Sex and Gender Differences in AKI.

Sex differences in AKI continue to be identified. Generally, women are protected from AKI when compared to men. Much of the protection exhibited in women is diminished after menopause. These sex and age effects have also been noted in animal models of AKI. Gonadal hormones, as modifiers of incidence, severity, and progression of AKI, have been offered as likely contributors to this sex and age effect. In animal models of AKI, estrogen and testosterone seem to modulate susceptibility. Questions remain however regarding cellular and molecular changes that are initiated by modulation of these hormones because both estrogen and testosterone have effects across cell types that play a role in AKI. Although findings have largely been informed by studies in males, molecular pathways that are involved in the initiation and progression of AKI may be modulated by gonadal hormones. Compounding the hormone-receptor effects are developmental effects of sex chromosomal complement and epigenetic influences that may confer sex-based baseline differences in gene and protein expression, and gene dosage effects of X inactivation and escape on molecular pathways. Elucidation of sex-based protection may afford a more complete view of AKI and potential therapeutic interventions. Furthermore, the effect on susceptibility to AKI in transgender patients, who receive life-altering and essential gender-affirming hormone therapy, requires greater attention. In this review, several potential contributors to the sex differences observed in humans and animal models are discussed.

Does Gender Affirming Hormone Therapy Increase the Risk of Kidney Disease?

Kidney health and manifestation of disease in transgender men, women, and nonbinary individuals are not well understood. Transgender individuals commonly receive gender-affirming hormone therapy (GAHT) to align their outward appearance with their gender. Recent attention to the differences in fundamental kidney parameters has identified that transgender individuals may manifest levels of these biomarkers differently than their cisgender counterparts. Improving understanding of the differences in biomarkers and in the development of kidney disease is essential to providing appropriate kidney care to this vulnerable population. In this review, we introduce the current information related to GAHT and kidney health and highlight the significant gaps in our understanding of how GAHT may affect kidney physiology and pathophysiology.

Does G Protein-Coupled Estrogen Receptor 1 Contribute to Cisplatin-Induced Acute Kidney Injury in Male Mice?

Nephrotoxicity is the dose-limiting side-effect of the chemotherapeutic agent cisplatin (Cp). Recent evidence points to renal protective actions of G protein-coupled estrogen receptor 1 (GPER1). In addition, it has been shown that GPER1 signaling elicits protective actions against acute ischemic injuries that involve multiple organ systems; however, the involvement of GPER1 signaling in Cp-induced acute kidney injury (AKI) remains unclear. This study tested whether genetic deletion of GPER1 exacerbates Cp-induced AKI in male mice. We subjected male mice, homozygous (homo) and heterozygous (het) knockout for the GPER1 gene, and wild-type (WT) littermates to Cp or saline injections and assessed markers for renal injury on the third day after injections. We also determined serum levels of proinflammatory markers in saline and Cp-treated mice. Given the protective role of heme oxygenase-1 (HO-1) in Cp-mediated apoptosis, we also investigated genotypic differences in renal HO-1 abundance, cell death, and proliferation by Western blotting, the TUNEL assay, and Ki67 immunostaining, respectively. Cp increased serum creatinine, urea, and neutrophil gelatinase-associated lipocalin (NGAL) levels, the renal abundance of kidney injury molecule-1, and NGAL in all groups. Cp-induced AKI resulted in comparable histological evidence of injury in all genotypes. WT and homo mice showed greater renal HO-1 abundance in response to Cp. Renal HO-1 abundance was lower in Cp-treated homo, compared to Cp-treated WT mice. Of note, GPER1 deletion elicited a remarkable increase in renal apoptosis; however, no genotypic differences in cell proliferation were observed. Cp augmented kidney Ki67-positive counts, regardless of the genotype. Overall, our data do not support a role for GPER1 in mediating Cp-induced renal injury. GPER1 deletion promotes renal apoptosis and diminishes HO-1 induction in response to Cp, suggesting that GPER1 may play cytoprotective and anti-apoptotic actions in AKI. GPER1-induced regulation of HO-1 and apoptosis may offer novel therapeutic targets for the treatment of AKI.

Thermoneutral Regulation and Acute Injury: Implications for Acute Kidney Injury.

