a similar elevation in systolic blood pressure (SBP), renin mRNA expression, and plasma angiotensin I content in WT and dbdb mice with RAS. Heart weight: Despite similar elevation in SBP, db/db RAS mice showed a greater increase in heart weight than WT RAS mice (163.7 +/- 6.4 mmHg vs 145.7 +/- 2.3; n=23).. Similar trends were observed on normalization of the heart weight to tibial length. Cardiac myocyte diameter: Cardiac myocyte diameter in WT RAS (n=26) was significantly higher than in WT sham (n=15; 16.4 ± 0.3 vs. 15.0 ± 0.3 μm, p=0.0031). Myocyte diameter was higher in dbdb RAS mice (n=19, 14.76 ± 0.35) than dbdb sham (15.94 ± 0.29 μm, p= 0.0126). However, myocyte diameter was similar in dbdb RAS and WT RAS mice (16.4 ± 0.3 vs. 15.9 ± 0.3 μm, p=0.3). Histology: Sham groups had no inflammation (INF) or necrosis (NEC). Interstitial fibrous tissue was increased in dbdb RAS mice (28% by area) vs WT RAS (19% by area). Dbdb RAS mice had more severe INF (severe in 18%, mild in 82%) than WT RAS (severe in 0%, jild in 12%). Severe NEC was found in 32.1% of dbdb compared to 11.5% of WT RAS. Conclusion: The histologic changes and hypertrophy of the heart superimpose in dbdb RAS compared to WT RAS. This indicates that the risk of cardiovascular morbidity and mortality increases synergisticly in the presence of DM II and RVH.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in developed countries. Kidney injuries related to CLL can occur via several mechanisms. Approximately 30%–40% of CLL patients with a kidney biopsy have monoclonal-protein related lesions. However, such lesions mostly involve glomeruli with only rare instances of tubule-interstitial disorders. There is only a single case report of a CLL patient who presented with crystalline light chain proximal tubulopathy (LCPT). Fanconi syndrome (FS) may provide clinical clues in these patients. We report a case of a CLL patient who presented with hypokalemia in the setting of advanced kidney failure in which serum monoclonal IgG kappa was detected. The patient demonstrated typical signs and symptoms of FS. The urinary retinol binding protein was also extremely elevated (638 times upper normal limit). A kidney biopsy revealed non-crystalline LCPT with CLL infiltration. FS in CLL is an extremely rare presentation, as only four cases had been previously reported. Of these, only a single case had crystalline LCPT. Our case is the first report of non-crystalline LCPT in a CLL patient. Obinutuzumab and Venetoclax were started to address the CLL, and an improvement in kidney function was noted thereafter.

Aim Th1 cells have potent pro‐inflammatory properties and have been shown to play an important role in the development and progression of acute renal injury to chronic renal disease. The aim of this study is to test the hypothesis that chronic renal damage in mice with renal artery stenosis (RAS) would be ameliorated in mice with homozygous deletion of the Th1 transcription factor T‐bet. Methods C57BL6/J (WT) and C57BL6/J mice with homozygous deletion of T‐bet (KO) were obtained from Jackson Laboratory. Renal artery stenosis surgery (RAS) was performed by placement of a polytetrafluoroethylene cuff on the right renal artery of KO (KO RAS group, n =5) and WT mice (WT RAS group, n =6). Sham (SH) surgery was performed with manipulation of the right renal artery without cuff placement in KO (KO SH group, n =4) and WT mice (WT SH group, n =5). Renal blood perfusion and oxygenation (R2*) were measured using 16.4T MRI. Blood pressure measurements were done using tail‐cuff method. Kidneys were harvested at 28 days after RAS for assessment of histology. Results Baseline intra‐renal perfusion in both KO RAS and WT RAS mice was reduced to a similar extent following cuff placement. In KO RAS group, baseline intra‐renal perfusion (795 ± 57 ml/100g/min) was reduced to 234 ± 22 ml/100g/min ( p <0.0001 vs baseline, mean reduction 71%) and in WT RAS group, baseline intra‐renal perfusion (836 ± 87 ml/100g/min) was reduced to 220 ± 21 ml/100g/min ( p =0.002 vs baseline, mean reduction 74%). Both KO RAS and WT RAS mice developed hypertension, which persisted throughout the 4 week study. WT RAS group showed significant elevation in cortical R2*, a measure of hypoxia, following RAS compared to their baseline ( p =0.002). KO RAS mice showed slightly higher R2* value, which was not significantly different from their baseline values. The weight of the stenotic kidney of the KO RAS group (105 ± 11.6 