Acute kidney injury (AKI) has demonstrated sex differences as illustrated in clinical and preclinical studies. In most cases, females show a significant resistance to AKI as manifested by renal indicators of injury, and thus much of the literature is derived from studies exclusively in males. Thermoneutral housing alters sex differences in acute injury of the liver, but has not been studied in the kidney. Thermoneutrality, the ambient temperature at which additional energy is not needed to maintain core body temperature, is regulated by mechanisms residing in mitochondria. Importantly, mitochondrial function plays an important role in induction and recovery of AKI. Mechanisms that regulate thermoneutrality include uncoupling proteins (UCPs) and one of its upstream regulators peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). PGC-1α has been extensively studied in AKI in males. UCP-2, a UCP expressed in the kidney, has been minimally studied in AKI in males. Expression of other UCPs in the kidney has not been well defined. No studies of either PGC-1α or UCPs have interrogated for a sex difference nor have they been investigated at thermoneutrality in the kidney. In this brief review, pathways of importance in thermoneutrality are described and related to pathways of importance in modulating susceptibility to AKI. Clarity in the understanding of the impact of thermoneutrality on AKI in altering susceptibility in females may expand our understanding of the critical role of mitochondrial function in this setting. Unique utilization of mitochondrial-based molecular pathways in females may then inform potential therapies.

UAB-UCSD O'Brien Center for Acute Kidney Injury Research.

Acute kidney injury (AKI) remains a significant clinical problem through its diverse etiologies, the challenges of robust measurements of injury and recovery, and its progression to chronic kidney disease (CKD). Bridging the gap in our knowledge of this disorder requires bringing together not only the technical resources for research but also the investigators currently endeavoring to expand our knowledge and those who might bring novel ideas and expertise to this important challenge. The University of Alabama at Birmingham-University of California-San Diego O'Brien Center for Acute Kidney Injury Research brings together technical expertise and programmatic and educational efforts to advance our knowledge in these diverse issues and the required infrastructure to develop areas of novel exploration. Since its inception in 2008, this O'Brien Center has grown its impact by providing state-of-the-art resources in clinical and preclinical modeling of AKI, a bioanalytical core that facilitates measurement of critical biomarkers, including serum creatinine via LC-MS/MS among others, and a biostatistical resource that assists from design to analysis. Through these core resources and with additional educational efforts, our center has grown its investigator base to include >200 members from 51 institutions. Importantly, this center has translated its pilot and catalyst funding program with a $37 return per dollar invested. Over 500 publications have resulted from the support provided with a relative citation ratio of 2.18 ± 0.12 (iCite). Through its efforts, this disease-centric O'Brien Center is providing the infrastructure and focus to help the development of the next generation of researchers in the basic and clinical science of AKI. This center creates the promise of the application at the bedside of the advances in AKI made by current and future investigators.

Renoprotective effect of Stat1 deletion in murine aristolochic acid nephropathy.

Injured tubule epithelium stimulates a profibrotic milieu that accelerates loss of function in chronic kidney disease (CKD). This study tested the role of signal transducer and activator of transcription 1 (STAT1) in the progressive loss of kidney function in aristolochic acid (AA) nephropathy, a model of CKD. Mean serum creatinine concentration increased in wild-type (WT) littermates treated with AA, whereas Stat1-/- mice were protected. Focal increases in the apical expression of kidney injury molecule (KIM)-1 were observed in the proximal tubules of WT mice with AA treatment but were absent in Stat1-/- mice in the treatment group as well as in both control groups. A composite injury score, an indicator of proximal tubule injury, was reduced in Stat1-/- mice treated with AA. Increased expression of integrin-ß6 and phosphorylated Smad2/3 in proximal tubules as well as interstitial collagen and fibronectin were observed in WT mice following AA treatment but were all decreased in AA-treated Stat1-/- mice. The data indicated that STAT1 activation facilitated the development of progressive kidney injury and interstitial fibrosis in AA nephropathy.

Protective role of HO-1 against acute kidney injury caused by cutaneous exposure to arsenicals.

Lewisite and many other similar arsenicals are warfare vesicants developed and weaponized for use in World Wars I and II. These chemicals, when exposed to the skin and other epithelial tissues, cause rapid severe inflammation and systemic damage. Here, we show that topically applied arsenicals in a murine model produce significant acute kidney injury (AKI), as determined by an increase in the AKI biomarkers NGAL and KIM-1. An increase in reactive oxygen species and ER stress proteins, such as ATF4 and CHOP, correlated with the induction of these AKI biomarkers. Also, TUNEL staining of CHOP-positive renal tubular cells suggests CHOP mediates apoptosis in these cells. A systemic inflammatory response characterized by a significant elevation in inflammatory mediators, such as IL-6, IFN-α, and COX-2, in the kidney could be the underlying cause of AKI. The mechanism of arsenical-mediated inflammation involves activation of AMPK/Nrf2 signaling pathways, which regulate heme oxygenase-1 (HO-1). Indeed, HO-1 induction with cobalt protoporphyrin (CoPP) treatment in arsenical-treated HEK293 cells afforded cytoprotection by attenuating CHOP-associated apoptosis and cytokine mRNA levels. These results demonstrate that topical exposure to arsenicals causes AKI and that HO-1 activation may serve a protective role in this setting.

Immunoglobulin light chains generate proinflammatory and profibrotic kidney injury.

Because of the less-than-robust response to therapy and impact on choice of optimal chemotherapy and prognosis, chronic kidney disease has drawn attention in the treatment of multiple myeloma, a malignant hematologic disorder that can produce significant amounts of monoclonal immunoglobulin free light chains (FLCs). These low-molecular-weight proteins are relatively freely filtered through the glomerulus and are reabsorbed by the proximal tubule. The present study demonstrated that during the process of metabolism of immunoglobulin FLCs, ROS activated the STAT1 pathway in proximal tubule epithelium. STAT1 activation served as the seminal signaling molecule that produced the proinflammatory molecule IL-1ß, as well as the profibrotic agent TGF-ß by this portion of the nephron. These effects occurred in vivo and were produced specifically by the generation of hydrogen peroxide by the VL domain of the light chain. To the extent that the experiments reflect the human condition, these studies offer insights into the pathogenesis of progressive kidney failure in the setting of lymphoproliferative disorders, such as multiple myeloma, that feature increased circulating levels of monoclonal immunoglobulin fragments that require metabolism by the kidney.

Unique sex- and age-dependent effects in protective pathways in acute kidney injury.

Sex and age influence susceptibility to acute kidney injury (AKI), with young females exhibiting lowest incidence. In these studies, we investigated mechanisms which may underlie the sex/age-based dissimilarities. Cisplatin (Cp)-induced AKI resulted in morphological evidence of injury in all groups. A minimal rise in plasma creatinine (PCr) was seen in Young Females, whereas in Aged Females, PCr rose precipitously. Relative to Young Males, Aged Males showed significantly, but temporally, comparably elevated PCr. Notably, Aged Females showed significantly greater mortality, whereas Young Females exhibited none. Tissue KIM-1 and plasma NGAL were significantly lower in Young Females than all others. IGFBP7 levels were modestly increased in both Young groups. IGFBP7 levels in Aged Females were significantly elevated at baseline relative to Aged Males, and increased linearly through day 3, when these levels were comparable in both Aged groups. Plasma cytokine levels similarly showed a pattern of protective effects preferentially in Young Females. Expression of the drug transporter MATE2 did not explain the sex/age distinctions. Heme oxygenase-1 (HO-1) levels (~28-kDa species) showed elevation at day 1 in all groups with highest levels seen in Young Males. Exclusively in Young Females, these levels returned to baseline on day 3, suggestive of a more efficient recovery. In aggregate, we demonstrate, for the first time, a distinctive pattern of response to AKI in Young Females relative to males which appears to be significantly altered in aging. These distinctions may offer novel targets to exploit therapeutically in both females and males in the treatment of AKI.

Leucine-rich repeat kinase 2 deficiency is protective in rhabdomyolysis-induced kidney injury.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common known genetic cause of Parkinson's disease, and LRRK2 is also linked to Crohn's and Hansen's disease. LRRK2 is expressed in many organs in mammals but is particularly abundant in the kidney. We find that LRRK2 protein is predominantly localized to collecting duct cells in the rat kidney, with much lower expression in other kidney cells. While genetic knockout (KO) of LRRK2 expression is well-tolerated in mice and rats, a unique age-dependent pathology develops in the kidney. The cortex and medulla of LRRK2 KO rat kidneys become darkly pigmented in early adulthood, yet aged animals display no overt signs of kidney failure. Accompanying the dark pigment we find substantial macrophage infiltration in LRRK2 KO kidneys, suggesting the presence of chronic inflammation that may predispose to kidney disease. Unexpectedly, the dark kidneys of the LRRK2 KO rats are highly resistant to rhabdomyolysis-induced acute kidney injury compared with wild-type rats. Biochemical profiling of the LRRK2 KO kidneys using immunohistochemistry, proteomic and lipidomic analyses show a massive accumulation of hemoglobin and lipofuscin in renal tubules that account for the pigmentation. The proximal tubules demonstrate a corresponding up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1) which is capable of mitigating acute kidney injury. The unusual kidney pathology of LRRK2 KO rats highlights several novel physiological roles for LRRK2 and provides indirect evidence for HO-1 expression as a protective mechanism in acute kidney injury in LRRK2 deficiency.