mg) was significantly lower than the KO SH mice (178 ± 14.1 mg, p =0.0021) but significantly higher than the WT RAS group (51.7 ± 7.9 mg, p =0.0010). On histologic and immune‐histochemical analysis, we observed that the stenotic kidney of the KO RAS group had significantly lower (%) cortical tubular atrophy (15% ± 6.7, p <0.0001) compared to WT RAS mice (81.7% ± 7). KO RAS group had significantly lower macrophage (MQ) ( p =0.0016) and T cell influx ( p =0.0052) compared to the WT RAS, as assessed by the % of cortical surface areas staining positive for F4/80 (MQ) and CD3 (T cell). When compared to their respective shams, KO RAS showed no significant differences in the % cortical tubular atrophy, MQ and CD3+ T cell influx. Conclusion We conclude that deficiency of Th1 transcription factor T‐bet, protects against chronic renal injury as evidenced by the less cortical atrophy in KO RAS compared to WT RAS group, despite a similar elevation in blood pressure and reduction in perfusion. This model may provide a novel therapeutic target to prevent renal disease progression in patients with renovascular hypertension. Support or Funding Information National Institutes of Health Grant AI 100911 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The objective of this study was to identify signaling pathways leading to the development of interstitial fibrosis and tubular atrophy in the stenotic kidney and compensatory hypertrophy/hyperplasia of the contralateral kidney in a murine 2 kidney 1 clip model of hypertensive nephrosclerosis. Mice were studied 2, 5, and 11 weeks after undergoing surgery to establish unilateral renal artery stenosis (RAS). RAS animals, but not sham controls, became hypertensive within 1 week. Compensatory enlargement of the contralateral kidney was associated with a transient increase in proliferation (as assessed by PCNA and Mib‐1), p‐ERK, caspase 3, p27, p53, and TGF‐β expression at early time points, with return to baseline levels by 11 weeks. Tubular atrophy in the stenotic kidney was associated with a persistent increase in p‐ERK, Mib‐1, p53, p21, and p27 expression through 11 weeks. Proliferative activity was observed in tubular epithelial cells and in inflammatory cells. The atrophic kidneys had little apoptotic activity (caspase 3, TUNEL staining). The atrophic kidneys showed progressive increases in TGF‐β and α‐SMA expression and extracellular matrix deposition throughout 11 weeks. Interstitial inflammation was associated with increased MCP‐1 expression. Mechanisms underlying this differential response to unilateral RAS await definition.

Renal artery stenosis (RAS) is a common disorder associated with both cardiovascular and renal morbidity and mortality. Recent studies have shown that stenting of the stenotic kidney fails to improve renal or cardiovascular outcomes, prompting a search for more effective therapies. Strain‐specific variability in renal outcomes is a well recognized problem associated with murine models of chronic injury. The objective of this study was to define strain specific differences in renal outcome and correlation with parameters of injury in a murine model of RAS. We used a 0.25 mm polytetrafluoroethylene cuff to establish RAS in mice (n=292 total). We examined the correlation between age, time following surgery, body weight, blood pressure, heart and kidney weights, ratio of STK/CLK weight, and plasma angiotensin level in 3 murine strains. Percent atrophy in the STK as a function of time following RAS surgery is summarized in the table below: Strain 1 Week 2 Week 4 Week 6 Week 129 21% 31% XX 45% C57BLKS/J 25% 51% 63% 72% C57BL/6J XX XX 39% 41% The right kidney weight and the STK/CLK weight ratio correlated with %atrophy in all strains, with the strongest correlation observed in C57BL/6J mice. In C57BL/6J mice, blood pressure correlated with %atrophy. CLK weight correlated with heart weight, with the strongest correlation observed in 129Sv mice. In BLKS mice, the time following surgery and age correlated with CLK weight and %atrophy in the STK; blood pressure correlated with plasma angiotensin level. We conclude that RAS produces severe chronic renal injury in the STK of all strains studied, with the most severe injury observed in C57BLKS/J mice.The STK/CLK weight ratio correlated most strongly with %atrophy and can be used to predict development of chronic renal damage in all strains. Hypertrophy of the CLK, as measured by CLK weight, strongly correlated with heart weight.