Heme Oxygenase-1 Regulates Myeloid Cell Trafficking in AKI.

Renal ischemia-reperfusion injury is mediated by a complex cascade of events, including the immune response, that occur secondary to injury to renal epithelial cells. We tested the hypothesis that heme oxygenase-1 (HO-1) expression, which is protective in ischemia-reperfusion injury, regulates trafficking of myeloid-derived immune cells in the kidney. Age-matched male wild-type (HO-1(+/+)), HO-1-knockout (HO-1(-/-)), and humanized HO-1-overexpressing (HBAC) mice underwent bilateral renal ischemia for 10 minutes. Ischemia-reperfusion injury resulted in significantly worse renal structure and function and increased mortality in HO-1(-/-) mice. In addition, there were more macrophages (CD45(+) CD11b(hi)F4/80(lo)) and neutrophils (CD45(+) CD11b(hi) MHCII(-) Gr-1(hi)) in HO-1(-/-) kidneys than in sham and HO-1(+/+) control kidneys subjected to ischemia-reperfusion. However, ischemic injury resulted in a significant decrease in the intrarenal resident dendritic cell (DC; CD45(+)MHCII(+)CD11b(lo)F4/80(hi)) population in HO-1(-/-) kidneys compared with controls. Syngeneic transplant experiments utilizing green fluorescent protein-positive HO-1(+/+) or HO-1(-/-) donor kidneys and green fluorescent protein-negative HO-1(+/+) recipients confirmed increased migration of the resident DC population from HO-1(-/-) donor kidneys, compared to HO-1(+/+) donor kidneys, to the peripheral lymphoid organs. This effect on renal DC migration was corroborated in myeloid-specific HO-1(-/-) mice subjected to bilateral ischemia. These mice also displayed impaired renal recovery and increased fibrosis at day 7 after injury. These results highlight an important role for HO-1 in orchestrating the trafficking of myeloid cells in AKI, which may represent a key pathway for therapeutic intervention.

Distinct populations of label-retaining cells in the adult kidney are defined temporally and exhibit divergent regional distributions.

DNA label-retention, or retention of a thymidine analog, is a characteristic of slow cycling cells and has been used to identify stem cells in several organ systems. Recent findings have demonstrated inconsistent localization of label-retaining cells (LRCs) in the kidney. Differences in the dose and timing of administration of deoxyuridine, the length of the chase period, and the species of animal used have made understanding the distinctions between these findings difficult. In the present studies, we utilized a dual loading scheme in the same animal to demonstrate that the cells labeled at different ages identified independent populations of LRC that distributed globally in an anti-parallel manner in the kidney. Loading with a DNA label in neonates identified LRC more often in the papilla, while administering the DNA label in adult mice identified LRC prominently in the cortex and the outer medulla. Furthermore, the tissue compartment distribution (epithelial-endothelial-interstitial) as well as the specific distribution within the nephron epithelia differed for these populations. These findings highlighted the complexity of the dynamics of cell proliferation in the kidney throughout the postnatal and adult period and call attention to the confusion associated with the term "label-retaining cells" for different timings of the loading and chase periods. This study indicated that the results of previous studies should be viewed as nonoverlapping and that further studies are needed to ascertain the role of each of these populations in the steady-state maintenance and injury recovery of the kidney.

Mechanism and prevention of acute kidney injury from cast nephropathy in a rodent model.

A common renal complication of multiple myeloma is "myeloma kidney," a condition also known as cast nephropathy. The renal lesions (casts) are directly related to the production of monoclonal immunoglobulin free light chains (FLCs), which coprecipitate with Tamm-Horsfall glycoprotein (THP) in the lumen of the distal nephron, obstructing tubular fluid flow. Here, we report that analysis of the binding interaction between FLCs and THP demonstrates that the secondary structure and key amino acid residues on the complementarity-determining region 3 (CDR3) of FLCs are critically important determinants of the molecular interaction with THP. The findings permitted development of a cyclized competitor peptide that demonstrated strong inhibitory capability in the binding of FLCs to THP in vitro. When used in a rodent model of cast nephropathy, this cyclized peptide construct served as an effective inhibitor of intraluminal cast formation and prevented the functional manifestations of acute kidney injury in vivo. These experiments provide proof of concept that intraluminal cast formation is integrally involved in the pathogenesis of acute kidney injury from cast nephropathy. Further, the data support a clinically relevant approach to the management of renal failure in the setting of multiple myeloma.

Paracrine effects of mesenchymal stem cells in cisplatin-induced renal injury require heme oxygenase-1.

Multipotent mesenchymal stem cells (MSC) have become a popular and promising therapeutic approach in many clinical conditions. MSC are beneficial in animal models of acute kidney injury (AKI), by mediating differentiation-independent paracrine properties, and have prompted ongoing clinical trials to evaluate the safety and efficacy of MSC. Heme oxygenase-1 (HO-1) is induced in response to stress including AKI and has important anti-apoptotic, anti-inflammatory, and proangiogenic properties in these settings. We therefore examined whether HO-1 plays a role in the beneficial effects of MSC in AKI. We isolated MSC from bone marrow of age-matched HO-1+/+ and HO-1-/- mice. Our studies indicate that while differentiation of MSC into osteo- and adipocytic lineages did not differ between cells isolated from HO-1+/+ and HO-1-/- mice, MSC from HO-1-/- mice had significantly lower angiogenic potential. Moreover, HO-1-/- MSC demonstrated reduced expression and secretion of several important growth and proangiogenic factors (stromal cell-derived factor-1, vascular endothelial growth factor-A, and hepatocyte growth factor) compared with MSC derived from HO-1+/+ mice. In addition, conditioned medium of HO-1+/+ MSC rescued functional and morphological changes associated with cisplatin-induced AKI, while the HO-1-/--conditioned medium was ineffectual. Our studies indicate that HO-1 plays an important role in MSC-mediated protection. The results expand understanding of the renoprotective effects of MSC and may provide novel strategies to better utilize MSC in various disease models.

Contribution of intrarenal cells to cellular repair after acute kidney injury: subcapsular implantation technique.

The kidney is capable of regeneration following injury, particularly following acute insults. Although the mechanisms underlying cellular regeneration are incompletely understood, emerging evidence suggests a role for cells of renal origin in the repair and replacement of damaged renal tubule cells. The overall hypothesis of this study is that native kidney cells that reside in a niche in the kidney provide robust contribution to the repair of kidney tubules following injury. To test this hypothesis, we utilized a model of renal ischemia-reperfusion injury that results in extensive morphological changes, particularly in the outer medulla. Renal tissue obtained from mice constitutively expressing Escherichia coli beta-galactosidase (ROSA26) was dissected from the cortex, outer medulla, or papilla and implanted under the renal capsule of the injured mice. Mice were allowed to recover for 7 days. Sections through the injured kidney demonstrated the presence of implant-derived cells in renal tubules in the outer medulla. The implanted renal region that exhibited the most robust response was the papilla, whereas tissue pieces from the cortex and outer medulla showed less contribution to recipient renal tubules. These results provide proof-of-principle evidence that renal-derived reparative cells reside in all regions of the kidney, perhaps more predominantly in the renal papilla. A greater understanding of the cell biology of renal repair by native kidney cells will provide further insight into the design of novel therapies in acute kidney injury, and the subcapsular implant technique described in this study may offer unique advantages to evaluate renal repair mechanisms.

Detection of early changes in renal function using 99mTc-MAG3 imaging in a murine model of ischemia-reperfusion injury.

Accurate determination of renal function in mice is a major impediment to the use of murine models in acute kidney injury. The purpose of this study was to determine whether early changes in renal function could be detected using dynamic gamma camera imaging in a mouse model of ischemia-reperfusion (I/R) injury. C57BL/6 mice (n = 5/group) underwent a right nephrectomy, followed by either 30 min of I/R injury or sham surgery of the remaining kidney. Dynamic renal studies (21 min, 10 s/frame) were conducted before surgery (baseline) and at 5, 24, and 48 h by injection of (99m)Tc-mercaptoacetyltriglycine (MAG3; approximately 1.0 mCi/mouse) via the tail vein. The percentage of injected dose (%ID) in the kidney was calculated for each 10-s interval after MAG3 injection, using standard region of interest analyses. A defect in renal function in I/R-treated mice was detected as early as 5 h after surgery compared with sham-treated mice, identified by the increased %ID (at peak) in the I/R-treated kidneys at 100 s (P < 0.01) that remained significantly higher than sham-treated mice for the duration of the scan until 600 s (P < 0.05). At 48 h, the renal scan demonstrated functional renal recovery of the I/R mice and was comparable to sham-treated mice. Our study shows that using dynamic imaging, renal dysfunction can be detected and quantified reliably as early as 5 h after I/R insult, allowing for evaluation of early treatment interventions.